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Sample records for hydroxymethylbilane synthase mutations

  1. Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS gene for analysis of flea (Ctenocephalides felis feeding patterns on dogs

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    Wang Chengming

    2012-01-01

    Full Text Available Abstract Background Precise data on quantitative kinetics of blood feeding of fleas, particularly immediately after contact with the host, are essential for understanding dynamics of flea-borne disease transmission and for evaluating flea control strategies. Standard methods used are inadequate for studies that simulate early events after real-life flea access to the host. Methods Here, we developed a novel quantitative polymerase chain reaction targeting mammalian DNA within fleas to quantify blood consumption with high sensitivity and specificity. We used primers and fluorescent probes that amplify the hydroxymethylbilane synthase (HMBS gene, an evolutionary divergent gene that is unlikely to be detected in insects by mammalian-specific primers and probes. To validate this assay, fleas were placed on dogs, allowed to distribute in the hair, and removed at specific time points with single-use combs. Fleas were then immediately homogenized by vigorous shaking with ceramic beads in guanidinium-based DNA preservation buffer for DNA extraction. Results The specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (Tm, and lack of amplification of bovine and porcine blood and of adult fleas reared from larvae fed with bovine blood. Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood. Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood. Saturation uptake of 118 and 100 nL blood per flea was found at 30 and 60 min on the dog, respectively. Conclusions The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of

  2. Understanding structure, function, and mutations in the mitochondrial ATP synthase

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    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  3. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance

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    Huang, Laurence; Crothers, Kristina; Atzori, Chiara

    2004-01-01

    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about...... in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim...... for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone....

  4. Mutations in the dihydropteroate synthase gene of Pneumocystis jiroveci isolates from Portuguese patients with Pneumocystis pneumonia

    DEFF Research Database (Denmark)

    Costa, M C; Helweg-Larsen, J; Lundgren, Bettina

    2003-01-01

    The aim of this study was to evaluate the frequency of mutations of the P. jiroveci dihydropteroate synthase (DHPS) gene in an immunocompromised Portuguese population and to investigate the possible association between DHPS mutations and sulpha exposure. In the studied population, DHPS gene...... mutations were not significantly more frequent in patients exposed to sulpha drugs compared with patients not exposed (P=0.390). The results of this study suggest that DHPS gene mutations are frequent in the Portuguese immunocompromised population but do not seem associated with previous sulpha exposure...

  5. Haplotype Analysis of Norwegian and Swedish Patients with Acute Intermittent Porphyria (AIP: Extreme Haplotype Heterogeneity for the Mutation R116W

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    Kjersti Tjensvoll

    2003-01-01

    Full Text Available Acute intermittent porphyria (AIP, the most common of the acute porphyrias, is caused by mutations in the gene encoding hydroxymethylbilane synthase (HMBS also called porphobilinogen deaminase (PBGD. The mutation spectrum in the HMBS gene is characterized by a majority of family specific mutations. Among the exceptions are R116W and W198X, with high prevalence in both the Dutch and Swedish populations. These two mutations were also detected in unrelated Norwegian patients. Thus, Norwegian and Swedish patients were haplotyped using closely linked flanking microsatellites and intragenic single nucleotide polymorphisms (SNPs to see if the high frequency of these two mutations is due to a founder effect. Twelve intragenic SNPs were determined by a method based on fluorescent restriction enzyme fingerprinting single-strand conformation polymorphism (F-REF-SSCP.

  6. Mutations and polymorphisms in the human N-acetylglutamate synthase (NAGS) gene.

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    Caldovic, Ljubica; Morizono, Hiroki; Tuchman, Mendel

    2007-08-01

    N-acetylglutamate synthase (NAGS) deficiency, an autosomal recessive disorder, is the last urea cycle disorder for which molecular testing became available. This is the first comprehensive report of 21 mutations that cause NAGS deficiency and of commonly found polymorphisms in the NAGS gene. Five mutations are reported here for the first time. A total of 10 disease-causing mutations are associated with acute neonatal hyperammonemia; the remaining mutations were found in patients with late onset disease. Residual enzymatic activities are included in this report and the deleterious effects of eight mutations were confirmed by expression studies. Mutations in the NAGS gene are distributed throughout its reading frame. No mutations have been found in exon 1, which encodes for the putative mitochondrial targeting signal and variable segment of NAGS. Three polymorphisms have been found. Early, accurate, and specific diagnosis of NAGS deficiency is critical since this condition can be successfully treated with N-carbamylglutamate (NCG, Carbaglu; Orphan Europe). Treatment with NCG should be initiated as soon as a patient is suspected of having NAGS deficiency. Molecular testing represents the most reliable method of diagnosis.

  7. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

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    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  8. Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids.

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    Kachanovsky, David E; Filler, Shdema; Isaacson, Tal; Hirschberg, Joseph

    2012-11-13

    Tomato (Solanum lycopersicum) fruit accumulate the red carotenoid pigment lycopene. The recessive mutation yellow-flesh (locus r) in tomato eliminates fruit carotenoids by disrupting the activity of the fruit-specific phytoene synthase (PSY1), the first committed step in the carotenoid biosynthesis pathway. Fruits of the recessive mutation tangerine (t) appear orange due to accumulation of 7,9,7',9'-tetra-cis-lycopene (prolycopene) as a result of a mutation in the carotenoid cis-trans isomerase. It was established 60 y ago that tangerine is epistatic to yellow-flesh. This uncharacteristic epistasis interaction defies a paradigm in biochemical genetics arguing that mutations that disrupt enzymes acting early in a biosynthetic pathway are epistatic to other mutations that block downstream steps in the same pathway. To explain this conundrum, we have investigated the interaction between tangerine and yellow-flesh at the molecular level. Results presented here indicate that allele r(2997) of yellow-flesh eliminates transcription of PSY1 in fruits. In a genetic background of tangerine, transcription of PSY1 is partially restored to a level sufficient for producing phytoene and downstream carotenoids. Our results revealed the molecular mechanism underlying the epistasis of t over r and suggest the involvement of cis-carotenoid metabolites in a feedback regulation of PSY1 gene expression.

  9. Evidence of positive selection for a glycogen synthase (GYS1) mutation in domestic horse populations.

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    McCoy, Annette M; Schaefer, Robert; Petersen, Jessica L; Morrell, Peter L; Slamka, Megan A; Mickelson, James R; Valberg, Stephanie J; McCue, Molly E

    2014-01-01

    A dominantly inherited gain-of-function mutation in the glycogen synthase (GYS1) gene, resulting in excess skeletal muscle glycogen, has been identified in more than 30 horse breeds. This mutation is associated with the disease Equine Polysaccharide Storage Myopathy Type 1, yet persists at high frequency in some breeds. Under historical conditions of daily work and limited feed, excess muscle glycogen may have been advantageous, driving the increase in frequency of this allele. Fine-scale DNA sequencing in 80 horses and genotype assays in 279 horses revealed a paucity of haplotypes carrying the mutant allele when compared with the wild-type allele. Additionally, we found increased linkage disequilibrium, measured by relative extended haplotype homozygosity, in haplotypes carrying the mutation compared with haplotypes carrying the wild-type allele. Coalescent simulations of Belgian horse populations demonstrated that the high frequency and extended haplotype associated with the GYS1 mutation were unlikely to have arisen under neutrality or due to population demography. In contrast, in Quarter Horses, elevated relative extended haplotype homozygosity was associated with multiple haplotypes and may be the result of recent population expansion or a popular sire effect. These data suggest that the GYS1 mutation underwent historical selection in the Belgian, but not in the Quarter Horse.

  10. Nonsense mutation inside anthocyanidin synthase gene controls pigmentation in yellow raspberry (Rubus idaeus L..

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    Muhammad Zubair Rafique

    2016-12-01

    Full Text Available Yellow raspberry fruits have reduced anthocyanin contents and offer unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase catalyzes the conversion of leucoanthocyanidin to anthocyanidin, a key committed step in biosynthesis of anthocyanins. Molecular analysis of the Ans gene enabled to identify an inactive ans allele in a yellow fruit raspberry (Anne. A 5-bp insertion in the coding region was identified and designated as ans+5. The insertion creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids wild type ANS protein. This mutation leads to loss of function of the encoded protein that might also result in transcriptional downregulation of Ans gene as a secondary effect i.e. nonsense-mRNA mediated decay. Further, this mutation results in loss of visible and detectable anthocyanin pigments. Functional characterization of raspberry Ans/ans alleles via complementation experiments in the Arabidopsis thaliana ldox mutant supports the inactivity of encoded protein through ans+5 and explains the proposed block in the anthocyanin biosynthetic pathway in raspberry. Taken together, our data shows that the mutation inside Ans gene in raspberry is responsible for yellow fruit phenotypes.

  11. Nonsense Mutation Inside Anthocyanidin Synthase Gene Controls Pigmentation in Yellow Raspberry (Rubus idaeus L.).

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    Rafique, Muhammad Z; Carvalho, Elisabete; Stracke, Ralf; Palmieri, Luisa; Herrera, Lorena; Feller, Antje; Malnoy, Mickael; Martens, Stefan

    2016-01-01

    Yellow raspberry fruits have reduced anthocyanin contents and offer unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase (Ans) catalyzes the conversion of leucoanthocyanidin to anthocyanidin, a key committed step in biosynthesis of anthocyanins. Molecular analysis of the Ans gene enabled to identify an inactive ans allele in a yellow fruit raspberry ("Anne"). A 5 bp insertion in the coding region was identified and designated as ans(+5). The insertion creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids wild-type ANS protein. This mutation leads to loss of function of the encoded protein that might also result in transcriptional downregulation of Ans gene as a secondary effect, i.e., nonsense-mediated mRNA decay. Further, this mutation results in loss of visible and detectable anthocyanin pigments. Functional characterization of raspberry Ans/ans alleles via complementation experiments in the Arabidopsis thaliana ldox mutant supports the inactivity of encoded protein through ans(+5) and explains the proposed block in the anthocyanin biosynthetic pathway in raspberry. Taken together, our data shows that the mutation inside Ans gene in raspberry is responsible for yellow fruit phenotypes.

  12. Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

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    Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison; Sanford, Lynn; Hamada, Jeffrey; Bettencourt, Conceição; Houlden, Henry; Chiapparini, Luisa; Zorzi, Giovanna; Kurian, Manju A; Nardocci, Nardo; Prokisch, Holger; Hayflick, Susan; Gout, Ivan; Tiranti, Valeria

    2014-01-02

    Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.

  13. Acetolactate synthase mutation conferring imidazolinone-specific herbicide resistance in Amaranthus hybridus.

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    Trucco, Federico; Hager, Aaron G; Tranel, Patrick J

    2006-03-01

    Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branched-chain amino acids in plants and is the target of several herbicides. ALS inhibitors have enjoyed popularity as herbicides due to numerous attributes, although their current adequacy in weed control programs is hampered by herbicide resistance. Most cases of ALS-inhibitor resistance have resulted from selection of an altered target site. The study herein reports on an alanine by threonine amino acid substitution at position 122 of ALS as the basis for imidazolinone-specific resistance in an A. hybridus population from Illinois. In vitro inhibition of enzymatic activity (I(50)) required 1000-fold greater concentration of imazethapyr in the resistant population compared with a susceptible control. This mutation represents the second ALS alteration associated with herbicide resistance in a natural A. hybridus population.

  14. Effects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P. carinii pneumonia

    DEFF Research Database (Denmark)

    Helweg-Larsen, J; Benfield, Thomas; Eugen-Olsen, Jesper

    1999-01-01

    BACKGROUND: Sulpha drugs are widely used for the treatment and long-term prophylaxis of Pneumocystis carinii pneumonia (PCP) in HIV-1-infected individuals. Sulpha resistance in many microorganisms is caused by point mutations in dihydropteroate synthase (DHPS), an enzyme that is essential...

  15. ATP Synthase Deficiency due to TMEM70 Mutation Leads to Ultrastructural Mitochondrial Degeneration and Is Amenable to Treatment

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    Anne K. Braczynski

    2015-01-01

    Full Text Available TMEM70 is involved in the biogenesis of mitochondrial ATP synthase and mutations in the TMEM70 gene impair oxidative phosphorylation. Herein, we report on pathology and treatment of ATP synthase deficiency in four siblings. A consanguineous family of Roma (Gipsy ethnic origin gave birth to 6 children of which 4 were affected presenting with dysmorphic features, failure to thrive, cardiomyopathy, metabolic crises, and 3-methylglutaconic aciduria as clinical symptoms. Genetic testing revealed a homozygous mutation (c.317-2A>G in the TMEM70 gene. While light microscopy was unremarkable, ultrastructural investigation of muscle tissue revealed accumulation of swollen degenerated mitochondria with lipid crystalloid inclusions, cristae aggregation, and exocytosis of mitochondrial material. Biochemical analysis of mitochondrial complexes showed an almost complete ATP synthase deficiency. Despite harbouring the same mutation, the clinical outcome in the four siblings was different. Two children died within 60 h after birth; the other two had recurrent life-threatening metabolic crises but were successfully managed with supplementation of anaplerotic amino acids, lipids, and symptomatic treatment during metabolic crisis. In summary, TMEM70 mutations can cause distinct ultrastructural mitochondrial degeneration and almost complete deficiency of ATP synthase but are still amenable to treatment.

  16. A transgenic Neospora caninum strain based on mutations of the dihydrofolate reductase-thymidylate synthase gene.

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    Pereira, Luiz Miguel; Baroni, Luciana; Yatsuda, Ana Patrícia

    2014-03-01

    Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (β-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii.

  17. Effects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P. carinii pneumonia

    DEFF Research Database (Denmark)

    Helweg-Larsen, J; Benfield, Thomas; Eugen-Olsen, J

    1999-01-01

    Sulpha drugs are widely used for the treatment and long-term prophylaxis of Pneumocystis carinii pneumonia (PCP) in HIV-1-infected individuals. Sulpha resistance in many microorganisms is caused by point mutations in dihydropteroate synthase (DHPS), an enzyme that is essential for folate biosynth......Sulpha drugs are widely used for the treatment and long-term prophylaxis of Pneumocystis carinii pneumonia (PCP) in HIV-1-infected individuals. Sulpha resistance in many microorganisms is caused by point mutations in dihydropteroate synthase (DHPS), an enzyme that is essential for folate...... biosynthesis. We assessed whether mutations in the DHPS gene of P. carinii were associated with exposure to sulpha drugs and influenced outcome from PCP....

  18. The clinical significance of aldosterone synthase deficiency: report of a novel mutation in the CYP11B2 gene

    Science.gov (United States)

    2014-01-01

    Background Aldosterone synthase (CYP11B2) deficiency is a rare autosomal recessive disorder, usually presenting with severe salt-wasting in infancy or stress-induced hyperkalaemia and postural hypotension in adulthood. Neonatal screening for congenital adrenal hyperplasia, another cause of salt wasting, using 17-hydroxyprogesterone measurement would fail to detect aldosterone synthase deficiency, a diagnosis which may be missed until the patient presents with salt-wasting crisis. Due to this potential life-threatening risk, comprehensive hormonal investigation followed by genetic confirmation for suspected patients would facilitate clinical management of the patient and assessment of the genetic implication in their offspring. Case presentation We describe a 33-year old Chinese man who presented in infancy with life-threatening hyponatraemia and failure to thrive, but remained asymptomatic on fludrocortisone since. Chromosomal analysis confirmed a normal male karyotype of 46, XY. Plasma steroid profile showed high plasma renin activity, low aldosterone level, and elevated 18-hydroxycorticosterone, compatible with type 2 aldosterone synthase deficiency. The patient was heterozygous for a novel CYP11B2 mutation: c.977C > A (p.Thr326Lys) in exon 3. He also carried a heterozygous mutation c.523_525delAAG (p.Lys175del) in exon 6, a known pathogenic mutation causing aldosterone synthase deficiency. Sequencing of CYP11B2 in his parents demonstrated that the mother was heterozygous for c.977C > A, and the father was heterozygous for c.523_525delAAG. Conclusion Although a rare cause of hyperreninaemic hypoaldosteronism, aldosterone synthase deficiency should be suspected and the diagnosis sought in patients who present with life-threatening salt-wasting in infancy, as it has a good long-term prognosis when adequate fludrocortisone replacement is instituted. To our knowledge, this is the first Chinese patient in which the molecular basis of aldosterone synthase

  19. A case of primary selective hypoaldosteronism carrying three mutations in the aldosterone synthase (Cyp11b2) gene.

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    Taranta, Anna; Bizzarri, Carla; Masotti, Andrea; Sciré, Giuseppe; Pampanini, Valentina; Cappa, Marco

    2012-05-25

    An infant with a clinical phenotype of early onset hypoaldosteronism has been screened for mutation analysis of the Cyp11b2 gene encoding aldosterone synthase enzyme. We have described a novel nonsense mutation in exon 3 (c.508C>T) that gave rise to a shorter protein (Q170X) and two known concurrent missense mutations (c.594A>C in exon 3 and c.1157T>C in exon 7) that led to substitution of glutamic acid for aspartic acid at amino acid position 198 (E198D) and of valine for alanine at amino acid position 386 (V386A). The father, who carried E198D plus V386A mutations, showed a fractional sodium excretion of 1.25% that was unmodified by dietary salt restriction, suggesting a mild haploinsufficiency. We examined by in silico analysis the effect of the mutations on the secondary and tertiary structures of aldosterone synthase to explain the inefficient enzymatic activity. The Q170X mutation produced a truncated protein, which was consequently associated with a loss of catalytic activity. As predicted by JPred web system and Dock 6.3 software, the concurrent expression of E198D and V386A mutations induced a significant secondary structure rearrangement and a shift of the heme group and the 18-hydroxycorticosterone substrate from their optimal placement.

  20. Late-onset cutaneous porphyria in a patient heterozygous for a uroporphyrinogen III synthase gene mutation.

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    Aguilera, P; Badenas, C; Whatley, S D; To-Figueras, J

    2016-12-01

    Deficiency of uroporphyrinogen III synthase (UROS) causes congenital erythropoietic porphyria (CEP). The disease, originating from the inheritance of mutations within the UROS gene, presents a recessive form of transmission. In a few patients, a late-onset CEP-like phenotype without UROS mutations appears to be associated with a myelodysplastic syndrome. We report a 60-year-old man with late-onset signs of cutaneous porphyria and accumulation in urine, plasma and faeces of type I porphyrin isomers characteristic of CEP. Analysis of DNA from peripheral leucocytes, skin and bone marrow aspirate showed that he was a heterozygous carrier of a Cys73Arg (c.217 T>C) mutation within UROS. Sequencing of cDNA from peripheral blood confirmed heterozygosity and expression of the normal allele. Measurement of UROS enzymatic activity in erythrocytes showed values ~70% of normal, indirectly indicating expression of the normal allele. Differently from other cases of late-onset uroporphyria, the patient did not present thrombocytopenia or any evidence of a myelodysplastic syndrome. Five years of clinical follow-up showed persistence of skin signs and increased excretion of porphyrins, independently of lifestyle factors or changes in medication regimes. We hypothesize acquired mosaicism (in the bone marrow) affecting the UROS gene. Thus, unstable cellular clones initiated overproduction of isomer I porphyrins leading to a CEP phenotype. This could be explained either by a clonal expansion of the porphyric (Cys73Arg) allele or by loss of function of the normal allele. Cellular turnover would facilitate release of uroporphyrins into circulation and subsequent skin lesions. This is the first case of a CEP heterozygous carrier presenting clinical manifestations.

  1. 16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases.

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    Conrad, J; Niu, L; Rudd, K; Lane, B G; Ofengand, J

    1999-06-01

    The gene for RsuA, the pseudouridine synthase that converts U516 to pseudouridine in 16S ribosomal RNA of Escherichia coli, has been deleted in strains MG1655 and BL21/DE3. Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in trans on a plasmid restored the pseudouridine. Therefore, rsuA is the only gene in E. coli with the ability to produce a protein capable of forming pseudouridine516. There was no effect on the growth rate of rsuA- MG1655 either in rich or minimal medium at either 24, 37, or 42 degrees C. Plasmid rescue of the BL21/DE3 rsuA- strain using pET15b containing an rsuA gene with aspartate102 replaced by asparagine or threonine demonstrated that neither mutant was active in vivo. This result supports a role for this aspartate, located in a unique GRLD sequence in this gene, at the catalytic center of the synthase. Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the lack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases. The conservation and distribution of this motif in nature was assessed.

  2. Effects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P. carinii pneumonia

    DEFF Research Database (Denmark)

    Helweg-Larsen, J; Benfield, Thomas; Eugen-Olsen, Jesper

    1999-01-01

    BACKGROUND: Sulpha drugs are widely used for the treatment and long-term prophylaxis of Pneumocystis carinii pneumonia (PCP) in HIV-1-infected individuals. Sulpha resistance in many microorganisms is caused by point mutations in dihydropteroate synthase (DHPS), an enzyme that is essential...... for folate biosynthesis. We assessed whether mutations in the DHPS gene of P. carinii were associated with exposure to sulpha drugs and influenced outcome from PCP. METHODS: We studied bronchoalveolar samples collected in 1989-99 from a prospective cohort of HIV-1-infected patients who had PCP. In 144...... patients with 152 episodes of PCP, we analysed portions of DHPS using PCR and direct sequencing. The relation between survival, P. carinii DHPS mutations, and other predictors of treatment failure was assessed by Kaplan-Meier and multivariate Cox regression analysis. FINDINGS: P. carinii DHPS mutations...

  3. Novel point mutation in the uroporphyrinogen III synthase gene causes congenital erythropoietic porphyria of a Japanese family.

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    Takamura, N; Hombrados, I; Tanigawa, K; Namba, H; Nagayama, Y; de Verneuil, H; Yamashita, S

    1997-06-13

    The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in a member of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Günther's disease. The patient was homozygous for a novel missense mutation: a G to T transition of nucleotide 7 that predicted a valine to phenylalanine substitution at residue 3 (V3F). The parents were heterozygous for the same mutation. The loss of UROIIIS activity was verified by an in vitro assay system. The corresponding mutated protein was expressed in Escherichia coli and no residual activity was observed. Further studies are needed to determine whether the mutations of the UROIIIS gene (UROS) have a specific profile in Japan compared to European or American countries.

  4. Functional modeling of vitamin responsiveness in yeast: a common pyridoxine-responsive cystathionine beta-synthase mutation in homocystinuria.

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    Kim, C E; Gallagher, P M; Guttormsen, A B; Refsum, H; Ueland, P M; Ose, L; Folling, I; Whitehead, A S; Tsai, M Y; Kruger, W D

    1997-12-01

    Cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder which results in extremely elevated levels of total plasma homocysteine (tHcy) and high risk of thromboembolic events. About half of all patients diagnosed with CBS deficiency respond to pyridoxine treatment with a significant lowering of tHcy levels. We examined 12 CBS-deficient patients from 10 Norwegian families for mutations in the CBS gene and identified mutations in 18 of the 20 CBS alleles. Five of the seven patients classified as pyridoxine-responsive contain the newly identified point mutation, G797A (R266K). This point mutation is tightly linked with a previously identified 'benign' 68 bp duplication of the intron 7-exon 8 boundary within the CBS gene. We tested the effect of all of the mutations identified on human CBS function utilizing a yeast system. Five of the six mutations had a distinguishable phenotype in yeast, indicating that they were in fact pathogenic. Interestingly, the G797A allele had no phenotype when the yeast were grown in high concentrations of pyridoxine, but a severe phenotype when grown in low concentrations, thus mirroring the behavior in humans. These studies show that the G797A mutation is an important cause of pyridoxine-responsive CBS deficiency and demonstrate the utility of yeast functional assays in the analysis of human mutations.

  5. Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families

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    Suchi, Mariko; Mizuno, Haruo; Tsuboi, Takashi [Nagoya City Univ. Medical School (Japan)] [and others

    1997-03-01

    Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5{prime}-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a {lambda}EMBL-3 human genomic library and report a single-copy gene spanning {approximately}15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5{prime} flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A- to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A ({nu} = .26) and 440 Gpoly ({nu} = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function. 76 refs., 5 figs., 7 tabs.

  6. Uroporphyrinogen III synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause congenital erythropoietic porphyria.

    Science.gov (United States)

    Solis, C; Aizencang, G I; Astrin, K H; Bishop, D F; Desnick, R J

    2001-03-01

    Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelated patients, and (d) a -90C to A transversion in a putative CP2 binding motif. Also, a -224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the -70C mutation altered GATA1 binding, whereas the adjacent -76A mutation did not. Similarly, the -90C mutation altered CP2 binding, whereas the -86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.

  7. Structural insights into E. coli porphobilinogen deaminase during synthesis and exit of 1-hydroxymethylbilane.

    Directory of Open Access Journals (Sweden)

    Navneet Bung

    2014-03-01

    Full Text Available Porphobilinogen deaminase (PBGD catalyzes the formation of 1-hydroxymethylbilane (HMB, a crucial intermediate in tetrapyrrole biosynthesis, through a step-wise polymerization of four molecules of porphobilinogen (PBG, using a unique dipyrromethane (DPM cofactor. Structural and biochemical studies have suggested residues with catalytic importance, but their specific role in the mechanism and the dynamic behavior of the protein with respect to the growing pyrrole chain remains unknown. Molecular dynamics simulations of the protein through the different stages of pyrrole chain elongation suggested that the compactness of the overall protein decreases progressively with addition of each pyrrole ring. Essential dynamics showed that domains move apart while the cofactor turn region moves towards the second domain, thus creating space for the pyrrole rings added at each stage. Residues of the flexible active site loop play a significant role in its modulation. Steered molecular dynamics was performed to predict the exit mechanism of HMB from PBGD at the end of the catalytic cycle. Based on the force profile and minimal structural changes the proposed path for the exit of HMB is through the space between the domains flanking the active site loop. Residues reported as catalytically important, also play an important role in the exit of HMB. Further, upon removal of HMB, the structure of PBGD gradually relaxes to resemble its initial stage structure, indicating its readiness to resume a new catalytic cycle.

  8. Mutation in the gene encoding 1-aminocyclopropane-1-carboxylate synthase 4 (CitACS4) led to andromonoecy in watermelon.

    Science.gov (United States)

    Ji, Gaojie; Zhang, Jie; Zhang, Haiying; Sun, Honghe; Gong, Guoyi; Shi, Jianting; Tian, Shouwei; Guo, Shaogui; Ren, Yi; Shen, Huolin; Gao, Junping; Xu, Yong

    2016-09-01

    Although it has been reported previously that ethylene plays a critical role in sex determination in cucurbit species, how the andromonoecy that carries both the male and hermaphroditic flowers is determined in watermelon is still unknown. Here we showed that the watermelon gene 1-aminocyclopropane-1-carboxylate synthase 4 (CitACS4), expressed specifically in carpel primordia, determines the andromonoecy in watermelon. Among four single nucleotide polymorphism (SNPs) and one InDel identified in the coding region of CitACS4, the C364W mutation located in the conserved box 6 was co-segregated with andromonoecy. Enzymatic analyses showed that the C364W mutation caused a reduced activity in CitACS4. We believe that the reduced CitACS4 activity may hamper the programmed cell death in stamen primordia, leading to the formation of hermaphroditic flowers. © 2016 Institute of Botany, Chinese Academy of Sciences.

  9. Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause Lenz-Majewski syndrome.

    Science.gov (United States)

    Sousa, Sérgio B; Jenkins, Dagan; Chanudet, Estelle; Tasseva, Guergana; Ishida, Miho; Anderson, Glenn; Docker, James; Ryten, Mina; Sa, Joaquim; Saraiva, Jorge M; Barnicoat, Angela; Scott, Richard; Calder, Alistair; Wattanasirichaigoon, Duangrurdee; Chrzanowska, Krystyna; Simandlová, Martina; Van Maldergem, Lionel; Stanier, Philip; Beales, Philip L; Vance, Jean E; Moore, Gudrun E

    2014-01-01

    Lenz-Majewski syndrome (LMS) is a syndrome of intellectual disability and multiple congenital anomalies that features generalized craniotubular hyperostosis. By using whole-exome sequencing and selecting variants consistent with the predicted dominant de novo etiology of LMS, we identified causative heterozygous missense mutations in PTDSS1, which encodes phosphatidylserine synthase 1 (PSS1). PSS1 is one of two enzymes involved in the production of phosphatidylserine. Phosphatidylserine synthesis was increased in intact fibroblasts from affected individuals, and end-product inhibition of PSS1 by phosphatidylserine was markedly reduced. Therefore, these mutations cause a gain-of-function effect associated with regulatory dysfunction of PSS1. We have identified LMS as the first human disease, to our knowledge, caused by disrupted phosphatidylserine metabolism. Our results point to an unexplored link between phosphatidylserine synthesis and bone metabolism.

  10. Congenital erythropoietic porphyria with two mutations of the uroporphyrinogen III synthase gene (Cys73Arg, Thr228Met).

    Science.gov (United States)

    Gucev, Zoran; Slavevska, Nevenka; Tasic, Velibor; Laban, Nevenka; Pop-Jordanova, Nada; Danilovski, Dragan; Woolf, Jacqueline; Cole, Duncan

    2011-05-01

    Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of uroporphyrinogen III synthase (UROS). We describe a 14-year-old girl with red urine since infancy, progressive blistering and scarring of the skin, and moderate hemolytic anemia. After years of skin damage, her face is mutilated; she has a bald patch on the scalp, hypertrichosis of the neck, areas of skin darkening, and limited joint movements of the hands. Total urine excretion and fecal total porphyrin were both markedly raised above normal levels. Sequencing of the UROS gene identified two mutations causing CEP (Cys73Arg, Thr228Met). The patient lesions are progressing. Bone marrow transplantation and/or gene therapy are proposed as the next steps in her treatment. In brief, we describe a CEP with confirmed two pathogenic mutations, severe phenotype and discuss the various treatment options available.

  11. Congenital erythropoietic porphyria with two mutations of the uroporphyrinogen III synthase gene (Cys73Arg, Thr228Met

    Directory of Open Access Journals (Sweden)

    Zoran Gucev

    2011-01-01

    Full Text Available Congenital erythropoietic porphyria (CEP is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of uroporphyrinogen III synthase (UROS. We describe a 14-year-old girl with red urine since infancy, progressive blistering and scarring of the skin, and moderate hemolytic anemia. After years of skin damage, her face is mutilated; she has a bald patch on the scalp, hypertrichosis of the neck, areas of skin darkening, and limited joint movements of the hands. Total urine excretion and fecal total porphyrin were both markedly raised above normal levels. Sequencing of the UROS gene identified two mutations causing CEP (Cys73Arg, Thr228Met. The patient lesions are progressing. Bone marrow transplantation and/or gene therapy are proposed as the next steps in her treatment. In brief, we describe a CEP with confirmed two pathogenic mutations, severe phenotype and discuss the various treatment options available.

  12. Modeling human Coenzyme A synthase mutation in yeast reveals altered mitochondrial function, lipid content and iron metabolism

    Directory of Open Access Journals (Sweden)

    Camilla Ceccatelli Berti

    2015-04-01

    Full Text Available Mutations in nuclear genes associated with defective coenzyme A biosynthesis have been identified as responsible for some forms of neurodegeneration with brain iron accumulation (NBIA, namely PKAN and CoPAN. PKAN are defined by mutations in PANK2, encoding the pantothenate kinase 2 enzyme, that account for about 50% of cases of NBIA, whereas mutations in CoA synthase COASY have been recently reported as the second inborn error of CoA synthesis leading to CoPAN. As reported previously, yeast cells expressing the pathogenic mutation exhibited a temperature-sensitive growth defect in the absence of pantothenate and a reduced CoA content. Additional characterization revealed decreased oxygen consumption, reduced activities of mitochondrial respiratory complexes, higher iron content, increased sensitivity to oxidative stress and reduced amount of lipid droplets, thus partially recapitulating the phenotypes found in patients and establishing yeast as a potential model to clarify the pathogenesis underlying PKAN and CoPAN diseases.

  13. Seven Novel Mutations in Bulgarian Patients with Acute Hepatic Porphyrias (AHP).

    Science.gov (United States)

    Dragneva, Sonya; Szyszka-Niagolov, Monika; Ivanova, Aneta; Mateva, Lyudmila; Izumi, Rumiko; Aoki, Yoko; Matsubara, Yoichi

    2014-01-01

    Acute intermittent porphyria (AIP), variegate porphyria (VP), and hereditary coproporphyria (HCP) are caused by mutations in the hydroxymethylbilane synthase (HMBS), protoporphyrinogen oxidase (PPOX), and coproporphyrinogen oxidase (CPOX) genes, respectively. This study aimed to identify mutations in seven Bulgarian families with AIP, six with VP, and one with HCP. A total of 33 subjects, both symptomatic (n = 21) and asymptomatic (n = 12), were included in this study. The identification of mutations was performed by direct sequencing of all the coding exons of the corresponding enzymes in the probands. The available relatives were screened for the possible mutations. A total of six different mutations in HMBS were detected in all seven families with AIP, three of which were previously described: c.76C>T [p.R26C] in exon 3, c.287C>T [p.S96F] in exon 7, and c.445C>T [p.R149X] in exon 9. The following three novel HMBS mutations were found: c.345-2A>C in intron 7-8, c.279-280insAT in exon 7, and c.887delC in exon 15. A total of three different novel mutations were identified in the PPOX gene in the VP families: c.441-442delCA in exon 5, c.917T>C [p.L306P] in exon 9, and c.1252T>C [p.C418R] in exon 12. A novel nonsense mutation, c.364G>T [p.E122X], in exon 1 of the CPOX gene was identified in the HCP family. This study, which identified mutations in Bulgarian families with AHP for the first time, established seven novel mutation sites. Seven latent carriers were also diagnosed and, therefore, were able to receive crucial counseling to prevent attacks.

  14. Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA).

    Science.gov (United States)

    Fernandez-Vizarra, Erika; Berardinelli, Angela; Valente, Lucia; Tiranti, Valeria; Zeviani, Massimo

    2009-01-01

    Myopathy, lactic acidosis and sideroblastic anaemia (MLASA) is a rare condition that combines early-onset myopathy with lactic acidosis and sideroblastic anaemia. MLASA has been associated with a missense mutation in pseudouridylate synthase 1 (PUS1), an enzyme located in both nucleus and mitochondria, which converts uridine into pseudouridine in several cytosolic and mitochondrial tRNA positions and increases the efficiency of protein synthesis in both compartments. We examined two Italian brothers with MLSA and sequenced the PUS1 gene. We found combined defects in mitochondrial respiratory chain complexes in muscle and fibroblast homogenates of both patients, and low levels of mtDNA translation products in fibroblast mitochondria. A novel, homozygous stop mutation was present in PUS1 (E220X). The stop mutation in PUS1 is likely to determine the loss of function of the protein, since it predicts the synthesis of a protein missing 208/427 amino acid residues on the C terminus, and was associated with low mtDNA translation.

  15. Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε.

    Science.gov (United States)

    Roy, Ankoor; Hutcheon, Marcus L; Duncan, Thomas M; Cingolani, Gino

    2012-10-01

    The bacterial ATP synthase (F(O)F(1)) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F(O)F(1) represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F(O)) and a peripheral catalytic complex (F(1)). The ATPase activity of isolated F(1) is fully expressed by the α(3)β(3)γ 'core', whereas single δ and ε subunits are required for structural and functional coupling of E. coli F(1) to F(O). In contrast to mitochondrial F(1)-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F(1)-ATPase catalytic core. Destabilizing the compact conformation of ε's C-terminal domain with a phosphomimetic mutation (εS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ∼3.15 Å resolution.

  16. Recurrent De Novo Mutations Affecting Residue Arg1 38 of Pyrroline-5-Carboxylate Synthase Cause a Progeroid Form of Autosomal-Dominant Cutis Laxa

    NARCIS (Netherlands)

    Fischer-Zirnsak, Bjoern; Escande-Beillard, Nathalie; Ganesh, Jaya; Tan, Yu Xuan; Al Bughaili, Mohammed; Lin, Angela E.; Sahai, Inderneel; Bahena, Paulina; Reichert, Sara L.; Loh, Abigail; Wright, Graham D.; Liu, Jaron; Rahikkala, Elisa; Pivnick, Eniko K.; Choudhri, Asim F.; Krueger, Ulrike; Zemojtel, Tomasz; van Ravenswaaij-Arts, Conny; Mostafavi, Roya; Stolte-Dijkstra, Irene; Symoens, Sofie; Pajunen, Leila; Al-Gazali, Lihadh; Meierhofer, David; Robinson, Peter N.; Mundlos, Stefan; Villarroel, Camilo E.; Byers, Peter; Masri, Amira; Robertson, Stephen P.; Schwarze, Ulrike; Callewaert, Bert; Reversade, Bruno; Kornak, Uwe

    2015-01-01

    Progeroid disorders overlapping with De Barsy syndrome (DBS) are collectively denoted as autosomal-recessive cutis laxa type 3 (ARCL3). They are caused by biallelic mutations in PYCR1 or ALDH18A1, encoding pyrroline-5-carboxylate reductase 1 and pyrroline-5-carboxylate synthase (P5CS), respectively,

  17. Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp(376)Glu mutation in the acetolactate synthase.

    Science.gov (United States)

    Sada, Yoshinao; Ikeda, Hajime; Yamato, Seiji; Kizawa, Satoru

    2013-09-01

    Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp376Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose-response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level. In whole-plant tests, resistance factors (RFs) based on 50% growth reduction (GR50) for imazosulfuron (ISF), bensulfuron-methyl (BSM), metsulfuron-methyl (MSM), bispyribac-sodium (BPS), and imazaquin (IMQ) were 176, 40, 14, 5.2 and 1.5, respectively. Thus, the accession having an Asp376Glu mutation in ALS2 was highly resistant to the three SU herbicides and moderately resistant to BPS, but was not substantially resistant to IMQ. This is slightly different from the earlier results reported from other weeds with an Asp376Glu mutation, in which the mutation confers resistance to broadly all the chemical classes of ALS-inhibiting herbicides. In enzymatic tests, ALS2 of S. juncoides was expressed in E. coli; the resultant ALS2 was subjected to an in vitro assay. RFs of the mutated ALS2 based on 50% enzymatic inhibition (I50) for ISF, BSM, MSM, BPS, and IMQ were 3699, 2438, 322, 80, and 4.8, respectively. The RFs of ALS2 were highly correlated with those of the whole-plant; this suggests that the Asp376Glu mutation in ALS2 is a molecular basis for the whole-plant resistance. The presence of two ALS genes in S. juncoides can at least partially explain why the whole-plant RFs were less than those of the expressed ALS2 enzymes.

  18. Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C.

    Science.gov (United States)

    Kabala, Anna Magdalena; Lasserre, Jean-Paul; Ackerman, Sharon H; di Rago, Jean-Paul; Kucharczyk, Roza

    2014-05-01

    Mutations in the human mitochondrial ATP6 gene encoding ATP synthase subunit a/6 (referred to as Atp6p in yeast) are at the base of neurodegenerative disorders like Neurogenic Ataxia and Retinitis Pigmentosa (NARP), Leigh syndrome (LS), Charcot-Marie-Tooth (CMT), and ataxia telangiectasia. In previous studies, using the yeast Saccharomyces cerevisiae as a model we were able to better define how several of these mutations impact the ATP synthase. Here we report the construction of yeast models of two other ATP6 pathogenic mutations, T9185C and T9191C. The first one was reported as conferring a mild, sometimes reversible, CMT clinical phenotype; the second one has been described in a patient presenting with severe LS. We found that an equivalent of the T9185C mutation partially impaired the functioning of yeast ATP synthase, with only a 30% deficit in mitochondrial ATP production. An equivalent of the mutation T9191C had much more severe effects, with a nearly complete block in yeast Atp6p assembly and an >95% drop in the rate of ATP synthesis. These findings provide a molecular basis for the relative severities of the diseases induced by T9185C and T9191C.

  19. Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.

    Science.gov (United States)

    Tian, Yong-Sheng; Xu, Jing; Peng, Ri-He; Xiong, Ai-Sheng; Xu, Hu; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Yao, Quan-Hong

    2013-09-01

    A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.

  20. Improvement of glyphosate resistance through concurrent mutations in three amino acids of the Ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase.

    Science.gov (United States)

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-12-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

  1. Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

    Science.gov (United States)

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-01-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops. PMID:21948846

  2. Enzymatic characterization and mutational studies of TruD--the fifth family of pseudouridine synthases.

    Science.gov (United States)

    Chan, Chio Mui; Huang, Raven H

    2009-09-01

    Pseudouridine (Psi) is formed through isomerization of uridine (U) catalyzed by a class of enzymes called pseudouridine synthases (PsiS). TruD is the fifth family of PsiS. Studies of the first four families (TruA, TruB, RsuA, and RluA) of PsiS reveal a conserved Asp and Tyr are critical for catalysis. However, in TruD family, the tyrosine is not conserved. In this study, we measured the enzymatic parameters for TruD in Escherichia coli, and carried out enzymatic assays for a series of single, double, and triple TruD mutants. Our studies indicate that a Glu, strictly conserved in only TruD family is likely to be the general base in TruD. We also proposed a possible distinct mechanism of TruD-catalyzed Psi formation compared to the first four families.

  3. Mutations in the Antifolate-Resistance-Associated Genes Dihydrofolate Reductase and Dihydropteroate Synthase in Plasmodium vivax Isolates from Malaria-Endemic Countries

    Science.gov (United States)

    Lu, Feng; Lim, Chae Seung; Nam, Deok Hwa; Kim, Kwonkee; Lin, Khin; Kim, Tong-Soo; Lee, Hyeong-Woo; Chen, Jun-Hu; Wang, Yue; Sattabongkot, Jetsumon; Han, Eun-Taek

    2010-01-01

    Parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) are known target enzymes of antifolate drugs used for the treatment and prophylaxis of persons with malaria. We sequenced the Plasmodium vivax dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) genes to examine the prevalence and extent of point mutations in isolates from malaria-endemic countries. Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117T) in the pvdhfr gene were found in isolates from Thailand (96.4%) and Myanmar (71.4%), but in only one isolate (1.0%) from Korea, where sulfadoxine-pyrimethamine has never been used. The pvdhfr point mutations correlated strongly with the pvdhps point mutations and ranged from single to triple mutations (S382A, A383G, and A553G), among isolates from Thailand, Myanmar, and Korea. These findings suggests that the prevalence of mutations in pvdhfr and pvdhps in P. vivax isolates from different malaria-endemic countries is associated with selection pressure imposed by sulfadoxine-pyrimethamine. PMID:20810806

  4. Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene.

    Science.gov (United States)

    Shaik, Abjal Pasha; Alsaeed, Abbas H; Sultana, Asma

    2012-01-01

    The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther's disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref NP_000366.1, NM_000375.2]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods. The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W (multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and these hits showed putative conserved domain, HemD superfamily (as on 14(th) Nov 2012). We then narrowed down the search by manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance in heme synthesis.

  5. N-acetylglutamate synthase deficiency: Novel mutation associated with neonatal presentation and literature review of molecular and phenotypic spectra

    Directory of Open Access Journals (Sweden)

    Eiman H. Al Kaabi

    2016-09-01

    Full Text Available The urea cycle is the main pathway for the disposal of excess nitrogen. Carbamoylphosphate synthetase 1 (CPS1, the first and rate-limiting enzyme of urea cycle, is activated by N-acetylglutamate (NAG, and thus N-acetylglutamate synthase (NAGS is an essential part of the urea cycle. Although NAGS deficiency is the rarest urea cycle disorder, it is the only one that can be specifically and effectively treated by a drug, N-carbamylglutamate, a stable structural analogous of NAG that activates CPS1. Here we report an infant with NAGS deficiency who presented with neonatal hyperammonemia. She was found to have a novel homozygous splice-site mutation, c.1097-2A>T, in the NAGS gene. We describe the clinical course of this infant, who had rapid response to N-carbamylglutamate treatment. In addition, we reviewed the clinical and molecular spectra of previously reported individuals with NAGS deficiency, which presents in most cases with neonatal hyperammonemia, and in some cases the presentation is later, with a broad spectrum of ages and manifestations. With this broad later-onset phenotypic spectrum, maintaining a high index of suspicion is needed for the early diagnosis of this treatable disease.

  6. N-acetylglutamate synthase deficiency: Novel mutation associated with neonatal presentation and literature review of molecular and phenotypic spectra.

    Science.gov (United States)

    Al Kaabi, Eiman H; El-Hattab, Ayman W

    2016-09-01

    The urea cycle is the main pathway for the disposal of excess nitrogen. Carbamoylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of urea cycle, is activated by N-acetylglutamate (NAG), and thus N-acetylglutamate synthase (NAGS) is an essential part of the urea cycle. Although NAGS deficiency is the rarest urea cycle disorder, it is the only one that can be specifically and effectively treated by a drug, N-carbamylglutamate, a stable structural analogous of NAG that activates CPS1. Here we report an infant with NAGS deficiency who presented with neonatal hyperammonemia. She was found to have a novel homozygous splice-site mutation, c.1097-2A>T, in the NAGS gene. We describe the clinical course of this infant, who had rapid response to N-carbamylglutamate treatment. In addition, we reviewed the clinical and molecular spectra of previously reported individuals with NAGS deficiency, which presents in most cases with neonatal hyperammonemia, and in some cases the presentation is later, with a broad spectrum of ages and manifestations. With this broad later-onset phenotypic spectrum, maintaining a high index of suspicion is needed for the early diagnosis of this treatable disease.

  7. Effects of point mutations in Plasmodium falciparum dihydrofolate reductase and dihydropterate synthase genes on clinical outcomes and in vitro susceptibility to sulfadoxine and pyrimethamine.

    Directory of Open Access Journals (Sweden)

    David J Bacon

    Full Text Available BACKGROUND: Sulfadoxine-pyrimethamine was a common first line drug therapy to treat uncomplicated falciparum malaria, but increasing therapeutic failures associated with the development of significant levels of resistance worldwide has prompted change to alternative treatment regimes in many national malaria control programs. METHODOLOGY AND FINDING: We conducted an in vivo therapeutic efficacy trial of sulfadoxine-pyrimethamine at two locations in the Peruvian Amazon enrolling 99 patients of which, 86 patients completed the protocol specified 28 day follow up. Our objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite susceptibility to sulfadoxine and pyrimethamine and to in vivo treatment outcomes. Inhibitory concentration 50 values of isolates increased with numbers of mutations (single [108N], sextuplet [BR/51I/108N/164L and 437G/581G] and septuplet (BR/51I/108N/164L and 437G/540E/581G with geometric means of 76 nM (35-166 nM, 582 nM (49-6890- nM and 4909 (3575-6741 nM nM for sulfadoxine and 33 nM (22-51 nM, 81 nM (19-345 nM, and 215 nM (176-262 nM for pyrimethamine. A single mutation present in the isolate obtained at the time of enrollment from either dihydrofolate reductase (164L or dihydropteroate synthase (540E predicted treatment failure as well as any other single gene alone or in combination. Patients with the dihydrofolate reductase 164L mutation were 3.6 times as likely to be treatment failures [failures 85.4% (164L vs 23.7% (I164; relative risk = 3.61; 95% CI: 2.14 - 6.64] while patients with the dihydropteroate synthase 540E were 2.6 times as likely to fail treatment (96.7% (540E vs 37.5% (K540; relative risk = 2.58; 95% CI: 1.88 - 3.73. Patients with both dihydrofolate reductase 164L and dihydropteroate synthase 540E mutations were 4.1 times as likely to be treatment failures [96.7% vs 23.7%; RR = 4.08; 95% CI: 2.45 - 7.46] compared to

  8. Study on structure-activity relationship of mutation-dependent herbicide resistance acetohydroxyacid synthase through 3D-QSAR and mutation

    Institute of Scientific and Technical Information of China (English)

    YU ZhiHong; NIU CongWei; BAN ShuRong; WEN Xin; XI Zhen

    2007-01-01

    Seventy-four sulfonylureas were synthesized and tested for their inhibitory activity against the whole enzyme of E. Coli acetohydroxyacid synthase (AHAS, EC 2.2.1.6) isoenzyme Ⅱ, and 3D-QSAR analyses were performed based on these inhibitory activities. The binding conformation of chlorimuron-ethyl, a commercial herbicide of AHAS, in the crystal structure of AHAS complex was extracted and used as template to build the initial three-dimensional structure of other sulfonylureas, and then all structures were fully geometry optimized. After systematic optimization of the alignment rule, molecular orientation, grid space and attenuation factor, two satisfactory models with excellent performances (CoMFA: q2 = 0.735, r2 = 0.954, n = 7, r 2pred = 0.832; CoMSIA: q2 = 0.721, r2 = 0.913, n = 8, r 2pred = 0.844) were established. By mapping the 3D contour maps of CoMFA and CoMSIA models into the possible inhibitory active site in the crystal structure of catalytic subunit of yeast AHAS, a plausible binding model for AHAS, with best fit QSAR in the literature so far, was proposed. Moreover, the results of 3D-QSAR were further utilized to interpret resistance of site-directed mutants. A relative activity index (RAI) for AHAS enzyme mutant was defined for the first time to relate the 3D-QSAR and resistance of mutants. This study, for the first time, demonstrated that combination of 3D-QSAR and enzyme mutation can be used to decipher the molecular basis of ligand-receptor interaction mechanism. This study refined our understanding of the ligand-receptor interaction and resistance mechanism in AHAS-sulfonylurea system, and provided basis for designing new potent herbicides to combat the herbicide resistance.

  9. Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: Molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Thoeny, B.; Leimbacher, W.; Blau, N.; Heizmann, C.W. (Univ. of Zurich (Switzerland)); Harvie, A.

    1994-05-01

    A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH[sub 4]), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH[sub 4]. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. The authors now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central form a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E.coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heteroxygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion ([Delta]14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120[yields]Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele [Delta]14bp exhibited no detectable activity. All three mutations result in reduced enzymatic activity when reconstituted in E. coli.

  10. Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations.

    Science.gov (United States)

    Montesinos, Isabel; Delforge, Marie-Luce; Ajjaham, Farida; Brancart, Françoise; Hites, Maya; Jacobs, Frederique; Denis, Olivier

    2017-01-01

    The PneumoGenius® real-time PCR assay is a new commercial multiplex real-time PCR method, which detects the Pneumocystis mitochondrial ribosomal large subunit (mtLSU) and two dihydropteroate synthase (DHPS) point mutations. To evaluate the clinical performance of this new real-time PCR assay we tested 120 extracted DNA samples from bronchoalveolar lavage specimens. These set of extracted DNA samples had already tested positive for Pneumocystis and patients had been classified in probable and unlikely PCP in a previous study. To evaluate de accuracy of the DHPS mutant's identification, an "in house" PCR and sequencing was performed. The sensitivity and specificity of PneumoGenius® PCR in discriminating between probable and unlikely Pneumocystis pneumonia (PCP) were 70% and 82% respectively. PneumoGenius® PCR was able to genotype more samples than "in house" DHPS PCR and sequencing. The same DHPS mutations were observed by both methods in four patients: two patients with a single mutation in position 171 (Pro57Ser) and two patients with a double mutation in position 165 (Thr55Ala) and in position 171 (Pro57Ser). A low rate of P. jirovecii (4.5%) harboring DHPS mutations was found, comparable to rates observed in other European countries. The PneumoGenius® real-time PCR is a suitable real-time PCR for PCP diagnosis and detection of DHPS mutants. The added value of DHPS mutation identification can assist in understanding the role of these mutations in prophylaxis failure or treatment outcome. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

    NARCIS (Netherlands)

    Gady, A.L.F.; Vriezen, W.; Wal, van de M.H.B.J.; Huang, P.; Bovy, A.G.; Visser, R.G.F.; Bachem, C.W.B.

    2012-01-01

    In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutatio

  12. Novel mutations in leukotriene C4 synthase and risk of cardiovascular disease based on genotypes from 50,000 individuals

    DEFF Research Database (Denmark)

    Freiberg, J J; Tybjærg-Hansen, Anne; Nordestgaard, B G

    2010-01-01

    Atherosclerosis is an inflammatory condition where cysteinyl leukotrienes have been identified to play an important role. Furthermore, cysteinyl leukotrienes may also affect thrombus formation. Using prospective, cross-sectional and case-control designs, we tested the hypothesis that hitherto unk...... unknown genetic variation, likely to affect the function of leukotriene C(4) synthase, is associated with risk of venous thromboembolism, ischemic stroke and myocardial infarction....

  13. Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16.

    Science.gov (United States)

    Sheu, Der-Shyan; Chen, Wen-Ming; Lai, Yung-Wei; Chang, Rey-Chang

    2012-05-01

    The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.

  14. Reduced response of Cystathionine Beta-Synthase (CBS) to S-Adenosylmethionine (SAM): Identification and functional analysis of CBS gene mutations in Homocystinuria patients.

    Science.gov (United States)

    Mendes, Marisa I S; Colaço, Henrique G; Smith, Desirée E C; Ramos, Rúben J J F; Pop, Ana; van Dooren, Silvy J M; Tavares de Almeida, Isabel; Kluijtmans, Leo A J; Janssen, Mirian C H; Rivera, Isabel; Salomons, Gajja S; Leandro, Paula; Blom, Henk J

    2014-03-01

    A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.

  15. Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder.

    Science.gov (United States)

    Coughlin, Curtis R; Scharer, Gunter H; Friederich, Marisa W; Yu, Hung-Chun; Geiger, Elizabeth A; Creadon-Swindell, Geralyn; Collins, Abigail E; Vanlander, Arnaud V; Coster, Rudy Van; Powell, Christopher A; Swanson, Michael A; Minczuk, Michal; Van Hove, Johan L K; Shaikh, Tamim H

    2015-08-01

    Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy. We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNA(Cys) was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  16. A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syndrome.

    Science.gov (United States)

    Bicknell, Louise S; Pitt, James; Aftimos, Salim; Ramadas, Ram; Maw, Marion A; Robertson, Stephen P

    2008-10-01

    There are several rare syndromes combining wrinkled, redundant skin and neurological abnormalities. Although phenotypic overlap between conditions has suggested that some might be allelic to one another, the aetiology for many of them remains unknown. A consanguineous New Zealand Maori family has been characterised that segregates an autosomal recessive connective tissue disorder (joint dislocations, lax skin) associated with neurological abnormalities (severe global developmental delay, choreoathetosis) without metabolic abnormalities in four affected children. A genome-screen performed under a hypothesis of homozygosity by descent for an ancestral mutation, identified a locus at 10q23 (Z = 3.63). One gene within the candidate interval, ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), was considered a plausible disease gene since a missense mutation had previously been shown to cause progressive neurodegeneration, cataracts, skin laxity, joint dislocations and metabolic derangement in a consanguineous Algerian family. A missense mutation, 2350C>T, was identified in ALDH18A1, which predicts the substitution H784Y. H784 is invariant across all phyla and lies within a previously unrecognised, conserved C-terminal motif in P5CS. In an in vivo assay of flux through this metabolic pathway using dermal fibroblasts obtained from an affected individual, proline and ornithine biosynthetic activity of P5CS was not affected by the H784Y substitution. These data suggest that P5CS may possess additional uncharacterised functions that affect connective tissue and central nervous system function.

  17. Endothelial Nitric Oxide Synthase T-786C Mutation, Prothrombin Gene Mutation (G-20210-A and Protein S Deficiency Could Lead to Myocardial Infarction in a Very Young Male Adult

    Directory of Open Access Journals (Sweden)

    Milka Klincheva

    2016-01-01

    Full Text Available INTRODUCTION: Myocardial infarction is a rare medical event in young people. The main reasons include congenital coronary abnormalities, coronary artery spasm, and coronary thrombosis due to hypercoagulable states (hereditary and acquired. AIM: We present a case of a young male adult with myocardial infarction caused by a combination of gene mutations and anticoagulation protein deficiency. CASE PRESENTATION: A 19 years old young man was admitted to our hospital complaining of chest pain during the last two weeks. The patient did not have any known cardiovascular risk factors, except a positive family anamnesis. Subacute inferior nonST segment myocardial infarction was diagnosed according to the patient’s history, electrocardiographic and laboratory findings. Coronary angiography revealed suboclusive thrombus in the proximal, medial and distal part of the right coronary artery (TIMI 2. Percutaneous coronary intervention was performed. Anticoagulant and antiagregant therapy (heparin, acetilsalicilic acid and clopidogrel according to protocol was started. The hospital stay was uneventful. Homozygous endothelial nitric oxid synthase (eNOS T-786-C mutation, heterozygote prothrombin gene mutation (G-20210-A, and protein S deficiency were verified from the thrombophilia testing. Other trombophilic tests were normal. Three months after discharge from hospital another coronary angiography was performed. It revealed normal coronary arteries. Four years after the attack, the patient is free of symptoms and another cardiovascular event. CONCLUSION: Combination of genetic mutations and anticoagulation protein deficiency could be a reasonable cause for myocardial infarction in a very young male adult without any other cardiovascular risk factors.

  18. Double-Mutated 5-Enol Pyruvylshikimate-3-phosphate Synthase Protein Expressed in MZHG0JG Corn (Zea mays L.) Has No Impact on Toxicological Safety and Nutritional Composition.

    Science.gov (United States)

    Matthews, Bethany A; Launis, Karen L; Bauman, Patricia A; Juba, Nicole C

    2017-09-27

    MZHG0JG corn will offer growers the flexibility to alternate between herbicides with two different modes of action in their weed-management programs, helping to mitigate and manage the evolution of herbicide resistance in weed populations. The proteins conferring herbicide tolerence in MZHG0JG corn, double-mutated 5-enol pyruvylshikimate-3-phosphate synthase protein (mEPSPS) and phosphinothricin acetyltransferase (PAT), as well as the MZHG0JG corn event, have been assessed by regulatory authorities globally and have been determined to be safe for humans, animals, and the environment. In addition to the safety data available for these proteins, further studies were conducted on MZHG0JG corn to assess levels of mEPSPS as compared to previously registered genetically modified (GM) corn. The results support the conclusion of no impact on toxicological safety or nutritional composition.

  19. N-acetylglutamate synthase deficiency: Novel mutation associated with neonatal presentation and literature review of molecular and phenotypic spectra

    OpenAIRE

    Eiman H. Al Kaabi; Ayman W. El-Hattab

    2016-01-01

    The urea cycle is the main pathway for the disposal of excess nitrogen. Carbamoylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of urea cycle, is activated by N-acetylglutamate (NAG), and thus N-acetylglutamate synthase (NAGS) is an essential part of the urea cycle. Although NAGS deficiency is the rarest urea cycle disorder, it is the only one that can be specifically and effectively treated by a drug, N-carbamylglutamate, a stable structural analogous of NAG that activates...

  20. Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells.

    Directory of Open Access Journals (Sweden)

    Yi Cao

    Full Text Available Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL, caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL, caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8 cerebellar cells. CbCln6(nclf/nclf cells and CbCln3(Δex7/8/Δex7/8 cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf and CbCln3(Δex7/8/Δex7/8 cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8 and Cln6(nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.

  1. Low prevalence of Pneumocystis pneumonia (PCP) but high prevalence of pneumocystis dihydropteroate synthase (dhps) gene mutations in HIV-infected persons in Uganda.

    Science.gov (United States)

    Taylor, Steve M; Meshnick, Steven R; Worodria, William; Andama, Alfred; Cattamanchi, Adithya; Davis, J Lucian; Yoo, Samuel D; Byanyima, Patrick; Kaswabuli, Sylvia; Goodman, Carol D; Huang, Laurence

    2012-01-01

    Pneumocystis jirovecii pneumonia (PCP) is an important opportunistic infection in patients infected with HIV, but its burden is incompletely characterized in those areas of sub-Saharan Africa where HIV is prevalent. We explored the prevalence of both PCP in HIV-infected adults admitted with pneumonia to a tertiary-care hospital in Uganda and of putative P. jirovecii drug resistance by mutations in fungal dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr). In 129 consecutive patients with sputum smears negative for mycobacteria, 5 (3.9%) were diagnosed with PCP by microscopic examination of Giemsa-stained bronchoalveolar lavage fluid. Concordance was 100% between Giemsa stain and PCR (dhps and dhfr). PCP was more prevalent in patients newly-diagnosed with HIV (11.4%) than in patients with known HIV (1.1%; p = 0.007). Mortality at 2 months after discharge was 29% overall: 28% among PCP-negative patients, and 60% (3 of 5) among PCP-positive patients. In these 5 fungal isolates and an additional 8 from consecutive cases of PCP, all strains harbored mutant dhps haplotypes; all 13 isolates harbored the P57S mutation in dhps, and 3 (23%) also harbored the T55A mutation. No non-synonymous dhfr mutations were detected. PCP is an important cause of pneumonia in patients newly-diagnosed with HIV in Uganda, is associated with high mortality, and putative molecular evidence of drug resistance is prevalent. Given the reliability of field diagnosis in our cohort, future studies in sub-Saharan Africa can investigate the clinical impact of these genotypes.

  2. A missense mutation in the cystathionine {beta}-synthase (CBS) gene associated with pyridoxine (B{sub 6}) responsive homocystinuria

    Energy Technology Data Exchange (ETDEWEB)

    Shih, V.E.; Fringer, J.M.; Mandell, R. [Massachusetts General Hospital, Boston, MA (United States)] [and others

    1994-09-01

    CBS deficiency is an autosomal recessive disorder characterized by homocystinuria and multisystem clinical disease. B{sub 6} responsive patients usually have a milder clinical phenotype than B{sub 6} nonresponsive patients. In our ongoing studies of the molecular defects in CBS deficiency, we reported a T-833 to C transition causing a substitution of threonine for isoleucine at position 278 (I278T). By PCR amplification and sequencing of exon 8 from genomic DNA we have now identified 2 index patients who are homozygous and 5 who are heterozygous for this mutation in a group of 32 patients with CBS deficiency. The mutation was detected in 7 of 10 unrelated families with in vivo B{sub 6} responsiveness, including one with a partial response, and in 0 of 22 B{sub 6} nonresponsive patients. The mutations on the other allele in the compound heterozygotes are still under investigation. We have now observed the I278T mutation in 9 of 20 independent alleles of varied ethnic backgrounds in the subgroup of B{sub 6} responsive patients. These findings, together with the previous report of this mutation in one allele of a B{sub 6} responsive patient, suggest that the I278T mutation is associated with B{sub 6} responsiveness in CBS deficiency. In compound heterozygotes, the degree of B{sub 6} responsiveness may also depend upon the nature of the other mutant allele and/or the interaction between the polypeptide subunits produced by the two mutant allelic genes.

  3. Mutation of Glu-361 in Human Endothelial Nitric-oxide Synthase Selectively Abolishes L-Arginine Binding without Perturbing the Behavior of Heme and Other Redox Centers

    Science.gov (United States)

    Chen, Pei-Feng; Tsai, Ah-Lim; Berka, Vladimir; Wu, Kenneth K.

    2010-01-01

    Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics. PMID:9045621

  4. Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in prenyl diphosphate synthase subunit 2.

    Science.gov (United States)

    Saiki, Ryoichi; Lunceford, Adam L; Shi, Yuchen; Marbois, Beth; King, Rhonda; Pachuski, Justin; Kawamukai, Makoto; Gasser, David L; Clarke, Catherine F

    2008-11-01

    Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10 provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q10 supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.

  5. Man o' War Mutation in UDP-α-D-Xylose Synthase Favors the Abortive Catalytic Cycle and Uncovers a Latent Potential for Hexamer Formation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Jr., Richard M.; Polizzi, Samuel J.; Kadirvelraj, Renuka; Howard, Wesley W.; Wood, Zachary A. [Georgia

    2015-03-17

    The man o’ war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-D-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ~16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies show that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-D-4-keto-xylose, is not reduced to the final product, UDP-α-D-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.

  6. Role of Mutations in Dihydrofolate Reductase DfrA (Rv2763c) and Thymidylate Synthase ThyA (Rv2764c) in Mycobacterium tuberculosis Drug Resistance

    KAUST Repository

    Koser, C. U.

    2010-09-17

    We would like to comment on a number of recent reports in this journal (6, 8, 12, 18) concerning Mycobacterium tuberculosis dihydrofolate reductase (DHFR), encoded by dfrA (Rv2763c). Around 36% of phenotypically para-aminosalicylic acid (PAS)-resistant M. tuberculosis strains harbor mutations in thyA (Rv2764c), which encodes a thymidylate synthase (20). In their effort to elucidate the remaining unknown resistance mechanism(s), Mathys et al. extended their sequence analysis to a number of additional genes, including dfrA (12). It was unclear whether the three dfrA mutations they identified in the PAS-resistant strains P-693 and P-3158 could contribute to PAS resistance on their own. Nonetheless, these findings are notable for two reasons. First, isoniazid (INH) has been shown to inhibit M. tuberculosis DHFR in vitro (1). Whether the same holds true for ethionamide, which shares a number of common resistance mechanisms with INH, was not tested (J. Blanchard, personal communication). In any case, the clinical relevance of DHFR-mediated INH resistance remains enigmatic. To date, only Ho et al. have addressed this question, but they did not identify any dfrA mutations in a screen of 127 INH-resistant clinical isolates (8). Consequently, Mathys et al. remain the first to describe mutations in this target (12). However, given that isolates with mutated DHFR are members of a cluster with baseline INH resistance, the importance of these mutations with respect to INH resistance remains unclear. Irrespective of their relevance in INH resistance, these dfrA mutations are noteworthy for a second reason. Contrary to previous wisdom, Forgacs et al. recently showed that M. tuberculosis is sensitive to the drug combination trimethoprim-sulfamethoxazole (TMP-SMX) (6, 18). DHFR is competitively inhibited by TMP, and consequently, mutations therein lead to resistance in a variety of organisms (9, 16, 19). The crystal structures of the wild-type M. tuberculosis DHFR in complex with

  7. Prevalence of cystathionine beta synthase gene mutation 852Ins68 as a possible risk for neural tube defects in eastern India.

    Science.gov (United States)

    Saxena, A K; Gupta, J; Pandey, S; Gangopadhaya, A N; Pandey, L K

    2011-10-07

    Cystathionine beta synthase gene (CβS) catalyzes the condensation of homocysteine with serine, forming cystathionine by the transsulfuration pathway. Disruption of CβS enzyme activity due to defective folic acid metabolism increases the risk factor for neural tube defects. We evaluated the CβS gene mutation in 25 children with neural tube defects (NTDs), including lumbosacral and thoracic myelomeningocele and open NTDs and mothers of cases, along with 25 healthy children and their mothers, serving as controls. Genomic DNA was isolated to assess the polymorphism of 852Ins68 in the CβS gene using PCR-RFLP analysis and nucleotide sequencing techniques. The 68-bp insertion was observed in one of the 25 NTD cases (lumbosacral myelomeningocele), and in two of the mothers of NTD cases. Statistical analysis was carried out using the Fischer exact probability test, which showed a lack of significance (P > 0.05), but the odds ratio of 2.08 with 95% confidence interval of 0.17-24.6 in NTDs mother was quite high because of the small sample size. However, the study was further extended to find out the involvement of specific nucleotide sequences, which again confirmed the 852Ins68 insertion and replacement of nucleotides (TCCAT to GGGG) in lumbosacral myelomeningocele (due to other category of NTDs), suggesting that it could be an independent risk factor for birth defects, including NTDs.

  8. Differential response of methionine metabolism in two grain legumes, soybean and azuki bean, expressing a mutated form of Arabidopsis cystathionine γ-synthase.

    Science.gov (United States)

    Hanafy, Moemen S; Rahman, Shaikh M; Nakamoto, Yumi; Fujiwara, Toru; Naito, Satoshi; Wakasa, Kyo; Ishimoto, Masao

    2013-02-15

    Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.

  9. Localization and expression of the Bacillus subtilis DL-endopeptidase LytF are influenced by mutations in LTA synthases and glycolipid anchor synthetic enzymes.

    Science.gov (United States)

    Kiriyama, Yuuka; Yazawa, Kazuya; Tanaka, Tatsuhito; Yoshikawa, Ritsuko; Yamane, Hisaya; Hashimoto, Masayuki; Sekiguchi, Junichi; Yamamoto, Hiroki

    2014-12-01

    Bacillus subtilis LytF plays a principal role in cell separation through its localization at the septa and poles on the vegetative cell surface. In this study, we found that a mutation in a major lipoteichoic acid (LTA) synthase gene--ltaS--results in a considerable reduction in the σ(D)-dependent transcription of lytF. The lytF transcription was also reduced in mutants that affected glycolipid anchor biosynthesis. Immunofluorescence microscopy revealed that both the numbers of cells expressing LytF and the LytF foci in these mutants were decreased. In addition, the transcriptional activity of lytF was almost abolished in the double (ltaS yfnI), triple (ltaS yfnI yqgS), and quadruple (ltaS yfnI yqgS yvgJ) mutants during vegetative growth. Cell separation defects in these mutants were partially restored with artificial expression of LytF. Interestingly, when lytF transcription was induced in the ltaS single or multiple mutants, LytF was localized not only at the septum, but also along the sidewall. The amounts of LytF bound to cell wall in the single (ltaS) and double (ltaS yfnI) mutants gradually increased as compared with that in the WT strain, and those in the triple (ltaS yfnI yqgS) and quadruple mutants were almost similar to that in the double mutant. Moreover, reduction of the lytF transcription and chained cell morphology in the ltaS mutant were completely restored with artificial induction of the yqgS gene. These results strongly suggest that LTA influences the temporal, σ(D)-dependent transcription of lytF and is an additional inhibitory component to the vegetative cell separation enzyme LytF.

  10. Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM

    DEFF Research Database (Denmark)

    Hansen, L; Arden, K C; Rasmussen, S B

    1997-01-01

    Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active ...

  11. A mitochondrial mutation at nt 9101 in the ATP synthase 6 gene associated with deficient oxidative phosphorylation in a family with leber hereditary optic neuroretinopathy

    Energy Technology Data Exchange (ETDEWEB)

    Lamninen, T.; Junoven, V.; Aula, P.; Savontaus, M.L. [Univ. of Turku (Finland); Majander, A.; Wikstroem, M. [Univ. of Helsinki (Finland)

    1995-05-01

    Leber hereditary optic neuroretinopathy (LHON) is a maternally inherited ocular disease resulting in bilateral optic atrophy in young adults. Several mtDNA point mutations have been proposed as being causative for LHON, all in complex I, III, or IV of the respiratory chain. The ND4/11778 mutation accounts for {approximately}50% of all LHON families, the ND1/3460 mutation is detected in {approximately}15% of cases, and {approximately}10% of LHON families have the ND6/14484 mutation. All these mutations are restricted to LHON families, and they change evolutionary conserved amino acids. Furthermore, these primary mutations have never been observed to occur simultaneously. Besides the primary mutations, several other replacement mutations have been found in LHON families. These mutations are also detected at low frequency in control individuals, and they change evolutionarily less conserved amino acids. 11 refs., 2 figs.

  12. Four new mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causing X-linked sideroblastic anemia: increased pyridoxine responsiveness after removal of iron overload by phlebotomy and coinheritance of hereditary hemochromatosis.

    Science.gov (United States)

    Cotter, P D; May, A; Li, L; Al-Sabah, A I; Fitzsimons, E J; Cazzola, M; Bishop, D F

    1999-03-01

    X-linked sideroblastic anemia (XLSA) in four unrelated male probands was caused by missense mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). All were new mutations: T647C, C1283T, G1395A, and C1406T predicting amino acid substitutions Y199H, R411C, R448Q, and R452C. All probands were clinically pyridoxine-responsive. The mutation Y199H was shown to be the first de novo XLSA mutation and occurred in a gamete of the proband's maternal grandfather. There was a significantly higher frequency of coinheritance of the hereditary hemochromatosis (HH) HFE mutant allele C282Y in 18 unrelated XLSA hemizygotes than found in the normal population, indicating a role for coinheritance of HFE alleles in the expression of this disorder. One proband (Y199H) with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and hematologic histories of two XLSA probands suggest that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the Y199H proband by phlebotomy resulted in higher hemoglobin concentrations during pyridoxine supplementation. The proband with the R452C mutation was symptom-free on occasional phlebotomy and daily pyridoxine. These studies indicate the value of combined phlebotomy and pyridoxine supplementation in the management of XLSA probands in order to prevent a downward spiral of iron toxicity and refractory anemia.

  13. Diversity of cystathionine ß-synthase haplotypes bearing the most common homocystinuria mutation c.833T>C: a possible role for gene conversion

    DEFF Research Database (Denmark)

    Vyletal, P; Sokolová, J; Cooper, DN;

    2007-01-01

    Homozygosity or compound heterozygosity for the c.833T>C transition (p.I278 T) in the cystathionine beta-synthase (CBS) gene represents the most common cause of pyridoxine-responsive homocystinuria in Western Eurasians. However, the frequency of the pathogenic c.833C allele, as observed in healthy...

  14. Pseudouridine synthases.

    Science.gov (United States)

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  15. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    Science.gov (United States)

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  16. Effects of Point Mutations in Plasmodium falciparum Dihydrofolate Reductase and Dihydropterate Synthase Genes on Clinical Outcomes and In Vitro Susceptibility to Sulfadoxine and Pyrimethamine

    Science.gov (United States)

    2009-08-01

    parasitological responses [23]. To distinguish between recrudescence and reinfection, malaria parasites collected on day of enrollment (day 0) and day of...Alejandro Llanos-Cuentas4, Coralith Garcia4, Lelv Solari4, Dennis Kyle5, Alan J. Magill3 1 Parasitology Program, Naval Medical Research Center...objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite

  17. The thanatos mutation in Arabidopsis thaliana cellulose synthase 3 (AtCesA3) has a dominant-negative effect on cellulose synthesis and plant growth.

    Science.gov (United States)

    Daras, Gerasimos; Rigas, Stamatis; Penning, Bryan; Milioni, Dimitra; McCann, Maureen C; Carpita, Nicholas C; Fasseas, Constantinos; Hatzopoulos, Polydefkis

    2009-01-01

    Genetic functional analyses of mutants in plant genes encoding cellulose synthases (CesAs) have suggested that cellulose deposition requires the activity of multiple CesA proteins. Here, a genetic screen has led to the identification of thanatos (than), a semi-dominant mutant of Arabidopsis thaliana with impaired growth of seedlings. Homozygous seedlings of than germinate and grow but do not survive. In contrast to other CesA mutants, heterozygous plants are dwarfed and display a radially swollen root phenotype. Cellulose content is reduced by approximately one-fifth in heterozygous and by two-fifths in homozygous plants, showing gene-dosage dependence. Map-based cloning revealed an amino acid substitution (P578S) in the catalytic domain of the AtCesA3 gene, indicating a critical role for this residue in the structure and function of the cellulose synthase complex. Ab initio analysis of the AtCesA3 subdomain flanking the conserved proline residue predicted that the amino acid substitution to serine alters protein secondary structure in the catalytic domain. Gene dosage-dependent expression of the AtCesA3 mutant gene in wild-type A. thaliana plants resulted in a than dominant-negative phenotype. We propose that the incorporation of a mis-folded CesA3 subunit into the cellulose synthase complex may stall or prevent the formation of functional rosette complexes.

  18. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    OpenAIRE

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic ch...

  19. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  20. Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2----q26.3.

    Science.gov (United States)

    Astrin, K H; Warner, C A; Yoo, H W; Goodfellow, P J; Tsai, S F; Desnick, R J

    1991-05-01

    Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%-53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2----q26.3.

  1. Methylenetetrahydrofolate reductase 677C>T and methionine synthase 2756A>G mutations: no impact on survival, cognitive functioning, or cognitive decline in nonagenarians

    DEFF Research Database (Denmark)

    Bathum, Lise; von Bornemann Hjelmborg, Jacob; Christiansen, Lene

    2007-01-01

    homocysteine level. METHODS: We examined the effect of the MTHFR 677C>T and MTR 2756A>G genotypes on baseline cognitive functioning, cognitive decline over 5 years measured in three assessments, and survival in a population-based cohort of 1581 nonagenarians. Cognitive functioning was assessed by using......BACKGROUND: Several reports have shown an association between homocysteine, cognitive functioning, and survival among the oldest-old. Two common polymorphisms in the genes coding for methylenetetrahydrofolate reductase (MTHFR 677C>T) and methionine synthase (MTR 2756A>G) have an impact on plasma...... the Mini-Mental State Examination (MMSE) and five brief cognitive tests (cognitive composite). RESULTS: There are no differences in MMSE score (p =.83) or in cognitive composite (p =.56) at intake as a function of genotype tested by analysis of variance, whereas sex and social group have a impact on MMSE...

  2. A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

    Science.gov (United States)

    Lassner, M W; Lardizabal, K; Metz, J G

    1996-02-01

    beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.

  3. Mutation of the gene promoter synthase nitric oxide eNOS T786С in patients with pseudarthrosis of long bones: relationship with lipid levels and endothelial function

    Directory of Open Access Journals (Sweden)

    Bezsmertnyi Yu.O.

    2013-06-01

    Full Text Available Objective: The prevalence of gene promoter polymorphism eNOS T786С and its relationship with the level of lipids and vascular endothelial function in patients with pseudoarthrosis of long bones was investigated. Materials and methods. In 118 patients with pseudoarthrosis of long bones frequency of mutation of gene promoter eNOS T786C was studied and its relationship to lipid levels and vascular endothelial function was determined. The control group consisted of 48 patients with consolidated diaphyseal fractures, representative by age, gender and location of the damage. Results and discussion. Pathological homozygotes eNOS 786-CC accumulate predominantly in patients with hypoplastic and atrophic type of pseudoarthrosis. The frequency of genotypes distribution in patients with normoplastic type of pseudoarthrosis corresponded to the same in people with consolidated fractures. In patients with genotype eNOS 786-CC more often individuals with aberrant lipid levels and structural and functional changes in the common carotid, brachial and femoral arteries are identified. Conclusions. In patients with pseudarthrosis of long bones tendency to decrease in the proportion of normal homozygotes (786-CT, increase in heterozygotes (786-CT and the proportion of individuals with abnormal genotype 786-CC is observed. The mutation in gene T786C eNOS was associated with the development of hypoplastic and aplastic types of pseudarthrosis, increasing the proportion of persons with aberrant lipid levels, endothelial dysfunction of the central and peripheral vessels.

  4. Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

    Science.gov (United States)

    Olsen, Rikke K J; Koňaříková, Eliška; Giancaspero, Teresa A; Mosegaard, Signe; Boczonadi, Veronika; Mataković, Lavinija; Veauville-Merllié, Alice; Terrile, Caterina; Schwarzmayr, Thomas; Haack, Tobias B; Auranen, Mari; Leone, Piero; Galluccio, Michele; Imbard, Apolline; Gutierrez-Rios, Purificacion; Palmfeldt, Johan; Graf, Elisabeth; Vianey-Saban, Christine; Oppenheim, Marcus; Schiff, Manuel; Pichard, Samia; Rigal, Odile; Pyle, Angela; Chinnery, Patrick F; Konstantopoulou, Vassiliki; Möslinger, Dorothea; Feichtinger, René G; Talim, Beril; Topaloglu, Haluk; Coskun, Turgay; Gucer, Safak; Botta, Annalisa; Pegoraro, Elena; Malena, Adriana; Vergani, Lodovica; Mazzà, Daniela; Zollino, Marcella; Ghezzi, Daniele; Acquaviva, Cecile; Tyni, Tiina; Boneh, Avihu; Meitinger, Thomas; Strom, Tim M; Gregersen, Niels; Mayr, Johannes A; Horvath, Rita; Barile, Maria; Prokisch, Holger

    2016-06-02

    Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

  5. Autopsy and postmortem examination case study on genetic risk factors for cardiac death: Polymorphisms of endothelial nitric oxide synthase gene Glu298asp variant and T-786c mutation, human paraoxonas

    Directory of Open Access Journals (Sweden)

    Ameno Kiyoshi

    2006-01-01

    Full Text Available Background/Aim. The Glu298Asp variant in exon 7 and T-786C mutation in the 5'-flanking region of the endothelial nitric oxide synthase (eNOS gene, paraoxonase I gene (PON1, and α2β- adrenergic receptor gene (α2β-AR have been reported to be genetic risk factors for coronary heart disease (CHD. The aim of this study was to investigate the effects of these four genetic polymorphisms on the probability of death due to CHD, using data obtained from medico-legal autopsies. Methods. Blood samples from three groups: healthy controls, dead cases with CHD and without CHD (the latter as a control for dead cases were used. After DNA extraction, genotyping was performed by polymerase chain reaction − restriction fragment length polymorphism (PCR-RFLP test. Results. The frequency of the T allele in Glu298Asp variant in the dead cases with CHD was significantly higher than that in the healthy control (p < 0.001, OR = 4.47 and that in the dead cases without CHD (p < 0.001, OR = 7.62. The gene frequency of PON1 was significantly different (p = 0.007 between dead cases with and without CHD, and was also significantly different (p = 0.025 between the healthy control and dead cases without CHD. The gene frequency of PON1 was not significantly different (p = 0.401 between the healthy controls and dead cases with CHD. Hence this gene was not associated with death due to CHD. The other polymorphisms (T- 786C mutation, α2β-AR also showed no effect on death due to CHD. Conclusion. The polymorphism of Glu298Asp eNOS gene in dead cases may be useful for determining the cause of death in CHD cases in the Japanese population.

  6. Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1{sup A903V} and CESA3{sup T942I} of cellulose synthase

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Darby; Corbin, Kendall; Wang, Tuo; Gutierrez, Ryan; Bertolo, Ana; Petti, Caroalberto; Smilgies, Detlef-M; Estevez, Jose Manuel; Bonetta, Dario; Urbanowicz, Breeanna; Ehrhardt, David; Somerville, Chris; Rose, Jocelyn; Hong, Mei; DeBolt, Seth

    2012-01-08

    The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1{sup A903V} and CESA3{sup T942I} in Arabidopsis thaliana. Using {sup 13}C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1{sup A903V} and CESA3{sup T942I} displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1{sup A903V} and CESA3{sup T942I} have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

  7. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    Science.gov (United States)

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  8. mtDNA T8993G mutation-induced F1F0-ATP synthase defect augments mitochondrial dysfunction associated with hypoxia/reoxygenation: the protective role of melatonin.

    Directory of Open Access Journals (Sweden)

    Wen-Yi Huang

    Full Text Available BACKGROUND: F1F0-ATP synthase (F1F0-ATPase plays important roles in regulating mitochondrial function during hypoxia, but the effect of F1F0-ATPase defect on hypoxia/reoxygenation (H/RO is unknown. The aim of this study was to investigate how mtDNA T8993G mutation (NARP-induced inhibition of F1F0-ATPase modulates the H/RO-induced mitochondrial dysfunction. In addition, the potential for melatonin, a potent antioxidant with multiple mitochondrial protective properties, to protect NARP cells exposed to H/RO was assessed. METHODS AND FINDINGS: NARP cybrids harboring 98% of mtDNA T8993G genes were established as an in vitro model for cells with F1F0-ATPase defect; their parental osteosarcoma 143B cells were studied for comparison. Treating the cells with H/RO using a hypoxic chamber resembles ischemia/reperfusion in vivo. NARP significantly enhanced apoptotic death upon H/RO detected by MTT assay and the trypan blue exclusion test of cell viability. Based on fluorescence probe-coupled laser scanning imaging microscopy, NARP significantly enhanced mitochondrial reactive oxygen species (mROS formation and mitochondrial Ca(2+ (mCa(2+ accumulation in response to H/RO, which augmented the depletion of cardiolipin, resulting in the retardation of mitochondrial movement. With stronger H/RO stress (either with longer reoxygenation duration, longer hypoxia duration, or administrating secondary oxidative stress following H/RO, NARP augmented H/RO-induced mROS formation to significantly depolarize mitochondrial membrane potential (ΔΨm, and enhance mCa(2+ accumulation and nitric oxide formation. Also, NARP augmented H/RO-induced mROS oxidized and depleted cardiolipin, thereby promoting permanent mitochondrial permeability transition, retarded mitochondrial movement, and enhanced apoptosis. Melatonin markedly reduced NARP-augmented H/RO-induced mROS formation and therefore significantly reduced mROS-mediated depolarization of ΔΨm and accumulation of mCa(2

  9. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

    Full Text Available Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  10. An investigation into eukaryotic pseudouridine synthases.

    Science.gov (United States)

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  11. 过表达拟南芥点突变乙酰羟酸合成酶基因改变植物对缬氨酸的抗性及增强缬氨酸合成%Overexpression of the PointMutated Acetohydroxyacid Synthase Alters Resis-tance to Valine and Enhances Production of Valine inArabidopsis

    Institute of Scientific and Technical Information of China (English)

    赵菲佚; 焦成瑾; 王太术; 田春芳; 谢尚强; 刘亚萍

    2015-01-01

    拟南芥乙酰羟酸合成酶(acetohydroxyacid synthase, AHAS)在支链氨基酸合成中具有重要的作用。为考察AHAS不同亚基关键位点突变对植物缬氨酸抗性与缬氨酸合成的影响,对AHAS大小亚基上特定位点进行体外突变,构建AHAS点突变过表达转基因植物,研究AHAS不同亚基点突变转基因植物对缬氨酸抗性及其合成的影响。研究结果表明: AHAS小亚基G88D突变解除了终端产物对该酶的反馈抑制作用,使转基因植物缬氨酸含量提高。大亚基E305D突变增强小亚基G88D突变效应,而大亚基E482D突变对G88D突变具有相反的作用。AHAS全酶E305DG88D双突变转基因植物较E482DG88D具有更强的缬氨酸抗性表型和更高的缬氨酸含量。这些结果提示AHAS大小亚基间存在着相互作用,大小亚基不同位点突变对AHAS全酶活性具有不同的影响。%Acetohydroxyacid synthase (AHAS) plays a pivotal role in the synthesis of brahched-chain amino acids (BCAAs) inArabidopsis. To investigate effects of various speciifc mutated sites harboring in the large and small subunits of AHAS on resistance to valine and production of valine inArabidopsis, transgenic plants overexpressing the point mutated AHAS were generated by site-directed mutagenesis, and the phenotype of re-sistance to valine and production of valine of the transgenic plants were evaluatedin planta. The results showed that the G88D mutation in the small unit of AHAS abolished the feedback-resistant of valine to AHAS and this mutation resulted in increase of valine in the transgenic plants. The E305D mutation in the large unit of AHAS strengthens the effect of the G88D mutation. Interestingly, the E482D mutation in the large unit of AHAS acts antagonistically on the G88D mutation in resistance to valine and production of valine in transgenic plants. Compared with the combined double E482DG88D AHAS mutant transgenic plants, the E305DG88D AHAS transgenic plants exhibited the

  12. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  13. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  14. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been...... in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...

  15. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    Science.gov (United States)

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of BPS was rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. 3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor substrate for PPS. The aryl moiety of the triketide intermediate may be trapped in the new pocket by hydrogen bond formation with the backbone, thereby acting as an inhibitor. PPS is a promising biotechnological tool for manipulating benzoate-primed biosynthetic pathways to produce novel compounds. PMID:19710020

  16. Mammalian N-acetylglutamate synthase.

    Science.gov (United States)

    Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

    2004-04-01

    N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

  17. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  18. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  19. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

    van Schie, C.C.N.; Haring, M.A.; Schuurink, R.C.; Bach, T.J.; Rohmer, M.

    2013-01-01

    Gibberellins are derived from the diterpene precursor geranylgeranyl diphophosphate (GGPP). GGPP is converted to ent-kaurene, which contains the basic structure of gibberellins, in the plastids by the combined actions of copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Generally, ge

  20. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences.

    Directory of Open Access Journals (Sweden)

    Andrew eCarroll

    2011-03-01

    Full Text Available The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized subfamilies and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair cellulose synthase function, and compare these sites to those observed in the closest cellulose synthase-like (Csl families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  1. The defective phosphoribosyl diphosphate synthase in a temperature-sensitive prs-2 mutant of Escherichia coli is compensated by increased enzyme synthesis

    DEFF Research Database (Denmark)

    Post, David A.; Switzer, Robert L.; Hove-Jensen, Bjarne

    1996-01-01

    at 25 degrees C. The mutant enzyme had nearly normal heat stability, as long as it was synthesized at 25 degrees C. In contrast, there was hardly any PRPP synthase activity or anti-PRPP synthase antibody cross-reactive material present in cells harbouring the glycine to serine alteration following...... synthase activity as a strain harbouring a plasmid-borne wild-type prs allele. In cells harbouring both mutations, the C -> T mutation appeared to compensate for the G -> A mutation by increasing the amount of a partially defective enzyme at the permissive temperature....

  2. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    Science.gov (United States)

    Del Campo, M; Kaya, Y; Ofengand, J

    2001-11-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

  3. N-acetylglutamate synthase deficiency and the treatment of hyperammonemic encephalopathy.

    Science.gov (United States)

    Elpeleg, Orly; Shaag, Avraham; Ben-Shalom, Efrat; Schmid, Tal; Bachmann, Claude

    2002-12-01

    Carbamylphosphate synthase is the first enzymatic reaction of the urea cycle. Its activator, N-acetylglutamate, is synthesized from acetyl-CoA and glutamate in a reaction catalyzed by N-acetylglutamate synthase (NAGS). We have identified the putative human NAGS gene and report the first mutation in this gene in a family with carbamylglutamate responsive hyperammonemia and normal activity of the urea cycle enzymes. Mutation analysis has a higher diagnostic specificity than the enzymatic assay in NAGS deficiency. A therapeutic trial with carbamylglutamate is recommended whenever hyperammonemia without an organic aciduria, increased orotate excretion, or diagnostic amino acidemia/uria is detected.

  4. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Xu, W.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States); Kozak, C.A. [National Institute of Health, Bethesda, MD (United States)

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  5. Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

    Science.gov (United States)

    Somerville, Chris R.; Scheible, Wolf

    2007-07-10

    Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  6. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat...

  7. A revisit to the natural history of homocystinuria due to cystathionine beta-synthase deficiency

    DEFF Research Database (Denmark)

    Skovby, Flemming; Gaustadnes, Mette; Mudd, S Harvey

    2010-01-01

    We review the evidence that in Denmark and probably certain other European countries the number of individuals identified with homocystinuria due to homozygosity for the widespread c.833T>C (p.I278T) mutation in the gene that encodes cystathionine beta-synthase (CBS) falls far short of the number...

  8. Biphenyl synthase, a novel type III polyketide synthase.

    Science.gov (United States)

    Liu, B; Raeth, T; Beuerle, T; Beerhues, L

    2007-05-01

    Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.

  9. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions GM3 synthase deficiency GM3 synthase ...

  10. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    Science.gov (United States)

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  11. Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

    Directory of Open Access Journals (Sweden)

    Mirian Perez Maluf

    2009-01-01

    Full Text Available In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

  12. Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase[S

    Science.gov (United States)

    Ternes, Philipp; Brouwers, Jos F. H. M.; van den Dikkenberg, Joep; Holthuis, Joost C. M.

    2009-01-01

    Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually. PMID:19454763

  13. Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase.

    Science.gov (United States)

    Carman, George M; Han, Gil-Soo

    2017-08-16

    The yeast Saccharomyces cerevisiae serves as a model eukaryote to elucidate the regulation of lipid metabolism. In exponentially growing yeast, a diverse set of membrane lipids are synthesized from the precursor phosphatidate via the liponucleotide intermediate CDP-diacylglycerol. As cells exhaust nutrients and progress into the stationary phase, phosphatidate is channeled via diacylglycerol to the synthesis of triacylglycerol. The CHO1-encoded phosphatidylserine synthase, which catalyzes the committed step in membrane phospholipid synthesis via CDP-diacylglycerol, and the PAH1-encoded phosphatidate phosphatase, which catalyzes the committed step in triacylglycerol synthesis are regulated throughout cell growth by genetic and biochemical mechanisms to control the balanced synthesis of membrane phospholipids and triacylglycerol. The loss of phosphatidate phosphatase activity (e.g., pah1Δ mutation) increases the level of phosphatidate and its conversion to membrane phospholipids by inducing Cho1 expression and phosphatidylserine synthase activity. The regulation of the CHO1 expression is mediated through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. Consequently, phosphatidate phosphatase activity regulates phospholipid synthesis through the transcriptional regulation of the phosphatidylserine synthase enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. The structural basis of Erwinia rhapontici isomaltulose synthase.

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

  15. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  16. Identification and characterization of a second isogene encoding γ-terpinene synthase in Thymus caespititius.

    Science.gov (United States)

    Mendes, Marta D; Barroso, José G; Oliveira, M Margarida; Trindade, Helena

    2014-07-15

    Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high polymorphism. They consist mostly of mono- and sesquiterpene, some of them with interest for the pharmaceutical and food industries. The search for terpene synthase genes was performed in three in vitro T. caespititius genotypes. For these plants, the expression of a previously described γ-terpinene synthase gene, Tctps2, was confirmed, occurring concomitantly with a new gene encoding an enzyme with similar activity, named Thymus caespititius terpene synthase 4 (Tctps4). The two isogenes were isolated and functionally characterized in the three plant genotypes. Alignment of the two Tctps revealed a transit peptide much shorter in Tctps4 than in Tctps2 (3-4 amino acids instead of 47). The Tctps4 open reading frame is shorter than Tctps2 (1665 bp versus 1794 bp). The amino acid sequence of both γ-terpinene synthases shared an 88% pairwise identity. The fact that T. caespititius carries two isogenes for γ-terpinene synthases, suggests gene duplication along the evolutionary process, followed by mutations leading to the differentiation of both genes. These mutations didn't compromise protein activity. A high accumulation of transcripts from both genes was found in shoots of in vitro plantlets, while in roots they could not be detected. Still, γ-terpinene levels in aerial parts were reduced, probably due to fast conversion into carvacrol and thymol, the main components from T. caespititius essential oils. This study is a contribution to the identification of terpene synthase genes in Lamiaceae.

  17. Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates

    Science.gov (United States)

    Caldwell, Harlan D.; Wood, Heidi; Crane, Debbie; Bailey, Robin; Jones, Robert B.; Mabey, David; Maclean, Ian; Mohammed, Zeena; Peeling, Rosanna; Roshick, Christine; Schachter, Julius; Solomon, Anthony W.; Stamm, Walter E.; Suchland, Robert J.; Taylor, Lacey; West, Sheila K.; Quinn, Tom C.; Belland, Robert J.; McClarty, Grant

    2003-01-01

    We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogen’s tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates — specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-γ resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-γ–mediated eradication and thus establish persistent infection. PMID:12782678

  18. Short-term efficacy of N-carbamylglutamate in a patient with N-acetylglutamate synthase deficiency.

    Science.gov (United States)

    Kim, Ja Hye; Kim, Yoo-Mi; Lee, Beom Hee; Cho, Ja Hyang; Kim, Gu-Hwan; Choi, Jin-Ho; Yoo, Han-Wook

    2015-07-01

    N-acetylglutamate synthase (NAGS) deficiency is a rare inborn error regarding the urea cycle, however, its diagnosis is important as it can be effectively treated by N-carbamylglutamate. We evaluated a patient with NAGS deficiency who harbored two novel NAGS mutations and who showed excellent responsiveness during 1 year of N-carbamylglutamate treatment.

  19. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  20. Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.

    Directory of Open Access Journals (Sweden)

    Roberta d'Emmanuele di Villa Bianca

    Full Text Available Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.

  1. Molecular evolution and functional divergence of soluble starch synthase genes in cassava (manihot esculenta crantz).

    Science.gov (United States)

    Yang, Zefeng; Wang, Yifan; Xu, Shuhui; Xu, Chenwu; Yan, Changjie

    2013-01-01

    Soluble starch synthases (SSs) are major enzymes involved in starch biosynthesis in plants. Cassava starch has many remarkable characteristics, which should be influenced by the evolution of SS genes in this starchy root crop. In this work, we performed a comprehensive phylogenetic and evolutionary analysis of the soluble starch synthases in cassava. Genome-wide identification showed that there are 9 genes encoding soluble starch synthases in cassava. All of the soluble starch synthases encoded by these genes contain both Glyco_transf_5 and Glycos_transf_1 domains, and a correlation analysis showed evidence of coevolution between these 2 domains in cassava SS genes. The SS genes in land plants can be divided into 6 subfamilies that were formed before the origin of seed plants, and species-specific expansion has contributed to the evolution of this family in cassava. A functional divergence analysis for this family provided statistical evidence for shifted evolutionary rates between the subfamilies of land plant soluble starch synthases. Although the main selective pressure acting on land plant SS genes was purifying selection, our results also revealed that point mutation with positive selection contributed to the evolution of 2 SS genes in cassava. The remarkable cassava starch characteristics might be the result of both the duplication and adaptive selection of SS genes.

  2. Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M. Brett; Ferreira, Antonio M.; Lee, Richard E.; Bashford, Donald; White, Stephen W. (SJCH)

    2013-04-08

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S{sub N}1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  3. Light- and metabolism-related regulation of the chloroplast ATP synthase has distinct mechanisms and functions.

    Science.gov (United States)

    Kohzuma, Kaori; Dal Bosco, Cristina; Meurer, Jörg; Kramer, David M

    2013-05-01

    The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

  4. Terpene synthases from Cannabis sativa.

    Science.gov (United States)

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  5. The Photoheterotrophic Growth of Bacteriochlorophyll Synthase-Deficient Mutant of Rhodobacter sphaeroides Is Restored by I44F Mutant Chlorophyll Synthase of Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Kim, Eui-Jin; Kim, Hyeonjun; Lee, Jeong K

    2016-05-28

    Chlorophyll synthase (ChlG) and bacteriochlorophyll synthase (BchG) have a high degree of substrate specificity. The BchG mutant of Rhodobacter sphaeroides, BG1 strain, is photosynthetically incompetent. When BG1 harboring chlG of Synechocystis sp. PCC 6803 was cultured photoheterotrophically, colonies arose at a frequency of approximately 10(-8). All the suppressor mutants were determined to have the same mutational change, ChlGI44F. The mutated enzyme ChlGI44F showed BchG activity. Remarkably, BchGF28I, which has the substitution of F at the corresponding 28(th) residue to I, showed ChlG activity. The Km values of ChlGI44F and BchGF28I for their original substrates, chlorophyllide (Chlide) a and bacteriochlorophyllide (Bchlide) a, respectively, were not affected by the mutations, but the Km values of ChlGI44F and BchGF28I for the new substrates Bchlide a and Chlide a, respectively, were more than 10-fold larger than those for their original substrates, suggesting the lower affinities for new substrates. Taken together, I44 and F28 are important for the substrate specificities of ChlG and BchG, respectively. The BchG activity of ChlGI44F and the ChlG activity of BchGF28I further suggest that ChlG and BchG are evolutionarily related enzymes.

  6. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    Science.gov (United States)

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-06-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase.

  7. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  8. Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

    Energy Technology Data Exchange (ETDEWEB)

    Somerville, Chris R.; Scieble, Wolf

    2000-10-11

    Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  9. Glucosylceramide synthase inhibition alleviates aberrations in synucleinopathy models

    Science.gov (United States)

    Sardi, S. Pablo; Viel, Catherine; Clarke, Jennifer; Treleaven, Christopher M.; Richards, Amy M.; Park, Hyejung; Olszewski, Maureen A.; Dodge, James C.; Marshall, John; Makino, Elina; Wang, Bing; Sidman, Richard L.; Cheng, Seng H.; Shihabuddin, Lamya S.

    2017-01-01

    Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson’s disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V and a A53T–α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T–SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T–SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a disease-modifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease. PMID:28223512

  10. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    Science.gov (United States)

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-12-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

  11. Enhancing production of bio-isoprene using hybrid MVA pathway and isoprene synthase in E. coli.

    Directory of Open Access Journals (Sweden)

    Jianming Yang

    Full Text Available The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the "upper pathway" which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation.

  12. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  13. Characteristics of inositol phosphorylceramide synthase and effects of aureobasidin A on growth and pathogenicity of Botrytis cinerea.

    Science.gov (United States)

    Wang, Xin-hui; Guo, Xing-Jun; Li, Hong-Ye; Gou, Ping

    2015-01-01

    Inositol phosphorylceramide (IPC) synthase is the key enzyme with highly conserved sequences, which is involved in fungal sphingolipid biosynthesis. The antibiotic aureobasidin A (AbA) induces the death of fungi through inhibiting IPC synthase activity. The mutations of AUR1 gene coding IPC synthase in fungi and protozoa causes a resistance to AbA. However, the mechanism of AbA resistance is still elusive. In this paper, we generated two mutants of Botrytis cinerea with AbA-resistance, BcAUR1a and BcAUR1b, through UV irradiation. BcAUR1a lost an intron and BcAUR1b had three amino acid mutations (L197P, F288S and T323A) in the AUR1 gene. AbA strongly inhibits the activity of IPC synthase in wild-type B. cinerea, which leads to distinct changes in cell morphology, including the delay in conidial germination, excessive branching near the tip of the germ tube and mycelium, and the inhibition of the mycelium growth. Further, AbA prevents the infection of wild-type B. cinerea in tomato fruits via reducing oxalic acid secretion and the activity of cellulase and pectinase. On the contrary, AbA has no effect on the growth and pathogenicity of the two mutants. Although both mutants show a similar AbA resistance, the molecular mechanisms might be different between the two mutants.

  14. New insight into the catalytic properties of rice sucrose synthase.

    Science.gov (United States)

    Huang, Yu-Chiao; Hsiang, Erh-Chieh; Yang, Chien-Chih; Wang, Ai-Yu

    2016-01-01

    Sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is a key enzyme in sucrose metabolism in higher plants. SuS belongs to family 4 of the glycosyltransferases (GT4) and contains an E-X7-E motif that is conserved in members of GT4 and two other GT families. To gain insight into the roles of this motif in rice sucrose synthase 3 (RSuS3), the two conserved glutamate residues (E678 and E686) in this motif and a phenylalanine residue (F680) that resides between the two glutamate residues were changed by site-directed mutagenesis. All mutant proteins maintained their tetrameric conformation. The mutants E686D and F680Y retained partial enzymatic activity and the mutants E678D, E678Q, F680S, and E686Q were inactive. Substrate binding assays indicated that UDP and fructose, respectively, were the leading substrates in the sucrose degradation and synthesis reactions of RSuS3. Mutations on E678, F680, and E686 affected the binding of fructose, but not of UDP. The results indicated that E678, F680, and E686 in the E-X7-E motif of RSuS3 are essential for the activity of the enzyme and the sequential binding of substrates. The sequential binding of the substrates implied that the reaction catalyzed by RSuS can be controlled by the availability of fructose and UDP, depending on the metabolic status of a tissue.

  15. Regulation of the PIS1-encoded Phosphatidylinositol Synthase in Saccharomyces cerevisiae by Zinc*

    Science.gov (United States)

    Han, Seung-Hee; Han, Gil-Soo; Iwanyshyn, Wendy M.; Carman, George M.

    2005-01-01

    In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism including an increase in phosphatidylinositol content (Iwanyshyn, W.M., Han, G.-S., and Carman, G.M. (2004) J. Biol. Chem. 279, 21976–21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Δ zrt2Δ mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the β-galactosidase activity driven by the PPIS1-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was due to a transcriptional mechanism. The zinc-mediated induction of the PPIS1-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Δ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between GST-Zap1p687–880 and a putative UASZRE in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UASZRE in the PIS1 promoter abolished the GST-Zap1p687–880-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene. PMID:15980062

  16. Congenital hyperreninemic hypoaldosteronism unlinked to the aldosterone synthase (CYP11B2) gene.

    Science.gov (United States)

    Kayes-Wandover, K M; Tannin, G M; Shulman, D; Peled, D; Jones, K L; Karaviti, L; White, P C

    2001-11-01

    Isolated hyperreninemic hypoaldosteronism presenting in infancy is usually caused by mutations in the CYP11B2 gene encoding aldosterone synthase. We studied five patients in four unrelated kindreds with hyperreninemic hypoaldosteronism, in whom we were unable to find such mutations. All presented in infancy with failure to thrive, hyponatremia, hyperkalemia, markedly elevated plasma renin activity, and low or inappropriately normal aldosterone levels. All had normal cortisol levels and no signs or symptoms of congenital adrenal hyperplasia. All responded to fludrocortisone treatment. There were no mutations detected in exons or splice junctions of CYP11B2. Linkage of the disorder to CYP11B2 was studied in two unrelated consanguineous patients and in an affected sib pair. The consanguineous patients were each heterozygous for at least one of three polymorphic microsatellite markers near CYP11B2, excluding linkage to CYP11B2. However, linkage of the disease to CYP11B2 could not be excluded in the affected sib pair. Genes involved in the regulation of aldosterone biosynthesis, including those encoding angiotensinogen, angiotensin-converting enzyme, and the AT1 angiotensin II receptor were similarly excluded from linkage. These results demonstrate the existence of an inherited form of hyperreninemic hypoaldosteronism distinct from aldosterone synthase deficiency. The affected gene(s) remain to be determined.

  17. Vascular complications of cystathionine β-synthase deficiency: future directions for homocysteine-to-hydrogen sulfide research

    OpenAIRE

    Richard S Beard; Bearden, Shawn E.

    2010-01-01

    Homocysteine (Hcy), a cardiovascular and neurovascular disease risk factor, is converted to hydrogen sulfide (H2S) through the transsulfuration pathway. H2S has attracted considerable attention in recent years for many positive effects on vascular health and homeostasis. Cystathionine β-synthase (CBS) is the first, and rate-limiting, enzyme in the transsulfuration pathway. Mutations in the CBS gene decrease enzymatic activity, which increases the plasma Hcy concentration, a condition called h...

  18. Biochemical investigation of a human pathogenic mutation in the nuclear ATP5E gene using yeast as a model

    Directory of Open Access Journals (Sweden)

    Elodie eSardin

    2015-04-01

    Full Text Available F1F0-ATP synthase is a key enzyme of the mitochondrial energetic metabolism responsible for the production of most cellular ATP in humans. Mayr et al. (Mayr JA, Havlickova V, Zimmermann F, Magler I, Kaplanova V, Jesina P, Pecinova A, Nuskova H, Koch J, Sperl W and Houstek J. 2010. Hum Mol Genet recently described a patient with a homozygote (Y12C mutation in the nuclear gene ATP5E encoding the ε-subunit of ATP synthase. To better define how it affects ATP synthase, we have modelled this mutation in the yeast Saccharomyces cerevisiae. A yeast equivalent of this mutation (Y11C had no significant effect on the growth of yeast on non-fermentable carbon sources (glycerol/ethanol or lactate, conditions under which the activity of the mitochondrial energy transducing system is absolutely essential. In addition, similar to what was observed in patient, this mutation in yeast has a minimal effect on the ATPase/synthase activities. On the contrary, this mutation which has been shown to have a strong impact on the assembly of the ATP synthase complex in humans, shows no significant impact on the assembly/stability of this complex in yeast, suggesting that biogenesis of this complex differs significantly.

  19. The tomato terpene synthase gene family

    NARCIS (Netherlands)

    Falara, V.; Akhtar, T.A.; Nguyen, T.T.H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R.C.; Pichersky, E.

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  20. Cloning of parsley flavone synthase I.

    Science.gov (United States)

    Martens, S; Forkmann, G; Matern, U; Lukacin, R

    2001-09-01

    A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.

  1. Inducible nitric oxide synthase in renal transplantation

    NARCIS (Netherlands)

    Joles, JA; Vos, IH; Grone, HJ; Rabelink, TJ

    2002-01-01

    The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the induc

  2. CF Mutation Panel

    Science.gov (United States)

    ... Testing; Cystic Fibrosis Transmembrane Conductance Regulator Mutation Analysis; CFTR Mutation Analysis Formal name: Cystic Fibrosis Gene Mutation ... an elevated immunoreactive trypsinogen (IRT) or positive sweat chloride test , to confirm the diagnosis of cystic fibrosis. ...

  3. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    Science.gov (United States)

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  4. A novel mitochondrial mutation m.8989G>C associated with neuropathy, ataxia, retinitis pigmentosa - the NARP syndrome.

    Science.gov (United States)

    Duno, Morten; Wibrand, Flemming; Baggesen, Kirsten; Rosenberg, Thomas; Kjaer, Niels; Frederiksen, Anja L

    2013-02-25

    The archetypal NARP syndrome is almost exclusively associated with the m.8993T>C/G mutation in the sixth subunit of the mitochondrial ATP synthase, whereas other mutations in the MT-ATP6 gene primarily associate with Leigh syndrome or Leber's hereditary optic neuropathy (LHON). We report a novel mitochondrial point mutation, m.8989G>C, in a patient presenting with neuropathy, ataxia and retinitis pigmentosa constituting the classical NARP phenotype. This mutation alters the amino acid right next to canonical NARP mutation. We suggest that classic NARP syndrome relates to a defined dysfunction of p.MT-ATP6.

  5. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...... endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle....

  6. Mutations in COX7B Cause Microphthalmia with Linear Skin Lesions, an Unconventional Mitochondrial Disease

    OpenAIRE

    2012-01-01

    Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possi...

  7. Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize.

    Directory of Open Access Journals (Sweden)

    Hao Wu

    Full Text Available The opaque-2 (o2 mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2 called "Quality Protein Maize" (QPM have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms.

  8. Phylogenomic and functional domain analysis of polyketide synthases in Fusarium

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Daren W.; Butchko, Robert A.; Baker, Scott E.; Proctor, Robert H.

    2012-02-01

    Fusarium species are ubiquitous in nature, cause a range of plant diseases, and produce a variety of chemicals often referred to as secondary metabolites. Although some fungal secondary metabolites affect plant growth or protect plants from other fungi and bacteria, their presence in grain based food and feed is more often associated with a variety of diseases in plants and in animals. Many of these structurally diverse metabolites are derived from a family of related enzymes called polyketide synthases (PKSs). A search of genomic sequence of Fusarium verticillioides, F. graminearum, F. oxysporum and Nectria haematococca (anamorph F. solani) identified a total of 58 PKS genes. To gain insight into how this gene family evolved and to guide future studies, we conducted a phylogenomic and functional domain analysis. The resulting genealogy suggested that Fusarium PKSs represent 34 different groups responsible for synthesis of different core metabolites. The analyses indicate that variation in the Fusarium PKS gene family is due to gene duplication and loss events as well as enzyme gain-of-function due to the acquisition of new domains or of loss-of-function due to nucleotide mutations. Transcriptional analysis indicate that the 16 F. verticillioides PKS genes are expressed under a range of conditions, further evidence that they are functional genes that confer the ability to produce secondary metabolites.

  9. N-acetylglutamate synthase: structure, function and defects.

    Science.gov (United States)

    Caldovic, Ljubica; Ah Mew, Nicholas; Shi, Dashuang; Morizono, Hiroki; Yudkoff, Marc; Tuchman, Mendel

    2010-01-01

    N-acetylglutamate (NAG) is a unique enzyme cofactor, essential for liver ureagenesis in mammals while it is the first committed substrate for de novo arginine biosynthesis in microorganisms and plants. The enzyme that produces NAG from glutamate and CoA, NAG synthase (NAGS), is allosterically inhibited by arginine in microorganisms and plants and activated in mammals. This transition of the allosteric effect occurred when tetrapods moved from sea to land. The first mammalian NAGS gene (from mouse) was cloned in 2002 and revealed significant differences from the NAGS ortholog in microorganisms. Almost all NAGS genes possess a C-terminus transferase domain in which the catalytic activity resides and an N-terminus kinase domain where arginine binds. The three-dimensional structure of NAGS shows two distinctly folded domains. The kinase domain binds arginine while the acetyltransferase domain contains the catalytic site. NAGS deficiency in humans leads to hyperammonemia and can be primary, due to mutations in the NAGS gene or secondary due to other mitochondrial aberrations that interfere with the normal function of the same enzyme. For either condition, N-carbamylglutamate (NCG), a stable functional analog of NAG, was found to either restore or improve the deficient urea-cycle function.

  10. The y1 gene of maize codes for phytoene synthase.

    Science.gov (United States)

    Buckner, B; Miguel, P S; Janick-Buckner, D; Bennetzen, J L

    1996-05-01

    The cloned y1 locus of maize was sequenced and found to encode phytoene synthase. Different "wild-type" alleles of the locus were found to differ by the insertion of transposable elements in their promoter and polyA addition regions, and by the length of a CCA tandem repeat series, without any obvious effect on function of the gene. A dominant Y1 ("wild-type") allele was observed to be expressed at highest levels in the seedling but also in the embryo and endosperm. The Mu3 transposable element insertion responsible for a pastel allele of y1, which gives lowered levels of carotenoids in the endosperm of kernels and seedlings grown at high temperatures, was located in the 5' end of the gene. Although the size of the transcript from this y1 mutation suggests that the Mu3 element provides the promoter for this allele, leaf tissue in this mutant line contained approximately normal amounts of y1 mRNA. A recessive allele of y1, which conditions normal levels of carotenoids in the embryo and seedling, but almost no carotenoids in the endosperm, was found to accumulate normal amounts of y1 mRNA in the seedling and embryo, while y1 transcripts were not detected in the endosperm.

  11. Conservation and Role of Electrostatics in Thymidylate Synthase.

    Science.gov (United States)

    Garg, Divita; Skouloubris, Stephane; Briffotaux, Julien; Myllykallio, Hannu; Wade, Rebecca C

    2015-11-27

    Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

  12. Cellulose Synthases and Synthesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Anne Endler; Staffan Persson

    2011-01-01

    Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.

  13. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  14. Building-block selectivity of polyketide synthases.

    Science.gov (United States)

    Liou, Grace F; Khosla, Chaitan

    2003-04-01

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

  15. Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

    Energy Technology Data Exchange (ETDEWEB)

    Baskaran, Sulochanadevi; Chikwana, Vimbai M.; Contreras, Christopher J.; Davis, Keri D.; Wilson, Wayne A.; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D. (Indiana-Med); (Des Moines U)

    2012-12-10

    Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V{sub max}/S{sub 0.5} for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V{sub max}/S{sub 0.5} for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells ({Delta}gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a 'toehold mechanism,' keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.

  16. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    Science.gov (United States)

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  17. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  18. Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase.

    Science.gov (United States)

    Srividya, Narayanan; Davis, Edward M; Croteau, Rodney B; Lange, B Markus

    2015-03-17

    Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

  19. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  20. Insights into Diterpene Cyclization from Structure of Bifunctional Abietadiene Synthase from Abies grandis

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Ke; Gao, Yang; Hoy, Julie A.; Mann, Francis M.; Honzatko, Richard B.; Peters, Reuben J. (Iowa State)

    2013-09-24

    Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 {angstrom} resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains ({alpha}, {beta}, and {gamma}). The class I active site is within the C-terminal {alpha} domain, and the class II active site is between the N-terminal {gamma} and {beta} domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg{sup 2+} complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This 'loop-in' conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the 'loop-out' conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.

  1. Insights into Diterpene Cyclization from Structure of Bifunctional Abietadiene Synthase from Abies grandis*

    Science.gov (United States)

    Zhou, Ke; Gao, Yang; Hoy, Julie A.; Mann, Francis M.; Honzatko, Richard B.; Peters, Reuben J.

    2012-01-01

    Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg2+ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities. PMID:22219188

  2. Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.

    Science.gov (United States)

    ben Bdira, Fredj; González, Esperanza; Pluta, Paula; Laín, Ana; Sanz-Parra, Arantza; Falcon-Perez, Juan Manuel; Millet, Oscar

    2014-11-01

    Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.

  3. Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants.

    Science.gov (United States)

    Gray, Dennis W; Breneman, Steven R; Topper, Lauren A; Sharkey, Thomas D

    2011-06-10

    2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ~90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K(+), whereas isoprene production is inhibited by K(+) such that, at physiologically relevant [K(+)], little or no isoprene emission should be detected from MBO-emitting trees. The K(m) of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site.

  4. S-Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics.

    Science.gov (United States)

    Módis, Katalin; Ju, YoungJun; Ahmad, Akbar; Untereiner, Ashley A; Altaany, Zaid; Wu, Lingyun; Szabo, Csaba; Wang, Rui

    2016-11-01

    Mammalian cells can utilize hydrogen sulfide (H2S) to support mitochondrial respiration. The aim of our study was to explore the potential role of S-sulfhydration (a H2S-induced posttranslational modification, also known as S-persulfidation) of the mitochondrial inner membrane protein ATP synthase (F1F0 ATP synthase/Complex V) in the regulation of mitochondrial bioenergetics. Using a biotin switch assay, we have detected S-sulfhydration of the α subunit (ATP5A1) of ATP synthase in response to exposure to H2S in vitro. The H2S generator compound NaHS induced S-sulfhydration of ATP5A1 in HepG2 and HEK293 cell lysates in a concentration-dependent manner (50-300μM). The activity of immunocaptured mitochondrial ATP synthase enzyme isolated from HepG2 and HEK293 cells was stimulated by NaHS at low concentrations (10-100nM). Site-directed mutagenesis of ATP5A1 in HEK293 cells demonstrated that cysteine residues at positions 244 and 294 are subject to S-sulfhydration. The double mutant ATP synthase protein (C244S/C294S) showed a significantly reduced enzyme activity compared to control and the single-cysteine-mutated recombinant proteins (C244S or C294S). To determine whether endogenous H2S plays a role in the basal S-sulfhydration of ATP synthase in vivo, we compared liver tissues harvested from wild-type mice and mice deficient in cystathionine-gamma-lyase (CSE, one of the three principal mammalian H2S-producing enzymes). Significantly reduced S-sulfhydration of ATP5A1 was observed in liver homogenates of CSE(-/-) mice, compared to wild-type mice, suggesting a physiological role for CSE-derived endogenous H2S production in the S-sulfhydration of ATP synthase. Various forms of critical illness (including burn injury) upregulate H2S-producing enzymes and stimulate H2S biosynthesis. In liver tissues collected from mice subjected to burn injury, we detected an increased S-sulfhydration of ATP5A1 at the early time points post-burn. At later time points (when systemic H2S

  5. Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

    Directory of Open Access Journals (Sweden)

    Atsuko Kanazawa

    2017-05-01

    Full Text Available In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf. The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid proton conductivity (gH+ in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.

  6. The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp. citri

    Institute of Scientific and Technical Information of China (English)

    Guo Jing; SonG Xue; Zou Li-fang; Zou Hua-song; CHen Gong-you

    2015-01-01

    Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. citri, carA and carB encode the smal and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hyper-sensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type III secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracel ular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp. citri.

  7. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  8. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives.

  9. Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley.

    Science.gov (United States)

    Howard, Thomas P; Fahy, Brendan; Leigh, Fiona; Howell, Phil; Powell, Wayne; Greenland, Andy; Trafford, Kay; Smith, Alison M

    2014-03-01

    The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC2F4 families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC2F4 families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high β-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent.

  10. Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley☆

    Science.gov (United States)

    Howard, Thomas P.; Fahy, Brendan; Leigh, Fiona; Howell, Phil; Powell, Wayne; Greenland, Andy; Trafford, Kay; Smith, Alison M.

    2014-01-01

    The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC2F4 families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC2F4 families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high β-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent. PMID:24748716

  11. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    OpenAIRE

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity ...

  12. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Science.gov (United States)

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

  13. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Berta Alquézar

    2017-08-01

    Full Text Available Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck genome sequence allowed us to characterize for the first time the terpene synthase (TPS family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  14. Multiple allelic forms of acetohydroxyacid synthase are responsible for herbicide resistance in Setaria viridis.

    Science.gov (United States)

    Laplante, Julie; Rajcan, Istvan; Tardif, François J

    2009-08-01

    In weed species, resistance to herbicides inhibiting acetohydroxyacid synthase (AHAS) is often conferred by genetic mutations at one of six codons in the AHAS gene. These mutations provide plants with various levels of resistance to different chemical classes of AHAS inhibitors. Five green foxtail [Setaria viridis (L.) Beauv.] populations were reported in Ontario with potential resistance to the AHAS-inhibiting herbicide imazethapyr. The objectives of this study were to confirm resistance, establish the resistance spectrum for each of the five populations, and determine its genetic basis. Dose response curves were generated for whole plant growth and enzyme activity, and the AHAS gene was sequenced. Resistance was confirmed by determining the resistance factor to imazethapyr in the five resistant green foxtail populations for whole plant dose response experiments (21- to 182-fold) and enzyme assays (15- to 260-fold). All five imazethapyr-resistant populations showed cross-resistance to nicosulfuron and flucarbazone while only three populations had cross-resistance to pyrithiobac. Sequence analyses revealed single base-pair mutations in the resistant populations of green foxtail. These mutations were coded for Thr, Asn, or Ile substitution at Ser(653). In addition, a new mutation was found in one population that coded for an Asp substitution at Gly(654). There is an agreement between the spectra of resistance observed and the type of resistance known to be conferred by these substitutions. Moreover, it indicates that, under similar selection pressure (imazethapyr), a variety of mutations can be selected for different populations, making the resistance pattern difficult to predict from herbicide exposure history.

  15. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  16. Mutations of mitochondrial genome in patients with carotid atherosclerosis

    Directory of Open Access Journals (Sweden)

    Margarita A Sazonova

    2015-03-01

    Full Text Available With aim of detection the spectrum of mitochondrial DNA mutations in patients with carotid atherosclerosis from Moscow Region, we used a Roche 454 high-throughput sequencing of the whole mitochondrial genome. We have found that the presence of a number of homoplasmic mitochondrial DNA mutations in genes of 16S ribosomal RNA, subunits 2, 4 and 5 NADH dehydrogenase, subunits 1 and 2 cytochrome C oxidase, subunit 6 ATP-synthase, tRNA- Leu 2 and cytochrome B differed between conventionally healthy participants of the study and patients with carotid atherosclerosis. We also found heteroplasmic mutations, including insertions one or several nucleotides, that occurred more frequently in mitochondrial DNA of conventionally healthy participants of the study or patients with atherosclerotic lesions.

  17. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    Science.gov (United States)

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  18. Hereditary sideroblastic anemia: pathophysiology and gene mutations.

    Science.gov (United States)

    Harigae, Hideo; Furuyama, Kazumichi

    2010-10-01

    Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. Ring sideroblasts are erythroblasts characterized by iron accumulation in perinuclear mitochondria due to impaired iron utilization. There are two forms of sideroblastic anemia, i.e., inherited and acquired sideroblastic anemia. Inherited sideroblastic anemia is a rare and heterogeneous disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur (Fe-S) cluster biogenesis, or Fe-S cluster transport, and mitochondrial metabolism. The most common inherited sideroblastic anemia is X-linked sideroblastic anemia (XLSA) caused by mutations of the erythroid-specific δ-aminolevulinate synthase gene (ALAS2), which is the first enzyme of heme biosynthesis in erythroid cells. Sideroblastic anemia due to SLC25A38 gene mutations, which is a mitochondrial transporter, is the next most common inherited sideroblastic anemia. Other forms of inherited sideroblastic anemia are very rare, and accompanied by impaired function of organs other than hematopoietic tissue, such as the nervous system, muscle, or exocrine glands due to impaired mitochondrial metabolism. Moreover, there are still significant numbers of cases with genetically undefined inherited sideroblastic anemia. Molecular analysis of these cases will contribute not only to the development of effective treatment, but also to the understanding of mitochondrial iron metabolism.

  19. Evolution and function of phytochelatin synthases.

    Science.gov (United States)

    Clemens, Stephan

    2006-02-01

    Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

  20. Torque generation mechanism of ATP synthase

    Science.gov (United States)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  1. The general base in the thymidylate synthase catalyzed proton abstraction.

    Science.gov (United States)

    Ghosh, Ananda K; Islam, Zahidul; Krueger, Jonathan; Abeysinghe, Thelma; Kohen, Amnon

    2015-12-14

    The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2'-deoxythymidine-5'-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic -OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton.

  2. Regulation of the PIS1-encoded phosphatidylinositol synthase in Saccharomyces cerevisiae by zinc.

    Science.gov (United States)

    Han, Seung-Hee; Han, Gil-Soo; Iwanyshyn, Wendy M; Carman, George M

    2005-08-12

    In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976-21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Delta zrt2Delta mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the beta-galactosidase activity driven by the P(PIS1)-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the P(PIS1)-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Delta mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p(687-880) and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p(687-880)-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.

  3. Maize Mutator transposon

    Institute of Scientific and Technical Information of China (English)

    Yijun WANG; Mingliang XU; Dexiang DENG; Yunlong BIAN

    2008-01-01

    Transposable elements are widely distributed in eukaryotes. Due to its high copy numbers, high forward mutation rate and preferential insertion into low-copy DNA sequences, among others, the Mutator system has been widely used as a mutagen in genomic research. The discovery, classification, transposition specificity and epige-netic regulation of Mutator transposons were described. The application of Mutator tagging in plant genomic research was also presented. The role of Mu-like elements in genome evolution was briefly depicted. Moreover, the direction of Mutator transposon research in the future was discussed.

  4. [Four cases of aldosterone synthase deficiency in childhood].

    Science.gov (United States)

    Collinet, E; Pelissier, P; Richard, O; Gay, C; Pugeat, M; Morel, Y; Stephan, J-L

    2012-11-01

    Neonatal salt-wasting syndromes are rare but potentially serious conditions. Isolated hypoaldosteronism is an autosomal recessive inherited disorder of terminal aldosterone synthesis, leading to selective aldosterone deficiency. Two different biochemical forms of this disease have been described, called aldosterone synthase deficiency or corticosterone methyl oxydase, types I and II. In type I, there is no aldosterone synthase activity and the 18 hydroxycorticosterone (18 OHB) level is low, whereas in type II, a residual activity of aldosterone synthase persists and 18 OHB is overproduced. We report on four patients with isolated hypoaldosteronism. In 2 of them, who were recently diagnosed with aldosterone synthase deficit, we discuss the symptoms and treatment. The 2 other patients are now adults. We discuss the long-term outcome, the quality of adult life, aldosterone synthase deficits, as well as the pathophysiology and molecular analysis.

  5. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  6. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

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    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  7. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway.

    Science.gov (United States)

    Taura, Futoshi; Tanaka, Shinji; Taguchi, Chiho; Fukamizu, Tomohide; Tanaka, Hiroyuki; Shoyama, Yukihiro; Morimoto, Satoshi

    2009-06-18

    Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.

  8. Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina.

    Science.gov (United States)

    Coppin, Evelyne; Silar, Philippe

    2007-08-01

    In the filamentous fungus Podospora anserina, many pigmentation mutations map to the median region of the complex locus '14', called segment '29'. The data presented in this paper show that segment 29 corresponds to a gene encoding a polyketide synthase, designated PaPKS1, and identifies two mutations that completely or partially abolish the activity of the PaPKS1 polypeptide. We present evidence that the P. anserina green pigment is a (DHN)-melanin. Using the powerful genetic system of PaPKS1 cloning, we demonstrate that in P. anserina trans-duplicated sequences are subject to the RIP process as previously demonstrated for the cis-duplicated regions.

  9. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  10. The nitric oxide synthase of mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Goin, J C; Boquet, M; Canteros, M G; Franchi, A M; Perez Martinez, S; Polak, J M; Viggiano, J M; Gimeno, M A

    1997-07-01

    Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

  11. Endothelial nitric oxide synthase in the microcirculation.

    Science.gov (United States)

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

  12. Phosphorylation regulates myo-inositol-3-phosphate synthase: a novel regulatory mechanism of inositol biosynthesis.

    Science.gov (United States)

    Deranieh, Rania M; He, Quan; Caruso, Joseph A; Greenberg, Miriam L

    2013-09-13

    myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using (32)Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS.

  13. Nucleotide variability in the 5-enolpyruvylshikimate-3-phosphate synthase gene from Eleusine indica (L.) Gaertn.

    Science.gov (United States)

    Chong, J L; Wickneswari, R; Ismail, B S; Salmijah, S

    2008-02-01

    This study reports the results of the partial DNA sequence analysis of the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant (R) and glyphosate-susceptible (S) biotypes of Eleusine indica (L.) Gaertn from Peninsular Malaysia. Sequencing results revealed point mutation at nucleotide position 875 in the R biotypes of Bidor, Chaah and Temerloh. In the Chaah R population, substitution of cytosine (C) to adenine (A) resulted in the change of threonine (Thr106) to proline (Pro106) and from C to thymidine (T) in the Bidor R population, leading to serine (Ser106) from Pro106. As for the Temerloh R, C was substituted by T resulting in the change of Pro106 to Ser106. A new mutation previously undetected in the Temerloh R was revealed with C being substituted with A, resulting in the change of Pro106 to Thr106 indicating multiple founding events rather than to the spread of a single resistant allele. There was no point mutation recorded at nucleotide position 875 previously demonstrated to play a pivotal role in conferring glyphosate resistance to E. indica for the Lenggeng, Kuala Selangor, Melaka R populations. Thus, there may be another resistance mechanism yet undiscovered in the resistant Lenggeng, Kuala Selangor and Melaka populations.

  14. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Science.gov (United States)

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  15. Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®

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    Vincent Kate

    2009-12-01

    Full Text Available Abstract Background TILLING (Targeting Induced Local Lesions IN Genomes is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes. Results We demonstrate that High Resolution Melting (HRM analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Conclusion Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence

  16. Extending a Single Residue Switch for Abbreviating Catalysis in Plant ent-Kaurene Synthases

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    Meirong Jia

    2016-11-01

    Full Text Available Production of ent-kaurene as a precursor for important signaling molecules such as the gibberellins seems to have arisen early in plant evolution, with corresponding cyclase(s present in all land plants (i.e., embryophyta. The relevant enzymes seem to represent fusion of the class II diterpene cyclase that produces the intermediate ent-copalyl diphosphate (ent-CPP and the subsequently acting class I diterpene synthase that produces ent-kaurene, although the bifunctionality of the ancestral gene is only retained in certain early diverging plants, with gene duplication and sub-functionalization leading to distinct ent-CPP synthases (CPSs and ent-kaurene synthases (KSs generally observed. This evolutionary scenario implies that plant KSs should have conserved structural features uniquely required for production of ent-kaurene relative to related enzymes that have alternative function. Notably, substitution of threonine for a conserved isoleucine has been shown to short-circuit the complex bicyclization and rearrangement reaction catalyzed by KSs after initial cyclization, leading to predominant production of ent-pimaradiene, at least in KSs from angiosperms. Here this effect is shown to extend to KSs from earlier diverging plants (i.e., bryophytes, including a bifunctional CPS/KS. In addition, attribution of the dramatic effect of this single residue switch on product outcome to electrostatic stabilization of the ent-pimarenyl carbocation intermediate formed upon initial cyclization by the hydroxyl introduced by threonine substitution has been called into question by the observation of similar effects from substitution of alanine. Here further mutational analysis and detailed product analysis is reported that supports the importance of electrostatic stabilization by a hydroxyl or water.

  17. Role of cysteine residues in pseudouridine synthases of different families.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  18. Subcellular localization of the homocitrate synthase in Penicillium chrysogenum.

    Science.gov (United States)

    Bañuelos, O; Casqueiro, J; Steidl, S; Gutiérrez, S; Brakhage, A; Martín, J F

    2002-01-01

    There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

  19. Three kinds of mutation

    CERN Document Server

    Buan, Aslak Bakke; Thomas, Hugh

    2010-01-01

    For a finite dimensional hereditary algebra, we consider: exceptional sequences in the category of finite dimensional modules, silting objects in the bounded derived category, and m-cluster tilting objects in the m-cluster category. There are mutation operations on both the set of m-cluster tilting objects and the set of exceptional sequences. It is also possible to define a mutation operation for silting objects. We compare these three different notions of mutation.

  20. Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism

    Directory of Open Access Journals (Sweden)

    Liu Ping-Li

    2012-11-01

    Full Text Available Abstract Background Chrysanthemyl diphosphate synthase (CDS is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown. Results Two duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity. Conclusion Positive selection associated with gene duplication played a major role in the evolution of CDS.

  1. Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Bo; Zhou, Jing; Kahen, Elliot; Terns, Rebecca M.; Terns, Michael P.; Li, Hong; (Inst. Mol. BioScience); (FSU); (Georgia)

    2009-09-29

    Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 {angstrom} of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

  2. Modification of human U4 RNA requires U6 RNA and multiple pseudouridine synthases.

    Science.gov (United States)

    Zerby, D B; Patton, J R

    1997-12-01

    Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore unmodified, U4 RNA. The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, since the activities in the extracts were micrococcal nuclease (MN) sensitive. Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glycerol gradient analysis we determined that exogenously added U4 RNA that is associated with U6 RNA (sedimentation velocity 16 S) was significantly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containing (5-FU) wild-type and mutant U4 RNAs, were used to investigate formation of Psi in U4 RNA. Deletions and point mutations in these 5-FU-containing U4 RNAs affected their ability to inhibit Psi synthase in vitro. With the aid of these potent inhibitors it was determined that at least two separate activities modify the uridines at these positions.

  3. Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA.

    Science.gov (United States)

    Liang, Bo; Zhou, Jing; Kahen, Elliot; Terns, Rebecca M; Terns, Michael P; Li, Hong

    2009-07-01

    Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 A of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

  4. The Pseudouridine Synthases Proceed through a Glycal Intermediate.

    Science.gov (United States)

    Veerareddygari, Govardhan Reddy; Singh, Sanjay K; Mueller, Eugene G

    2016-06-29

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2'. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases.

  5. Peroxisomal and mitochondrial citrate synthase in CAM plants.

    Science.gov (United States)

    Zafra, M F; Segovia, J L; Alejandre, M J; García-Peregrín, E

    1981-12-01

    Citrate synthase wa studied for the first time in peroxisomes and mitochondria of crassulacean acid metabolism plants. Cellular organelles were isolated from Agave americana leaves by sucrose density gradient centrifugation and characterized by the use of catalase and cytochrome oxidase as marker enzymes, respectively. 48,000 X g centrifugation caused the breakdown of the cellular organelles. The presence of a glyoxylate cycle enzyme (citrate synthase) and a glycollate pathway enzyme (catalase) in the same organelles, besides the absence of another glyoxalate cycle enzyme (malate synthase) is reported for the first time, suggesting that peroxisomal and glyoxysomal proteins are synthesized at the same time and housed in he same organelle.

  6. Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis

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    D'Hulst Christophe

    2008-09-01

    Full Text Available Abstract Background The biochemical mechanisms that determine the molecular architecture of amylopectin are central in plant biology because they allow long-term storage of reduced carbon. Amylopectin structure imparts the ability to form semi-crystalline starch granules, which in turn provides its glucose storage function. The enzymatic steps of amylopectin biosynthesis resemble those of the soluble polymer glycogen, however, the reasons for amylopectin's architectural distinctions are not clearly understood. The multiplicity of starch biosynthetic enzymes conserved in plants likely is involved. For example, amylopectin chain elongation in plants involves five conserved classes of starch synthase (SS, whereas glycogen biosynthesis typically requires only one class of glycogen synthase. Results Null mutations were characterized in AtSS2, which codes for SSII, and mutant lines were compared to lines lacking SSIII and to an Atss2, Atss3 double mutant. Loss of SSII did not affect growth rate or starch quantity, but caused increased amylose/amylopectin ratio, increased total amylose, and deficiency in amylopectin chains with degree of polymerization (DP 12 to DP28. In contrast, loss of both SSII and SSIII caused slower plant growth and dramatically reduced starch content. Extreme deficiency in DP12 to DP28 chains occurred in the double mutant, far more severe than the summed changes in SSII- or SSIII-deficient plants lacking only one of the two enzymes. Conclusion SSII and SSIII have partially redundant functions in determination of amylopectin structure, and these roles cannot be substituted by any other conserved SS, specifically SSI, GBSSI, or SSIV. Even though SSIII is not required for the normal abundance of glucan chains of DP12 to DP18, the enzyme clearly is capable of functioning in production such chains. The role of SSIII in producing these chains cannot be detected simply by analysis of an individual mutation. Competition between

  7. Gestational mutations in radiation carcinogenesis

    Science.gov (United States)

    Meza, R.; Luebeck, G.; Moolgavkar, S.

    Mutations in critical genes during gestation could increase substantially the risk of cancer. We examine the consequences of such mutations using the Luebeck-Moolgavkar model for colorectal cancer and the Lea-Coulson modification of the Luria-Delbruck model for the accumulation of mutations during gestation. When gestational mutation rates are high, such mutations make a significant contribution to cancer risk even for adult tumors. Furthermore, gestational mutations ocurring at distinct times during emryonic developmemt lead to substantially different numbers of mutated cells at birth, with early mutations leading to a large number (jackpots) of mutated cells at birth and mutation occurring late leading to only a few mutated cells. Thus gestational mutations could confer considerable heterogeneity of the risk of cancer. If the fetus is exposed to an environmental mutagen, such as ionizing radiation, the gestational mutation rate would be expected to increase. We examine the consequences of such exposures during gestation on the subsequent development of cancer.

  8. Polysaccharide storage myopathy phenotype in quarter horse-related breeds is modified by the presence of an RYR1 mutation.

    Science.gov (United States)

    McCue, M E; Valberg, S J; Jackson, M; Borgia, L; Lucio, M; Mickelson, J R

    2009-01-01

    In this study we examined a family of Quarter Horses with Polysaccharide Storage Myopathy (PSSM) with a dominant mutation in the skeletal muscle glycogen synthase (GYS1) gene. A subset of horses within this family had a more severe and occasionally fatal PSSM phenotype. The purpose of this study was to identify a modifying gene(s) for the severe clinical phenotype. A genetic association analysis was used to identify RYR1 as a candidate modifying gene. A rare, known equine RYR1 mutation, associated with malignant hyperthermia (MH), was found to segregate in this GYS1 PSSM family. Retrospective analysis of patient records (n=179) demonstrated that horses with both the GYS1 and RYR1 mutations had a more severe clinical phenotype than horses with the GYS1 mutation alone. A treadmill trial (n=8) showed that serum creatine kinase activity was higher and exercise intolerance greater in horses with both mutations compared to the GYS1 mutation alone.

  9. A stilbene synthase from Japanese red pine (Pinus densiflora): implications for phytoalexin accumulation and down-regulation of flavonoid biosynthesis.

    Science.gov (United States)

    Kodan, Atsushi; Kuroda, Hiroyuki; Sakai, Fukumi

    2002-03-05

    Stilbene synthase (STS) and chalcone synthase (CHS) are plant-specific polyketide synthases that play key roles in the stilbenoid and flavonoid biosyntheses, respectively. We have recently isolated from Pinus densiflora three STS cDNAs (PDSTS1, PDSTS2, and PDSTS3) and one CHS cDNA (PDCHSX). We then heterologously expressed these cDNAs in Escherichia coli and characterized their properties. An unusual STS isozyme, PDSTS3, lacks the common C-terminal extension of STS because of a frame-shift mutation and shows the highest pinosylvin-forming activity among the STSs tested. Pinosylvin was shown to be a potent inhibitor of PDCHSX (K(i) = 6 microM) as well as PDSTS2 (K(i) = 13 microM), which presumably maintains the balance between the stilbenoid and flavonoid biosyntheses. PDSTS3 was insensitive to product inhibition. We identified PDSTS3 in the pine seedlings as well as full-length STS. The data provide evidence that PDSTS3 is involved in the potential regulation of the stilbenoid and flavonoid biosynthetic pathways in pine trees.

  10. Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p.

    Science.gov (United States)

    Maceluch, J; Kmieciak, M; Szweykowska-Kulińska, Z; Jarmołowski, A

    2001-01-01

    Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

  11. Asp residues of βDELSEED-motif are required for peptide binding in the Escherichia coli ATP synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Tayou, Junior; Laughlin, Thomas F

    2015-04-01

    This study demonstrates the requirement of Asp-380 and Asp-386 in the βDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants βD380A/βD386A, βD380Q/βD386Q, or βD380R/βD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of βD380 and βD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of βDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, βD380 and βD386, are essential for proper peptide binding and inhibition of ATP synthase. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...... in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than...... in the longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather...

  13. Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

    NARCIS (Netherlands)

    Banzhaf, M.; van den Berg van Saparoea, B.; Terrak, M.; Fraipont, C.; Egan, A.; Philippe, J.; Zapun, A.; Breukink, E.; Nguyen-Distèche, M.; den Blaauwen, T.; Vollmer, W.

    2012-01-01

    Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan

  14. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Directory of Open Access Journals (Sweden)

    Zulfiqar Ahmad

    Full Text Available We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  15. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Science.gov (United States)

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  16. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... 1Department of Environmental Biotechnology, Bharathidasan University, ... script and UA cloning vector (QIAGEN PCR Cloning Kit) was used to clone ..... Expression of a Arabidopsis sucrose synthase gene indicates a role.

  17. Mutation and premating isolation.

    Science.gov (United States)

    Woodruff, R C; Thompson, J N

    2002-11-01

    While premating isolation might be traceable to different genetic mechanisms in different species, evidence supports the idea that as few as one or two genes may often be sufficient to initiate isolation. Thus, new mutation can theoretically play a key role in the process. But it has long been thought that a new isolation mutation would fail, because there would be no other individuals for the isolation-mutation-carrier to mate with. We now realize that premeiotic mutations are very common and will yield a cluster of progeny carrying the same new mutant allele. In this paper, we discuss the evidence for genetically simple premating isolation barriers and the role that clusters of an isolation mutation may play in initiating allopatric, and even sympatric, species divisions.

  18. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function

    OpenAIRE

    Haines, Ricci J.; Corbin, Karen D.; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C.

    2012-01-01

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regu...

  19. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  20. Nitric Oxide Synthases in Heart Failure

    Science.gov (United States)

    Carnicer, Ricardo; Crabtree, Mark J.; Sivakumaran, Vidhya

    2013-01-01

    Abstract Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal. 18, 1078–1099. PMID:22871241

  1. Linking pseudouridine synthases to growth, development and cell competition.

    Science.gov (United States)

    Tortoriello, Giuseppe; de Celis, José F; Furia, Maria

    2010-08-01

    Eukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H/ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA-like molecules. In humans, loss of pseudouridine synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing disc, an advantageous model system for studies of cell growth and differentiation. In this organ, depletion of the mfl-encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a key role in the development of the loss of function mutant phenotype. Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine synthase loss of function phenotype is caused by defects in Notch signalling.

  2. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    Science.gov (United States)

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  3. Bifunctional activity of deoxyhypusine synthase/hydroxylase from Trichomonas vaginalis.

    Science.gov (United States)

    Quintas-Granados, Laura Itzel; Carvajal Gamez, Bertha Isabel; Villalpando, Jose Luis; Ortega-Lopez, Jaime; Arroyo, Rossana; Azuara-Liceaga, Elisa; Álvarez-Sánchez, María Elizbeth

    2016-04-01

    The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ((3)H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity.

  4. Mutation rates among RNA viruses

    OpenAIRE

    Drake, John W.; Holland, John J.

    1999-01-01

    The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viru...

  5. Synergistic activation of defense responses in Arabidopsis by simultaneous loss of the GSL5 callose synthase and the EDR1 protein kinase.

    Science.gov (United States)

    Wawrzynska, Anna; Rodibaugh, Natalie L; Innes, Roger W

    2010-05-01

    Loss-of-function mutations in the EDR1 gene of Arabidopsis confer enhanced resistance to Golovinomyces cichoracearum (powdery mildew). Disease resistance mediated by the edr1 mutation is dependent on an intact salicylic acid (SA) signaling pathway, but edr1 mutant plants do not constitutively express the SA-inducible gene PR-1 and are not dwarfed. To identify other components of the EDR1 signaling network, we screened for mutations that enhanced the edr1 mutant phenotype. Here, we describe an enhancer of edr1 mutant, eed3, which forms spontaneous lesions in the absence of pathogen infection, constitutively expresses both SA- and methyl jasmonate (JA)-inducible defense genes, and is dwarfed. Positional cloning of eed3 revealed that the mutation causes a premature stop codon in GLUCAN SYNTHASE-LIKE 5 (GSL5, also known as POWDERY MILDEW RESISTANT 4), which encodes a callose synthase required for pathogen-induced callose production. Significantly, gsl5 single mutants do not constitutively express PR-1 or AtERF1 (a JA-inducible gene) and are not dwarfed. Thus, loss of both EDR1 and GSL5 function has a synergistic effect. Our data suggest that EDR1 and GSL5 negatively regulate SA and JA production or signaling by independent mechanisms and that negative regulation of defense signaling by GSL5 may be independent of callose production.

  6. Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

    Science.gov (United States)

    Lahuta, Lesław B; Pluskota, Wioletta E; Stelmaszewska, Joanna; Szablińska, Joanna

    2014-09-01

    The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

  7. Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii

    Directory of Open Access Journals (Sweden)

    Huan Zhao

    2017-03-01

    Full Text Available Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS and cycloartenol synthase (CAS are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR approach. The SgSQS cDNA has a 1254 bp open reading frame (ORF encoding 417 amino acids, and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression.

  8. Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain

    DEFF Research Database (Denmark)

    Zhang, Yiming; Liu, Guodong; Engqvist, Martin K. M.

    2015-01-01

    DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase...... further increased the maximum specific growth rate to 0.069 h-1. Conclusions: In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing...... expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain....

  9. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  10. PS Dependent APP Cleavage Regulates Glucosylceramide Synthase and is Affected in Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Marcus O.W. Grimm

    2014-06-01

    Full Text Available Background: Gangliosides were found to be associated with Alzheimer's disease (AD. Here we addressed a potential function of γ-secretase (presenilin dependent cleavage of the amyloid-precursor-protein (APP in the regulation of ganglioside de novo synthesis. Methods: To identify a potential role of γ-secretase and APP in ganglioside de novo synthesis we used presenilin (PS deficient and APP deficient cells and mouse brains, mutated PS as well as transgenic mice and AD post mortem brains. Changes in glucosylceramide synthase (GCS activity were identified by incorporation of radiolabeled UDP-glucose in glucosylceramide, changes in gene expression via real-time PCR and Western blot analysis. Alterations in ganglioside levels were determined by thin layer chromatography and mass spectrometry. Results: We found that PS and APP deficiency, in vitro and in vivo, resulted in increased GCS gene expression, elevated enzyme activity and thus increased glucosylceramide and total ganglioside level. Using a specific γ-secretase inhibitor revealed that PS proteolytic activity alters ganglioside homeostasis. By the use of mutated PS causing early onset AD in cell culture and transgenic mice we found that GCS is increased in AD, further substantiated by the use of AD post mortem brains, suffering from sporadic AD. Conclusion: APP processing regulates ganglioside de novo synthesis and is affected in AD.

  11. Branching patterns in leaf starches from Arabidopsis mutants deficient in diverse starch synthases.

    Science.gov (United States)

    Zhu, Fan; Bertoft, Eric; Szydlowski, Nicolas; d'Hulst, Christophe; Seetharaman, Koushik

    2015-01-12

    This is the first report on the cluster structure of transitory starch from Arabidopsis leaves. In addition to wild type, the molecular structures of leaf starch from mutants deficient in starch synthases (SS) including single enzyme mutants ss1-, ss2-, or ss3-, and also double mutants ss1-ss2- and ss1-ss3- were characterized. The mutations resulted in increased amylose content. Clusters from whole starch were isolated by partial hydrolysis using α-amylase of Bacillus amyloliquefaciens. The clusters were then further hydrolyzed with concentrated α-amylase of B. amyloliquefaciens to produce building blocks (α-limit dextrins). Structures of the clusters and their building blocks were characterized by chromatography of samples before and after debranching treatment. While the mutations increased the size of clusters, the reasons were different as reflected by the composition of their unit chains and building blocks. In general, all mutants contained more of a-chains that preferentially increased the number of small building blocks with only two chains. The clusters of the double mutant ss1-ss3- were very large and possessed also more of large building blocks with four or more chains. The results from transitory starch are compared with those from agriculturally important crops in the context that to what extent the Arabidopsis can be a true biotechnological reflection for starch modifications through genetic means.

  12. Relationship Between Polymorphism of Cystathionine beta Synthase Gene and Congenital Heart Disease in Chinese Nuclear Families

    Institute of Scientific and Technical Information of China (English)

    XIAO-MING SONG; XIAO-YING ZHENG; WEN-LI ZHU; LEI HUANG; YONG LI

    2006-01-01

    Objective To study the relationship between polymorphism of cystathionine beta synthase (CBS) gene and development of congenital heart disease (CHD). Methods One hundred and twenty-seven CHD case-parent triads were recruited from Liaoning Province as patient group, and 129 healthy subjects without family history of birth defect were simultaneously recruited as control group together with their biological parents. For all subjects the polymorphism of CBS gene G919A locus was examined by PCR-ARMS method. Results The frequencies of three genotypes (w/w, w/m, and m/m) in control group were 27.2%, 58.4%, and 14.4%, respectively, with no significant difference in gender. A significant difference in the allele frequency was found between CHD patients and controls, the wild allele frequency was 67.9% in patients and 55.7% in controls.CHD parents' genotype distribution was significantly different from that in controls. Further comparison of each type of CHD showed that genotype frequencies in several CHD subtypes were significantly different from those in their corresponding controls. The results of TDT analysis showed that no allele transmission disequilibrium existed in CHD nuclear families.Conclusions CBS gene G919A mutation is associated with the development of CHD, and the mutated allele may decrease the risk of CHD.

  13. Reduced expression of citrate synthase leads to excessive superoxide formation and cell apoptosis.

    Science.gov (United States)

    Cai, Quanxiang; Zhao, Mengmeng; Liu, Xiang; Wang, Xiaochun; Nie, Yao; Li, Ping; Liu, Tingyan; Ge, Ruli; Han, Fengchan

    2017-02-16

    A/J mice are a mouse model of age-related hearing loss. It has been demonstrated that a mutation in gene of citrate synthase (CS) contributes to the early onset of hearing loss occurring at about one month of age. To understand the effects of a decreased CS activity that results from the mutation in Cs gene on hearing loss in A/J mice, human kidney cell line (293T) was transiently transfected with short hairpin RNA for Cs (shRNA-Cs) to reduce expression of CS. In comparison with those of cells transfected with a scrambled sequence (shRNA-NC), the oxygen consumption rate and adenosine trisphosphate (ATP) production level were decreased in 293T cells transfected with shRNA-Cs. Meanwhile, excessive superoxide production was induced as determined by mitochondrial superoxide formation assay (MitoSOX) and superoxide dismutase 2 (SOD2) detection. Moreover, the expression levels of BIP (binding immunoglobulin protein) and CHOP (CCAAT/enhancer-binding protein-homologous protein), markers of endoplasmic reticulum stress, were upregulated. Furthermore, apoptosis related molecule caspase-3 and the mitochondrial membrane potential were reduced. It is therefore concluded that downregulation of Cs expression in 293T cells leads to low level of ATP production, excessive superoxide formation and cell apoptosis, which implies a possible mechanism for hearing loss in A/J mice.

  14. Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes.

    Science.gov (United States)

    Johannesen, J; Pie, A; Pociot, F; Kristiansen, O P; Karlsen, A E; Nerup, J

    2001-06-01

    Exposure of human pancreatic islets to a mixture of cytokines induces expression of the inducible nitric oxide synthase (iNOS), impairs beta-cell function, and induces apoptosis. We performed a mutational scanning of all 27 exons of the human NOS2 gene and linkage transmission disequilibrium testing of identified NOS2 polymorphisms in a Danish nationwide type 1 diabetes mellitus (IDDM) family collection. Mutational screening was performed using PCR-amplified exons, followed by single stranded conformation polymorphism and verification of potential polymorphisms by sequencing. The transmission disequilibrium test was performed in an IDDM family material comprising 257 Danish families; 154 families were affected sibling pair families, and 103 families were simplex families. In total, 10 polymorphisms were identified in 8 exons, of which 4 were tested in the family material. A C/T single nucleotide polymorphism in exon 16 resulting in an amino acid substitution, Ser(608)Leu, showed linkage to IDDM in human leukocyte antigen DR3/4-positive affected offspring (P = 0.008; corrected P = 0.024). No other distorted transmission patterns were found for any other tested single nucleotide polymorphism or constructed haplotypes with the exception of those including data from exon 16. In conclusion, linkage of the human NOS2 gene to IDDM in a subset of patients supports a pathogenic role of nitric oxide in human IDDM.

  15. Mutations in GABRB3

    DEFF Research Database (Denmark)

    Møller, Rikke S; Wuttke, Thomas V; Helbig, Ingo

    2017-01-01

    OBJECTIVE: To examine the role of mutations in GABRB3 encoding the β3 subunit of the GABAA receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes. METHODS: We performed massive parallel sequencing...... of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs. RESULTS: We identified 22 patients with heterozygous mutations in GABRB3, including 3...... probands from multiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy with myoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies...

  16. AIP mutations and gigantism.

    Science.gov (United States)

    Rostomyan, Liliya; Potorac, Iulia; Beckers, Pablo; Daly, Adrian F; Beckers, Albert

    2017-06-01

    AIP mutations are rare in sporadic acromegaly but they are seen at a higher frequency among certain specific populations of pituitary adenoma patients (pituitary gigantism cases, familial isolated pituitary adenoma (FIPA) kindreds, and patients with macroadenomas who are diagnosed ≤30 years). AIP mutations are most prevalent in patients with pituitary gigantism (29% of this group were found to have mutations in AIP gene). These data support targeted genetic screening for AIP mutations/deletions in these groups of pituitary adenoma patients. Earlier diagnosis of AIP-related acromegaly-gigantism cases enables timely clinical evaluation and treatment, thereby improving outcomes in terms of excessive linear growth and acromegaly comorbidities. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. Mapping Mutations on Phylogenies

    DEFF Research Database (Denmark)

    Nielsen, Rasmus

    2005-01-01

    This chapter provides a short review of recent methodologies developed for mapping mutations on phylogenies. Mapping of mutations, or character changes in general, using the maximum parsimony principle has been one of the most powerful tools in phylogenetics, and it has been used in a variety...... of different applications, for example, in the detection of correlated evolution and to identify selection acting on DNA sequences. However, many uses of parsimony mappings have been criticized because they focus on only one of many possible mappings and/or because they do not incorporate statistical...... uncertainty in the mapping. Recently developed probabilistic methods can incorporate statistical uncertainty in the character mappings. In these methods, focus is on a probability distribution of mutational mappings instead of a single estimate of the mutational mapping....

  18. PRRT2 gene mutations

    Science.gov (United States)

    Gardiner, Alice R.; Bhatia, Kailash P.; Stamelou, Maria; Dale, Russell C.; Kurian, Manju A.; Schneider, Susanne A.; Wali, G.M.; Counihan, Tim; Schapira, Anthony H.; Spacey, Sian D.; Valente, Enza-Maria; Silveira-Moriyama, Laura; Teive, Hélio A.G.; Raskin, Salmo; Sander, Josemir W.; Lees, Andrew; Warner, Tom; Kullmann, Dimitri M.; Wood, Nicholas W.; Hanna, Michael

    2012-01-01

    ABSTRACT Objective: The proline-rich transmembrane protein (PRRT2) gene was recently identified using exome sequencing as the cause of autosomal dominant paroxysmal kinesigenic dyskinesia (PKD) with or without infantile convulsions (IC) (PKD/IC syndrome). Episodic neurologic disorders, such as epilepsy, migraine, and paroxysmal movement disorders, often coexist and are thought to have a shared channel-related etiology. To investigate further the frequency, spectrum, and phenotype of PRRT2 mutations, we analyzed this gene in 3 large series of episodic neurologic disorders with PKD/IC, episodic ataxia (EA), and hemiplegic migraine (HM). Methods: The PRRT2 gene was sequenced in 58 family probands/sporadic individuals with PKD/IC, 182 with EA, 128 with HM, and 475 UK and 96 Asian controls. Results: PRRT2 genetic mutations were identified in 28 out of 58 individuals with PKD/IC (48%), 1/182 individuals with EA, and 1/128 individuals with HM. A number of loss-of-function and coding missense mutations were identified; the most common mutation found was the p.R217Pfs*8 insertion. Males were more frequently affected than females (ratio 52:32). There was a high proportion of PRRT2 mutations found in families and sporadic cases with PKD associated with migraine or HM (10 out of 28). One family had EA with HM and another large family had typical HM alone. Conclusions: This work expands the phenotype of mutations in the PRRT2 gene to include the frequent occurrence of migraine and HM with PKD/IC, and the association of mutations with EA and HM and with familial HM alone. We have also extended the PRRT2 mutation type and frequency in PKD and other episodic neurologic disorders. PMID:23077024

  19. AHAS herbicide resistance endowing mutations: effect on AHAS functionality and plant growth.

    Science.gov (United States)

    Yu, Qin; Han, Heping; Vila-Aiub, Martin M; Powles, Stephen B

    2010-09-01

    Twenty-two amino acid substitutions at seven conserved amino acid residues in the acetohydroxyacid synthase (AHAS) gene have been identified to date that confer target-site resistance to AHAS-inhibiting herbicides in biotypes of field-evolved resistant weed species. However, the effect of resistance mutations on AHAS functionality and plant growth has been investigated for only a very few mutations. This research investigates the effect of various AHAS resistance mutations in Lolium rigidum on AHAS functionality and plant growth. The enzyme kinetics of AHAS from five purified L. rigidum populations, each homozygous for the resistance mutations Pro-197-Ala, Pro-197-Arg, Pro-197-Gln, Pro-197-Ser or Trp-574-Leu, were characterized and the pleiotropic effect of three mutations on plant growth was assessed via relative growth rate analysis. All these resistance mutations endowed a herbicide-resistant AHAS and most resulted in higher extractable AHAS activity, with no-to-minor changes in AHAS kinetics. The Pro-197-Arg mutation slightly (but significantly) increased the K(m) for pyruvate and remarkably increased sensitivity to feedback inhibition by branched chain amino acids. Whereas the Pro-197-Ser and Trp-574-Leu mutations exhibited no significant effects on plant growth, the Pro-197-Arg mutation resulted in lower growth rates. It is clear that, at least in L. rigidum, these five AHAS resistance mutations have no major impact on AHAS functionality and hence probably no plant resistance costs. These results, in part, explain why so many Pro-197 AHAS resistance mutations in AHAS have evolved and why the Pro-197-Ser and the Trp-574-Leu AHAS resistance mutations are frequently found in many weed species.

  20. Subquivers of mutation-acyclic quivers are mutation-acyclic

    CERN Document Server

    Warkentin, Matthias

    2011-01-01

    Quiver mutation plays a crucial role in the definition of cluster algebras by Fomin and Zelevinsky. It induces an equivalence relation on the set of all quivers without loops and two-cycles. A quiver is called mutation-acyclic if it is mutation-equivalent to an acyclic quiver. The aim of this note is to show that full subquivers of mutation-acyclic quivers are mutation-acyclic.

  1. Genetic characterization of the acetohydroxyacid synthase (AHAS) gene responsible for resistance to imidazolinone in chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Thompson, Courtney; Tar'an, Bunyamin

    2014-07-01

    A point mutation in the AHAS1 gene leading to resistance to imidazolinone in chickpea was identified. The resistance is inherited as a single gene. A KASP marker targeting the mutation was developed. Weed control in chickpea (Cicer arietinum L.) is challenging due to poor crop competition ability and limited herbicide options. A chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified, but the genetic inheritance and the mechanism were unknown. In many plant species, resistance to IMI is caused by point mutation(s) in the acetohydroxyacid synthase (AHAS) gene resulting in an amino acid substitution preventing herbicide attachment to the molecule. The main objective of this research was to characterize the resistance to IMI herbicides in chickpea. Two homologous AHAS genes namely AHAS1 and AHAS2 sharing 80 % amino acid sequence similarity were identified in the chickpea genome. Cluster analysis indicated independent grouping of AHAS1 and AHAS2 across legume species. A point mutation in the AHAS1 gene at C675 to T675 resulting in an amino acid substitution from Ala205 to Val205 confers the resistance to IMI in chickpea. A KASP marker targeting the point mutation was developed and effectively predicted the response to IMI herbicides in a recombinant inbred (RI) population of chickpea. The RI population was used in molecular mapping where the major locus for the reaction to IMI herbicide was mapped to chromosome 5. Segregation analysis across an F2 population and RI population demonstrated that the resistance is inherited as a single gene in a semi-dominant fashion. The simple genetic inheritance and the availability of KASP marker generated in this study would speed up development of chickpea varieties with resistance to IMI herbicides.

  2. Two residues determine the product profile of the class II diterpene synthases TPS14 and TPS21 of Tripterygium wilfordii

    DEFF Research Database (Denmark)

    Hansen, Nikolaj Lervad; Nissen, Jakob N.; Hamberger, Björn Robert

    2017-01-01

    The medicinal plant Tripterygium wilfordii (Celastraceae) contains a pair of class II diterpene synthases (diTPS) of specialized labdane-type metabolism that, despite remarkably close homology, form strikingly different products. TwTPS21 catalyzes bicyclization of the linear C20 precursor...... residue gave mixed product profiles. Two mutants, TwTPS14:Y265H and TwTPS21:A325V, also produced ent-copalyl diphosphate, highlighting the evolutionary potential of enzymes of this family to drive rapid diversification of plant diterpene biosynthesis through neo-functionalization. Our study contributes...... to the understanding of structure-function relation in plant class II diTPSs and complements previous mutational studies of Arabidopsis ent-copalyl diphosphate synthase with additional examples from the specialized metabolism of T. wilfordii....

  3. Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe.

    Science.gov (United States)

    Perysinakis, A; Kinghorn, J R; Drainas, C

    1995-06-01

    Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamine-oxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as Km values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.

  4. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  5. Properties of peroxisomal and mitochondrial citrate synthase from Agave americana.

    Science.gov (United States)

    Segovia, J L; Zafra, M F; Alejandre, M J; García-Peregrín, E

    1982-09-01

    Adenine nucleotides were tested as effectors of peroxisomal and mitochondrial citrate synthase from Agave americana leaves in the presence of different concentrations of acetyl-CoA and oxalacetate substrates. ATP inhibited both enzyme activities but with a different inhibition profile. 1.0-7.5 mM ADP did not inhibit the peroxisomal citrate synthase in the presence of high substrate concentrations, while the mitochondrial enzyme was strongly inhibited by 1.0 mM ADP in the same conditions. Likewise, a different pattern was obtained with AMP on both peroxisomal and mitochondrial activities. The rate of citrate formation as function of acetyl-CoA and oxalacetate concentration was also studied in both fractions. Maximal velocity was highest in the peroxisomal fraction, whether acetyl-CoA or oxalacetate were the variable substrates. These differences indicate that peroxisomal and mitochondrial citrate synthases seem to be two different isoenzymes.

  6. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    Science.gov (United States)

    Robinson, M L; Carman, G M

    1982-01-01

    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  7. Mutational spectrum drives the rise of mutator bacteria.

    Directory of Open Access Journals (Sweden)

    Alejandro Couce

    Full Text Available Understanding how mutator strains emerge in bacterial populations is relevant both to evolutionary theory and to reduce the threat they pose in clinical settings. The rise of mutator alleles is understood as a result of their hitchhiking with linked beneficial mutations, although the factors that govern this process remain unclear. A prominent but underappreciated fact is that each mutator allele increases only a specific spectrum of mutational changes. This spectrum has been speculated to alter the distribution of fitness effects of beneficial mutations, potentially affecting hitchhiking. To study this possibility, we analyzed the fitness distribution of beneficial mutations generated from different mutator and wild-type Escherichia coli strains. Using antibiotic resistance as a model system, we show that mutational spectra can alter these distributions substantially, ultimately determining the competitive ability of each strain across environments. Computer simulation showed that the effect of mutational spectrum on hitchhiking dynamics follows a non-linear function, implying that even slight spectrum-dependent fitness differences are sufficient to alter mutator success frequency by several orders of magnitude. These results indicate an unanticipated central role for the mutational spectrum in the evolution of bacterial mutation rates. At a practical level, this study indicates that knowledge of the molecular details of resistance determinants is crucial for minimizing mutator evolution during antibiotic therapy.

  8. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

    Directory of Open Access Journals (Sweden)

    Yan Ping Lim

    2016-06-01

    Full Text Available Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

  9. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

    OpenAIRE

    2010-01-01

    Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphib...

  10. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    OpenAIRE

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control...

  11. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...... PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  12. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  13. Microsatellite instability in colorectal cancer and association with thymidylate synthase and dihydropyrimidine dehydrogenase expression

    Directory of Open Access Journals (Sweden)

    Kruhøffer Mogens

    2009-01-01

    Full Text Available Abstract Background Microsatellite instability (MSI refers to mutations in short motifs of tandemly repeated nucleotides resulting from replication errors and deficient mismatch repair (MMR. Colorectal cancer with MSI has characteristic biology and chemosensitivity, however the molecular basis remains unclarified. The association of MSI and MMR status with outcome and with thymidylate synthase (TS and dihydropyrimidine dehydrogenase (DPD expression in colorectal cancer were evaluated. Methods MSI in five reference loci, MMR enzymes (hMSH2, hMSH6, hMLH1 and hPMS2, thymidylate synthase (TS and dihydropyrimidine dehydrogenase (DPD expression were assessed in paraffin embedded tumor specimens, and associated with outcome in 340 consecutive patients completely resected for colorectal cancer stages II-IV and subsequently receiving adjuvant 5-fluorouracil therapy. Results MSI was found in 43 (13.8% tumors. Absence of repair protein expression was assessed in 52 (17.0% tumors, which had primarily lost hMLH1 in 39 (12.7%, hMSH2 in 5 (1.6%, and hMSH6 in 8 (2.6% tumors. In multivariate analysis MSI (instable compared to MSS (stable tumors were significantly associated with lower risk of recurrence (hazard ratio (HR = 0.3; 95% CI: 0.2–0.7; P = 0.0007 and death (HR = 0.4; 95% CI: 0.2–0.9; P = 0.02 independently of the TS and DPD expressions. A direct relationship between MSI and TS intensity (P = 0.001 was found, while there was no significant association with DPD intensity (P = 0.1. Conclusion The favourable outcome of MSI colorectal carcinomas is ascribed mainly to the tumor biology and to a lesser extent to antitumor response to 5-fluorouracil therapy. There is no evidence that differential TS or DPD expression may account for these outcome characteristics.

  14. N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment

    Directory of Open Access Journals (Sweden)

    Caldovic L

    2011-08-01

    Full Text Available Nicholas Ah Mew, Ljubica CaldovicCenter for Genetic Medicine Research, Children’s Research Institute, Children’s National Medical Center, Washington DC, USAAbstract: The conversion of ammonia into urea by the human liver requires the coordinated function of the 6 enzymes and 2 transporters of the urea cycle. The initial and rate-limiting enzyme of the urea cycle, carbamylphosphate synthetase 1 (CPS1, requires an allosteric activator, N-acetylglutamate (NAG. The formation of this unique cofactor from glutamate and acetyl Coenzyme-A is catalyzed by N-acetylglutamate synthase (NAGS. An absence of NAG as a consequence of NAGS deficiency may compromise flux through CPS1 and result in hyperammonemia. The NAGS gene encodes a 528-amino acid protein, consisting of a C-terminal catalytic domain, a variable segment, and an N-terminal mitochondrial targeting signal. Only 22 mutations in the NAGS gene have been reported to date, mostly in the catalytic domain. NAGS is primarily expressed in the liver and intestine. However, it is also surprisingly expressed in testis, stomach and spleen, and during early embryonic development at levels not concordant with the expression of other urea cycle enzymes, CPS1, or ornithine transcarbamylase. The purpose of NAGS expression in these tissues, and its significance to NAGS deficiency is as yet unknown. Inherited NAGS deficiency is the rarest of the urea cycle disorders, and we review the currently reported 34 cases. Treatment of NAGS deficiency with N-carbamyglutamate, a stable analog of NAG, can restore deficient urea cycle function and normalize blood ammonia in affected patients.Keywords: urea cycle, urea cycle disorder, N-acetyl-L-glutamate, N-acetylglutamate synthase, hyperammonemia, N-carbamyl-L-glutamate

  15. Metalloproteins and phytochelatin synthase may confer protection against zinc oxide nanoparticle induced toxicity in Caenorhabditis elegans.

    Science.gov (United States)

    Polak, Natasa; Read, Daniel S; Jurkschat, Kerstin; Matzke, Marianne; Kelly, Frank J; Spurgeon, David J; Stürzenbaum, Stephen R

    2014-03-01

    Zinc oxide nanoparticles (ZnONPs) are used in large quantities by the cosmetic, food and textile industries. Here we exposed Caenorhabditis elegans wild-type and a metal sensitive triple knockout mutant (mtl-1;mtl-2;pcs-1) to ZnONPs (0-50mg/L) to study strain and exposure specific effects on transcription, reactive oxygen species generation, the biomolecular phenotype (measured by Raman microspectroscopy) and key endpoints of the nematode life cycle (growth, reproduction and lifespan). A significant dissolution effect was observed, where dissolved ZnO constituted over 50% of total Zn within a two day exposure to the test medium, suggesting that the nominal exposure to pure ZnONPs represents in vivo, at best, a mixture exposure of ionic zinc and nanoparticles. Nevertheless, the analyses provided evidence that the metallothioneins (mtl-1 and mtl-2), the phytochelatin synthase (pcs-1) and an apoptotic marker (cep-1) were transcriptionally activated. In addition, the DCFH-DA assay provided in vitro evidence of the oxidative potential of ZnONPs in the metal exposure sensitive triple mutant. Raman spectroscopy highlighted that the biomolecular phenotype changes significantly in the mtl-1;mtl-2;pcs-1 triple knockout worm upon ZnONP exposure, suggesting that these metalloproteins are instrumental in the protection against cytotoxic damage. Finally, ZnONP exposure was shown to decrease growth and development, reproductive capacity and lifespan, effects which were amplified in the triple knockout. By combining diverse toxicological strategies, we identified that individuals (genotypes) housing mutations in key metalloproteins and phytochelatin synthase are more susceptible to ZnONP exposure, which underlines their importance to minimize ZnONP induced toxicity.

  16. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

    Science.gov (United States)

    Belkheir, Asma K; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

  17. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    Science.gov (United States)

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  18. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    Science.gov (United States)

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-26

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

  19. The Remarkable Character of Porphobilinogen Synthase.

    Science.gov (United States)

    Jaffe, Eileen K

    2016-11-15

    Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn(2+); a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb(2+). PBGSs that do not require the soft metal ion Zn(2+) at the active site instead are suspected of using the hard metal Mg(2+). The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg(2+) as an allosteric activator. Mg(2+) functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do

  20. Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae.

    Science.gov (United States)

    Lukacin, Richard; Schreiner, Stephan; Silber, Katrin; Matern, Ulrich

    2005-02-01

    Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75-50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.

  1. Silting mutation in triangulated categories

    CERN Document Server

    Aihara, Takuma

    2010-01-01

    In representation theory of algebras the notion of `mutation' often plays important roles, and two cases are well known, i.e. `cluster tilting mutation' and `exceptional mutation'. In this paper we focus on `tilting mutation', which has a disadvantage that it is often impossible, i.e. some of summands of a tilting object can not be replaced to get a new tilting object. The aim of this paper is to take away this disadvantage by introducing `silting mutation' for silting objects as a generalization of `tilting mutation'. We shall develope a basic theory of silting mutation. In particular, we introduce a partial order on the set of silting objects and establish the relationship with `silting mutation' by generalizing the theory of Riedmann-Schofield and Happel-Unger. We show that iterated silting mutation act transitively on the set of silting objects for local, hereditary or canonical algebras. Finally we give a bijection between silting subcategories and certain t-structures.

  2. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  3. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  4. Are There Mutator Polymerases?

    Directory of Open Access Journals (Sweden)

    Miguel Garcia-Diaz

    2003-01-01

    Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

  5. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  6. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B;

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  7. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  8. Biosynthesis of polyketides by trans-AT polyketide synthases.

    Science.gov (United States)

    Piel, Jörn

    2010-07-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that have recently been recognized as one of the major group of proteins involved in the production of bioactive polyketides. 436 references are cited.

  9. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    Directory of Open Access Journals (Sweden)

    David O. Hooks

    2014-06-01

    Full Text Available Polyhydroxyalkanoate (PHA is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC. Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

  10. Functional Characterization of Sesquiterpene Synthase from Polygonum minus

    Directory of Open Access Journals (Sweden)

    Su-Fang Ee

    2014-01-01

    Full Text Available Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS has a complete open reading frame (ORF of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

  11. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    Science.gov (United States)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data. PMID:27721652

  12. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highl

  13. Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

    Science.gov (United States)

    Le Gal, Solène; Robert-Gangneux, Florence; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Damiani, Céline; Totet, Anne; Gangneux, Jean-Pierre; Nevez, Gilles

    2013-05-01

    Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

  14. Cloning and expression pattern of chitin synthase (CHS) gene in ...

    African Journals Online (AJOL)

    USER

    2010-08-16

    Aug 16, 2010 ... African Journal of Biotechnology Vol. 9(33), pp. 5297-5308, 16 ... Chitin synthase (CHS) plays an important role in biosynthesis of chitin .... strand cDNA Synthesis kit, 5'/3' RACE kit and pMD18-T vector were purchased from ...

  15. Analysis of an Arabidopsis heat-sensitive mutant reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll turnover.

    Science.gov (United States)

    Lin, Yao-Pin; Lee, Tsung-yuan; Tanaka, Ayumi; Charng, Yee-yung

    2014-10-01

    Chlorophylls, the most abundant pigments in the photosynthetic apparatus, are constantly turned over as a result of the degradation and replacement of the damage-prone reaction center D1 protein of photosystem II. Results from isotope labeling experiments suggest that chlorophylls are recycled by reutilization of chlorophyllide and phytol, but the underlying mechanism is unclear. In this study, by characterization of a heat-sensitive Arabidopsis mutant we provide evidence of a salvage pathway for chlorophyllide a. A missense mutation in CHLOROPHYLL SYNTHASE (CHLG) was identified and confirmed to be responsible for a light-dependent, heat-induced cotyledon bleaching phenotype. Following heat treatment, mutant (chlg-1) but not wild-type seedlings accumulated a substantial level of chlorophyllide a, which resulted in a surge of phototoxic singlet oxygen. Immunoblot analysis suggested that the mutation destabilized the chlorophyll synthase proteins and caused a conditional blockage of esterification of chlorophyllide a after heat stress. Accumulation of chlorophyllide a after heat treatment occurred during recovery in the dark in the light-grown but not the etiolated seedlings, suggesting that the accumulated chlorophyllides were not derived from de novo biosynthesis but from de-esterification of the existing chlorophylls. Further analysis of the triple mutant harboring the CHLG mutant allele and null mutations of CHLOROPHYLLASE1 (CLH1) and CLH2 indicated that the known chlorophyllases are not responsible for the accumulation of chlorophyllide a in chlg-1. Taken together, our results show that chlorophyll synthase acts in a salvage pathway for chlorophyll biosynthesis by re-esterifying the chlorophyllide a produced during chlorophyll turnover.

  16. Msx1 Mutations

    Science.gov (United States)

    Wang, Y.; Kong, H.; Mues, G.; D’Souza, R.

    2011-01-01

    Mutations in the transcription factors PAX9 and MSX1 cause selective tooth agenesis in humans. In tooth bud mesenchyme of mice, both proteins are required for the expression of Bmp4, which is the key signaling factor for progression to the next step of tooth development. We have previously shown that Pax9 can transactivate a 2.4-kb Bmp4 promoter construct, and that most tooth-agenesis-causing PAX9 mutations impair DNA binding and Bmp4 promoter activation. We also found that Msx1 by itself represses transcription from this proximal Bmp4 promoter, and that, in combination with Pax9, it acts as a potentiator of Pax9-induced Bmp4 transactivation. This synergism of Msx1 with Pax9 is significant, because it is currently the only documented mechanism for Msx1-mediated activation of Bmp4. In this study, we investigated whether the 5 known tooth-agenesis-causing MSX1 missense mutations disrupt this Pax9-potentiation effect, or if they lead to deficiencies in protein stability, protein-protein interactions, nuclear translocation, and DNA-binding. We found that none of the studied molecular mechanisms yielded a satisfactory explanation for the pathogenic effects of the Msx1 mutations, calling for an entirely different approach to the investigation of this step of odontogenesis on the molecular level. PMID:21297014

  17. Mutations in galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Reichardt, J.K.V. [Univ. of Southern California School of Medicine, Los Angeles, CA (United States)

    1995-10-01

    This Letter raises four issues concerning two papers on galactosemia published in the March 1995 of the Journal. First, table 2 in the paper by Elsas et al. incorrectly attributes seven galactose-l-phosphate uridyl transferase (GALT) mutations (S135L, L195P, K285N, N314D, R333W, R333G, and K334R). The table also fails to mention that others have reported the same two findings attributed to {open_quotes}Leslie et al.; Elsas et al. and in press{close_quotes} and {open_quotes}Leslie et al.; Elsas et al.{close_quotes} The first finding on the prevalence of the Q188R galactosemia mutation in the G/G Caucasian population has also been described by Ng et al., and the second finding on the correlation of the N314D GALT mutation with the Duarte variant was reported by Lin et al. Second, Elsas et al. suggest that the E203K and N314D mutations may {open_quotes}produce intra-allelic complementation when in cis{close_quotes}. This speculation is supported by the activity data of individual III-2 but is inconsistent with the activities of three other individuals I-1, II-1, and III-1 of the same pedigree. The GALT activity measured in these three individuals suggests a dominant negative effect of E203K in E203K-N314D chromosomes, since they all have less than normal activity. Thus, the preponderance of the data in this paper is at odds with the authors speculation. It is worth recalling that Lin et al. also identified four N314D GALT mutations on 95 galactosemic chromosomes examined. A similar situation also appears to be the case in proband III-1 (with genotype E203K-N314D/IVSC) in the Elsas et al. paper. 9 refs.

  18. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  19. Phytochelatin synthase: of a protease a peptide polymerase made.

    Science.gov (United States)

    Rea, Philip A

    2012-05-01

    Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.

  20. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice.

    Science.gov (United States)

    Tsutsui, Masato; Tanimoto, Akihide; Tamura, Masahito; Mukae, Hiroshi; Yanagihara, Nobuyuki; Shimokawa, Hiroaki; Otsuji, Yutaka

    2015-01-01

    Nitric oxide (NO) is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs), all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling), lung abnormalities (accelerated pulmonary fibrosis), and bone abnormalities (increased bone mineral density and bone turnover). These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  1. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice

    Directory of Open Access Journals (Sweden)

    Masato Tsutsui

    2015-01-01

    Full Text Available Nitric oxide (NO is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs, all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling, lung abnormalities (accelerated pulmonary fibrosis, and bone abnormalities (increased bone mineral density and bone turnover. These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  2. Evolutionary Stability Against Multiple Mutations

    CERN Document Server

    Ghatak, Anirban; Shaiju, A J

    2012-01-01

    It is known (see e.g. Weibull (1995)) that ESS is not robust against multiple mutations. In this article, we introduce robustness against multiple mutations and study some equivalent formulations and consequences.

  3. BRAF mutations in conjunctival melanoma

    DEFF Research Database (Denmark)

    Larsen, Ann-Cathrine; Dahl, Christina; Dahmcke, Christina M.

    2016-01-01

    Purpose: To investigate incidence, clinicopathological features and prognosis of BRAF-mutated conjunctival melanoma in Denmark. Furthermore, to determine BRAF mutations in paired premalignant lesions and evaluate immunohistochemical BRAF V600E oncoprotein detection. Methods: Data from 139 patients...

  4. N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase.

    Science.gov (United States)

    Reinbothe, S; Nelles, A; Parthier, B

    1991-06-01

    Photoautotrophic cells of Euglena gracilis can be adapted to N-(phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40-fold accumulation of the target enzyme. 5-enolpyruvylshikimate-3-phosphate synthase, in cells adapted to 6 mM N-(phosphonomethyl)glycine. The other is connected with a herbicide-insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N-(phosphonomethyl)glycine excreted shikimate and shikimate 3-phosphate into the medium: the amounts depended on the actual concentration of the herbicide. Two-dimensional gel electrophoresis and determination of 5-enolpyruvylshikimate-3-phosphate synthase activity in crude extracts, as well as after separation by non-denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59-kDa protein. Overproduction of this 59-kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5-kDa polypeptide which appeared in adapted cells, as shown by cell-free translation in the wheat germ system. In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N-(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5-enolpyruvylshikimate-3-phosphate synthase, which could not be inhibited by even 2 mM N-(phosphonomethyl)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3-phosphate into the growth medium containing N-(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N-(phosphonomethyl)glycine.

  5. Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

    Directory of Open Access Journals (Sweden)

    Catalina Sanz

    Full Text Available Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

  6. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  7. Chaperone therapy for homocystinuria: the rescue of CBS mutations by heme arginate.

    Science.gov (United States)

    Melenovská, Petra; Kopecká, Jana; Krijt, Jakub; Hnízda, Aleš; Raková, Kateřina; Janošík, Miroslav; Wilcken, Bridget; Kožich, Viktor

    2015-03-01

    Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.

  8. PIK3CA Mutation in Colorectal Cancer: Relationship with Genetic and Epigenetic Alterations

    Directory of Open Access Journals (Sweden)

    Katsuhiko Nosho

    2008-06-01

    Full Text Available Somatic PIK3CA mutations are often present in colorectal cancer. Mutant PIK3CA activates AKT signaling, which up-regulates fatty acid synthase (FASN. Microsatellite instability (MSI and CpG island methylator phenotype (CIMP are important molecular classifiers in colorectal cancer. However, the relationship between PIK3CA mutation, MSI and CIMP remains uncertain. Using Pyrosequencing technology, we detected PIK3CA mutations in 91 (15% of 590 population-based colorectal cancers. To determine CIMP status, we quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight. PIK3CA mutation was significantly associated with mucinous tumors [P = .0002; odds ratio (OR = 2.44], KRAS mutation (P < .0001; OR = 2.68, CIMP-high (P = .03; OR = 2.08, phospho–ribosomal protein S6 expression (P = .002; OR = 2.19, and FASN expression (P = .02; OR = 1.85 and inversely with p53 expression (P = .01; OR = 0.54 and β-catenin (CTNNB1 alteration (P = .004; OR = 0.43. In addition, PIK3CA G-to-A mutations were associated with MGMT loss (P = .001; OR = 3.24 but not with MGMT promoter methylation. In conclusion, PIK3CA mutation is significantly associated with other key molecular events in colorectal cancer, and MGMT loss likely contributes to the development of PIK3CA G>A mutation. In addition, Pyrosequencing is useful in detecting PIK3CA mutation in archival paraffin tumor tissue. PIK3CA mutational data further emphasize heterogeneity of colorectal cancer at the molecular level.

  9. [Identification and characterization of intraspecific variability of the sucrose synthase gene Sus4 of potato (Solanum tuberosum)].

    Science.gov (United States)

    Boris, K V; Ryzhova, N N; Kochieva, E Z

    2011-02-01

    Nucleotide and amino acid variability of fragments of the Sus4 gene encoding the sucrose synthase enzyme was studied in 24 potato cultivars selected in Russia and other countries and differing in starch content in tubers. Both SNPs and indels were detected in a chosen Sus4 gene fragment including the sequence from exon 3 to exon 6 and corresponding to the main part of the sucrose synthase domain. Four types of Sus4 sequences were revealed depending on the presence of an insertion in introns 4 and 5 and of the mononucleotide octamer (T)8 in intron 5. Differentiation of these sequences was confirmed by statistical methods. Sixteen amino acid substitutions were identified in the translated sequence, of which eleven were nonsynonymous. Specific varietal nucleotide and amino acid substitutions were also revealed, which can be used in future for marking potato cultivars/genotypes. No direct associations between the mutational changes and the starch content were found in the potato cultivars studied by us.

  10. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

    Science.gov (United States)

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2016-02-01

    ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.

  11. Structure of the Mycobacterium tuberculosis Flavin Dependent Thymidylate Synthase (MtbThyX) at 2.0 Å Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Sampathkumar, Parthasarathy; Turley, Stewart; Ulmer, Jonathan E.; Rhie, Ho Gun; Hopkins Sibley, Carol; Hol, Wim G.J. (Kyunghee); (UWASH)

    2010-07-20

    A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M. tuberculosis ThyX (MtbThyX) enzyme complements the E. coli {chi}2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.

  12. Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy

    Science.gov (United States)

    Sander, Mikael; Chavoshan, Bahman; Harris, Shannon A.; Iannaccone, Susan T.; Stull, James T.; Thomas, Gail D.; Victor, Ronald G.

    2000-01-01

    Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin–glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to α-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD. PMID:11087833

  13. Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis.

    Science.gov (United States)

    Seung, David; Lu, Kuan-Jen; Stettler, Michaela; Streb, Sebastian; Zeeman, Samuel C

    2016-09-23

    Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.

  14. Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis.

    Science.gov (United States)

    Zhong, Ruiqin; Morrison, W Herbert; Freshour, Glenn D; Hahn, Michael G; Ye, Zheng-Hua

    2003-06-01

    Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.

  15. Acute Intermittent Porphyria in Argentina: An Update

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    Gabriela Nora Cerbino

    2015-01-01

    Full Text Available Porphyrias are a group of metabolic diseases that arise from deficiencies in the heme biosynthetic pathway. A partial deficiency in hydroxymethylbilane synthase (HMBS produces a hepatic disorder named Acute Intermittent Porphyria (AIP; the acute porphyria is more frequent in Argentina. In this paper we review the results obtained for 101 Argentinean AIP families and 6 AIP families from foreign neighbour countries studied at molecular level at Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP. Thirty-five different mutations were found, of which 14 were described for the first time in our population. The most prevalent type of mutations was the missense mutations (43% followed by splice defects (26% and small deletions (20%. An odd case of a double heterozygous presentation of AIP in a foreign family from Paraguay is discussed. Moreover, it can be noted that 38 new families were found carrying the most frequent mutation in Argentina (p.G111R, increasing to 55.66% the prevalence of this genetic change in our population and adding further support to our previous hypothesis of a founder effect for this mutation in Argentina. Identification of patients with an overt AIP is important because treatment depends on an accurate diagnosis, but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death.

  16. A product of the bicistronic Drosophila melanogaster gene CG31241, which also encodes a trimethylguanosine synthase, plays a role in telomere protection.

    Science.gov (United States)

    Komonyi, Orban; Schauer, Tamas; Papai, Gabor; Deak, Peter; Boros, Imre M

    2009-03-15

    Although telomere formation occurs through a different mechanism in Drosophila compared with other organisms, telomere associations result from mutations in homologous genes, indicating the involvement of similar pathways in chromosome end protection. We report here that mutations of the Drosophila melanogaster gene CG31241 lead to high frequency chromosome end fusions. CG31241 is a bicistronic gene that encodes trimethylguanosine synthase (TGS1), which forms the m3G caps of noncoding small RNAs, and a novel protein, DTL. We show that although TGS1 has no role in telomere protection, DTL is localized at specific sites, including the ends of polytene chromosomes, and its loss results in telomere associations. Mutations of ATM- and Rad3-related (ATR) kinase suppress telomere fusions in the absence of DTL. Thus, genetic interactions place DTL in an ATR-related pathway in telomere protection. In contrast to ATR kinase, mutations of ATM (ataxia telangiectasia mutated) kinase, which acts in a partially overlapping pathway of telomere protection, do not suppress formation of telomere associations in the absence of DTL. Thus, uncovering the role of DTL will help to dissect the evolutionary conserved pathway(s) controlling ATM-ATR-related telomere protection.

  17. Kin Selection - Mutation Balance

    DEFF Research Database (Denmark)

    Dyken, J. David Van; Linksvayer, Timothy Arnold; Wade, Michael J.

    2011-01-01

    Abstract Social conflict, in the form of intraspecific selfish "cheating" has been observed in a number of natural systems. However, a formal, evolutionary genetic theory of social cheating that provides an explanatory, predictive framework for these observations is lacking. Here we derive the kin...... selection-mutation balance, which provides an evolutionary null hypothesis for the statics and dynamics of cheating. When social interactions have linear fitness effects and Hamilton´s rule is satisfied, selection is never strong enough to eliminate recurrent cheater mutants from a population, but cheater...... lineages are transient and do not invade. Instead, cheating lineages are eliminated by kin selection but are constantly reintroduced by mutation, maintaining a stable equilibrium frequency of cheaters. The presence of cheaters at equilibrium creates a "cheater load" that selects for mechanisms of cheater...

  18. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    Science.gov (United States)

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  19. Mutation of Auslander generators

    CERN Document Server

    Lada, Magdalini

    2009-01-01

    Let $\\Lambda$ be an artin algebra with representation dimension equal to three and $M$ an Auslander generator of $\\Lambda$. We show how, under certain assumptions, we can mutate $M$ to get a new Auslander generator whose endomorphism ring is derived equivalent to the endomorphism ring of $M$. We apply our results to selfinjective algebras with radical cube zero of infinite representation type, where we construct an infinite set of Auslander generators.

  20. The contribution of coevolving residues to the stability of KDO8P synthase.

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    Sharon H Ackerman

    Full Text Available BACKGROUND: The evolutionary tree of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P synthase (KDO8PS, a bacterial enzyme that catalyzes a key step in the biosynthesis of bacterial endotoxin, is evenly divided between metal and non-metal forms, both having similar structures, but diverging in various degrees in amino acid sequence. Mutagenesis, crystallographic and computational studies have established that only a few residues determine whether or not KDO8PS requires a metal for function. The remaining divergence in the amino acid sequence of KDO8PSs is apparently unrelated to the underlying catalytic mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The multiple alignment of all known KDO8PS sequences reveals that several residue pairs coevolved, an indication of their possible linkage to a structural constraint. In this study we investigated by computational means the contribution of coevolving residues to the stability of KDO8PS. We found that about 1/4 of all strongly coevolving pairs probably originated from cycles of mutation (decreasing stability and suppression (restoring it, while the remaining pairs are best explained by a succession of neutral or nearly neutral covarions. CONCLUSIONS/SIGNIFICANCE: Both sequence conservation and coevolution are involved in the preservation of the core structure of KDO8PS, but the contribution of coevolving residues is, in proportion, smaller. This is because small stability gains or losses associated with selection of certain residues in some regions of the stability landscape of KDO8PS are easily offset by a large number of possible changes in other regions. While this effect increases the tolerance of KDO8PS to deleterious mutations, it also decreases the probability that specific pairs of residues could have a strong contribution to the thermodynamic stability of the protein.

  1. Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

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    Ju-Chen Chia

    Full Text Available Phytochelatin synthase (PCS uses the substrates glutathione (GSH, γGlu-Cys-Gly and a cadmium (Cd-bound GSH (Cd∙GS2 to produce the shortest phytochelatin product (PC2, (γGlu-Cys2-Gly through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218 by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

  2. The assembly of the plasmodial PLP synthase complex follows a defined course.

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    Ingrid B Müller

    Full Text Available BACKGROUND: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP. For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers. METHODOLOGY/PRINCIPAL FINDINGS: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK, amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

  3. Polymorphism of Methionine Synthase Gene in Nuclear Families of Congenital Heart Disease

    Institute of Scientific and Technical Information of China (English)

    WEN-LI ZHU; JUN CHENG; JING-JING DAO; RU-BING ZHAO; LI-YING YAN; SHU-QING LI; AND YONG LI

    2004-01-01

    Objective To investgate the relation of methionine synthase (MS) gene variation with congenital heart disease (CHD) phenotype. Methods One hundred and ninety three CHD patients (94 males and 99 females) and their biological parents (nuclear families) in Liaoning Province were selected as the case group, and another 104 normal persons (60 males and 44 females) and their parents without family history of birth defects as the control group. For all subjects the polymorphism of MS gene A2756G locus was examined by PCR-RFLP method. Results In offspring of the control group the frequencies of MS genotype (+/-) and allele (+) were 10.7% and 5.3%, without existence of homozygote. The MS genotype distribution and allele frequencies of CHD patients and their mothers were not significantly different from the control (P > 0.05). The frequency of allele (+)in case fathers (5.0 %) was apparently lower than that in the control (9.1%, P=0.060), and the odds ratio (OR) was 0.53 (95% CI: 0.25-1.09). There was no difference in parents' genotype combination between the two groups, and in genotype distribution among different types of CHD. Analysis of genetic transmission indicated that mutation allele (+) existed transmission disequilibrium in CHD nuclear families. The percentage of allele (+) transmitted from parents was lower than that allele (-)with OR 0.26 (95% CI: 0.11-0.60). Conclusion MS gene variation in parents is associated with occurrence of CHD in offspring, and mutation allele (+) in parents may be related with the decrease of CHD risk in offspring.

  4. Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.

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    Pei-Rung Wu

    Full Text Available BACKGROUND: Human endothelial nitric oxide synthase (eNOS requires calcium-bound calmodulin (CaM for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50 of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q or at C-terminal (B34Q lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding.

  5. Dissecting the roles of a strictly conserved tyrosine in substrate recognition and catalysis by pseudouridine 55 synthase.

    Science.gov (United States)

    Phannachet, Kulwadee; Elias, Youssef; Huang, Raven H

    2005-11-29

    Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases (PsiS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile for the PsiS-catalyzed formation of pseudouridine (Psi). However, superposition of the representative structures from these four families of enzymes identifies two additional amino acids, a lysine or an arginine (K/R) and a tyrosine (Y), from a K/RxY motif that are structurally conserved in the active site. We have created a series of Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Psi55S) mutants in which the conserved Y is mutated to other amino acids. A new crystal structure of the T. maritima Psi55S Y67F mutant in complex with a 5FU-RNA at 2.4 A resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhPsi), the same product previously seen in wild-type Psi55S-5FU-RNA complex structures. HPLC analysis confirmed efficient formation of 5FhPsi by both Psi55S Y67F and Y67L mutants but to a much lesser extent by the Y67A mutant when 5FU-RNA substrate was used. However, both HPLC analysis and a tritium release assay indicated that these mutants had no detectable enzymatic activity when the natural RNA substrate was used. The combined structural and mutational studies lead us to propose that the side chain of the conserved tyrosine in these four families of PsiS plays a dual role within the active site, maintaining the structural integrity of the active site through its hydrophobic phenyl ring and acting as a general base through its OH group for the proton abstraction required in the last step of PsiS-catalyzed formation of Psi.

  6. Sex and deleterious mutations.

    Science.gov (United States)

    Gordo, Isabel; Campos, Paulo R A

    2008-05-01

    The evolutionary advantage of sexual reproduction has been considered as one of the most pressing questions in evolutionary biology. While a pluralistic view of the evolution of sex and recombination has been suggested by some, here we take a simpler view and try to quantify the conditions under which sex can evolve given a set of minimal assumptions. Since real populations are finite and also subject to recurrent deleterious mutations, this minimal model should apply generally to all populations. We show that the maximum advantage of recombination occurs for an intermediate value of the deleterious effect of mutations. Furthermore we show that the conditions under which the biggest advantage of sex is achieved are those that produce the fastest fitness decline in the corresponding asexual population and are therefore the conditions for which Muller's ratchet has the strongest effect. We also show that the selective advantage of a modifier of the recombination rate depends on its strength. The quantification of the range of selective effects that favors recombination then leads us to suggest that, if in stressful environments the effect of deleterious mutations is enhanced, a connection between sex and stress could be expected, as it is found in several species.

  7. Septin mutations in human cancers

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    Elias T Spiliotis

    2016-11-01

    Full Text Available Septins are GTP-binding proteins that are evolutionarily and structurally related to the RAS oncogenes. Septin expression levels are altered in many cancers and new advances point to how abnormal septin expression may contribute to the progression of cancer. In contrast to the RAS GTPases, which are frequently mutated and actively promote tumorigenesis, little is known about the occurrence and role of septin mutations in human cancers. Here, we review septin missense mutations that are currently in the Catalog of Somatic Mutations in Cancer (COSMIC database. The majority of septin mutations occur in tumors of the large intestine, skin, endometrium and stomach. Over 25% of the annotated mutations in SEPT2, SEPT4 and SEPT9 belong to large intestine tumors. From all septins, SEPT9 and SEPT14 exhibit the highest mutation frequencies in skin, stomach and large intestine cancers. While septin mutations occur with frequencies lower than 3%, recurring mutations in several invariant and highly conserved amino acids are found across different septin paralogs and tumor types. Interestingly, a significant number of these mutations occur in the GTP-binding pocket and septin dimerization interfaces. Future studies may determine how these somatic mutations affect septin structure and function, whether they contribute to the progression of specific cancers and if they could serve as tumor-specific biomarkers.

  8. Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic.

    Science.gov (United States)

    Balachandran, Naresh; To, Frederick; Berti, Paul J

    2017-01-31

    3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with Ki = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes 1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KM,MnKM,PEPKM,E4P/kcat) versus log(Ki), revealed a slope, m, of 0.34, with r(2) = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.

  9. Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.

    Science.gov (United States)

    Petković, Hrvoje; Sandmann, Axel; Challis, Iain R; Hecht, Hans-Jürgen; Silakowski, Barbara; Low, Lindsey; Beeston, Nicola; Kuscer, Enej; Garcia-Bernardo, Jose; Leadlay, Peter F; Kendrew, Steven G; Wilkinson, Barrie; Müller, Rolf

    2008-02-07

    The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.

  10. Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Bulfer, Stacie L.; Scott, Erin M.; Couture, Jean-François; Pillus, Lorraine; Trievel, Raymond C.; (Michigan); (UCSD)

    2010-01-12

    Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed {alpha}/{beta} topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

  11. Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis.

    Science.gov (United States)

    Nakazawa, Masami; Nishimura, Masaaki; Inoue, Kengo; Ueda, Mitsuhiro; Inui, Hiroshi; Nakano, Yoshihisa; Miyatake, Kazutaka

    2011-01-01

    The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.

  12. Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

    Science.gov (United States)

    Luo, Chuping; Liu, Xuehui; Zhou, Huafei; Wang, Xiaoyu; Chen, Zhiyi

    2015-01-01

    Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.

  13. Pyrroline-5-carboxylate synthase and proline biosynthesis: From osmotolerance to rare metabolic disease

    Science.gov (United States)

    Pérez-Arellano, Isabel; Carmona-Álvarez, Francisco; Martínez, Ana I; Rodríguez-Díaz, Jesús; Cervera, Javier

    2010-01-01

    Pyrroline-5-carboxylate synthase (P5CS) is a bifunctional enzyme that exhibits glutamate kinase (GK) and γ-glutamyl phosphate reductase (GPR) activities. The enzyme is highly relevant in humans because it belongs to a combined route for the interconversion of glutamate, ornithine and proline. The deficiency of P5CS activity in humans is associated with a rare, inherited metabolic disease. It is well established that some bacteria and plants accumulate proline in response to osmotic stress. The alignment of P5CSs from different species and analysis of the solved structures of GK and GPR reveal high sequence and structural conservation. The information acquired from different mutant enzymes with increased osmotolerant properties, together with the position of the insertion found in the longer human isoform, permit the delimitation of the regulatory site of GK and P5CS and the proposal of a model of P5CS architecture. Additionally, the GK moiety of the human enzyme has been modeled and the known clinical mutations and polymorphisms have been mapped. PMID:20091669

  14. Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase.

    Directory of Open Access Journals (Sweden)

    Anne eJunker

    2013-07-01

    Full Text Available Putrescine N-methyltransferases (PMTs are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-L-methionine (SAM as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs, which are ubiquitous enzymes of polyamine metabolism. SPDS use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in Datura stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom.

  15. N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment.

    Science.gov (United States)

    Ah Mew, Nicholas; Caldovic, Ljubica

    2011-01-01

    The conversion of ammonia into urea by the human liver requires the coordinated function of the 6 enzymes and 2 transporters of the urea cycle. The initial and rate-limiting enzyme of the urea cycle, carbamylphosphate synthetase 1 (CPS1), requires an allosteric activator, N-acetylglutamate (NAG). The formation of this unique cofactor from glutamate and acetyl Coenzyme-A is catalyzed by N-acetylglutamate synthase (NAGS). An absence of NAG as a consequence of NAGS deficiency may compromise flux through CPS1 and result in hyperammonemia. The NAGS gene encodes a 528-amino acid protein, consisting of a C-terminal catalytic domain, a variable segment, and an N-terminal mitochondrial targeting signal. Only 22 mutations in the NAGS gene have been reported to date, mostly in the catalytic domain. NAGS is primarily expressed in the liver and intestine. However, it is also surprisingly expressed in testis, stomach and spleen, and during early embryonic development at levels not concordant with the expression of other urea cycle enzymes, CPS1, or ornithine transcarbamylase. The purpose of NAGS expression in these tissues, and its significance to NAGS deficiency is as yet unknown. Inherited NAGS deficiency is the rarest of the urea cycle disorders, and we review the currently reported 34 cases. Treatment of NAGS deficiency with N-carbamyglutamate, a stable analog of NAG, can restore deficient urea cycle function and normalize blood ammonia in affected patients.

  16. The effect of dietary modulation of sulfur amino acids on cystathionine β synthase-deficient mice.

    Science.gov (United States)

    Kruger, Warren D; Gupta, Sapna

    2016-01-01

    Cystathionine β synthase (CBS) is a key enzyme in the methionine and cysteine metabolic pathway, acting as a metabolic gatekeeper to regulate the flow of fixed sulfur from methionine to cysteine. Mutations in the CBS gene cause clinical CBS deficiency, a disease characterized by elevated plasma total homocysteine (tHcy) and methionine and decreased plasma cysteine. The treatment goal for CBS-deficient patients is to normalize the metabolic values of these three metabolites using a combination of vitamin therapy and dietary manipulation. To better understand the effectiveness of nutritional treatment strategies, we have performed a series of long-term dietary manipulation studies using our previously developed Tg-I278T Cbs(-/-) mouse model of CBS deficiency and sibling Tg-I278T Cbs(+/-) controls. Tg-I278T Cbs(-/-) mice have undetectable levels of CBS activity, extremely elevated plasma tHcy, modestly elevated plasma methionine, and low plasma cysteine. They exhibit several easily assayable phenotypes, including osteoporosis, loss of fat mass, reduced life span, and facial alopecia. The diets used in these studies differed in the amounts of sulfur amino acids or sulfur amino acid precursors. In this review, we will discuss our findings and their relevance to CBS deficiency and the concept of gene-diet interaction. © 2015 New York Academy of Sciences.

  17. Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii.

    Science.gov (United States)

    Donald, R G; Roos, D S

    1994-02-01

    To investigate the feasibility of genomic transgene expression and gene targeting in Toxoplasma gondii, parasites have been transfected with constructs differing in the length of contiguous genomic sequence spanning the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously reported that vectors derived from a DHFR-TS cDNA 'minigene' containing mutations in the DHFR coding sequence confer pyrimethamine resistance to transfected parasites (Donald and Roos, 1993). Stably resistant parasite clones arise at high frequency, generally by virtue of transgene integration into parasite chromosomes at locations scattered throughout the genome. In contrast, using a vector which contains 8 kb of contiguous genomic sequence (vs. homologous recombination. Homologous recombination appears to occur at even higher frequency when a 16 kb genomic clone is used. Circular plasmids were more efficient than linearized molecules at producing homologous recombination in this system, integrating by reciprocal crossing-over to produce a duplication of the DHFR-TS locus. Double crossing-over (or gene conversion) was also observed at low frequency, resulting in complete allelic replacement in this haploid stage of the parasite. The ability to produce either homologous or non-homologous recombinants, by the selection of appropriate transformation constructs, has considerable genetic potential.

  18. The PPLA motif of glycogen synthase kinase 3beta is required for interaction with Fe65.

    Science.gov (United States)

    Lee, Eun Jeoung; Hyun, Sunghee; Chun, Jaesun; Shin, Sung Hwa; Lee, Kyung Eun; Yeon, Kwang Hum; Park, Tae Yoon; Kang, Sang Sun

    2008-07-31

    Glycogen synthase kinase 3beta (GSK 3 beta) is a serine/ threonine kinase that phosphorylates substrates such as beta-catenin and is involved in a variety of biological processes, including embryonic development, metabolism, tumorigenesis, and cell death. Here, we present evidence that human GSK 3beta is associated with Fe65, which has the characteristics of an adaptor protein, possessing a WW domain, and two phosphotyrosine interaction domains, PID1 and PID2. The GSK 3beta catalytic domain also contains a putative WW domain binding motif ((371)PPLA(374)), and we observed, using a pull down approach and co-immuno-precipitation, that it interacts physically with Fe65 via this motif. In addition, we detected co-localization of GSK 3beta and Fe65 by confocal microscopy, and this co-localization was disrupted by mutation of the putative WW domain binding motif of GSK 3beta.Finally, in transient transfection assays interaction of GSK 3 beta (wt) with Fe65 induced substantial cell apoptosis, whereas interaction with the GSK 3beta AALA mutant ((371)AALA(374)) did not, and we noted that phosphorylation of the Tyr 216 residue of the GSK 3beta AALA mutant was significantly reduced compared to that of GSK 3beta wild type. Thus, our observations indicate that GSK 3beta binds to Fe65 through its (371)PPLA(374) motif and that this interaction regulates apoptosis and phosphorylation of Tyr 216 of GSK 3beta.

  19. A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase.

    Science.gov (United States)

    Brock, Joseph S; Hamberg, Mats; Balagunaseelan, Navisraj; Goodman, Michael; Morgenstern, Ralf; Strandback, Emilia; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z

    2016-01-26

    Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.

  20. Mucopolysaccharidosis IVA mutations in Chinese patients: 16 novel mutations.

    Science.gov (United States)

    Wang, Zheng; Zhang, Weimin; Wang, Yun; Meng, Yan; Su, Liang; Shi, Huiping; Huang, Shangzhi

    2010-08-01

    Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disease caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS) and transmitted as an autosomal recessive trait. This is the first systematic mutation screen in Chinese MPS IVA patients. Mutation detections in 24 unrelated Chinese MPS IVA patients were performed by PCR and direct sequencing of exons or the mRNA of GALNS. A total of 42 mutant alleles were identified, belonging to 27 different mutations. Out of the 27 mutations, 16 were novel, including 2 splicing mutations (c.567-1G>T and c.634-1G>A), 2 nonsense mutations (p.W325X and p.Q422X) and 12 missense mutations (p.T88I, p.H142R, p.P163H, p.G168L, p.H236D, p.N289S, p.T312A, p.G316V, p.A324E, p.L366P, p.Q422K and p.F452L). p.G340D was found to be a common mutation in the Chinese MPS IVA patients, accounting for 16.7% of the total number of mutant alleles. The results show that the mutations in Chinese MPS IVA patients are also family specific but have a different mutation spectrum as compared to those of other populations.

  1. Calreticulin Mutations in Myeloproliferative Neoplasms

    Directory of Open Access Journals (Sweden)

    Noa Lavi

    2014-10-01

    Full Text Available With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph− myeloproliferative neoplasms (MPNs in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (ET and primary myelofibrosis (PMF. At the end of 2013, two studies identified recurrent mutations in the gene encoding calreticulin (CALR using whole-exome sequencing. These mutations were revealed in the majority of ET and PMF patients with non-mutated JAK2 or MPL but not in polycythemia vera patients. Somatic 52-bp deletions (type 1 mutations and recurrent 5-bp insertions (type 2 mutations in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acids of the protein calreticulin were detected and found always to generate frameshift mutations. All detected mutant calreticulin proteins shared a novel amino acid sequence at the C-terminal. Mutations in CALR are acquired early in the clonal history of the disease, and they cause activation of JAK/STAT signaling. The CALR mutations are the second most frequent mutations in Ph− MPN patients after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. The characteristics of the CALR mutations as well as their diagnostic, clinical, and pathogenesis implications are discussed in this review.

  2. Novel Compound Heterozygous CBS Mutations Cause Homocystinuria in a Han Chinese Family.

    Science.gov (United States)

    Gong, Bo; Liu, Liping; Li, Zhiwei; Ye, Zimeng; Xiao, Ying; Zeng, Guangqun; Shi, Yi; Wang, Yumeng; Feng, Xiaoyun; Li, Xiulan; Hao, Fang; Liu, Xiaoqi; Qu, Chao; Li, Yuanfeng; Mu, Guoying; Yang, Zhenglin

    2015-12-15

    The cystathionine β-synthase (CBS) gene has been shown to be related to homocystinuria. This study was aimed to detect the mutations in CBS in a Han Chinese family with homocystinuria. A four-generation family from Shandong Province of China was recruited in this study. All available members of the family underwent comprehensive medical examinations. Genomic DNA was collected from peripheral blood of all the participants. The coding sequence of CBS was amplified by polymerase chain reaction (PCR), followed by direct DNA sequencing. Among all the family members, three affected individuals showed typical clinical features of homocystinuria. Two novel compound heterozygous mutations in the CBS gene, c.407T > C (p. L136P) and c.473C > T (p.A158V), were identified by sequencing analysis in this family. Both of the two missense mutations were detected in the three patients. Other available normal individuals, including the patients' parents, grand parents, her younger sister and brother in this family either carried one of the two mutations, or none. In addition, the two mutations were not found in 600 ethnically matched normal controls. This study provides a mutation spectrum of CBS resulting in homocystinuriain a Chinese population, which may shed light on the molecular pathogenesis and clinical diagnosis of CBS-associated homocystinuria.

  3. Sucrose Synthase Expression during Cold Acclimation in Wheat 1

    Science.gov (United States)

    Crespi, Martin D.; Zabaleta, Eduardo J.; Pontis, Horacio G.; Salerno, Graciela L.

    1991-01-01

    When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4°C) above 0°C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23°C to 4°C. The level of SS diminished when plants were moved back to 23°C. Northern blots of poly(A)+ RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress. ImagesFigure 1Figure 2Figure 3 PMID:16668270

  4. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian;

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS...... classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...

  5. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    DEFF Research Database (Denmark)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with ...... was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s-1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s-1 μM-1 for TgTPS2. The kinetic parameters were in agreement with previously published data....

  6. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

  7. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine (AEC)

  8. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    expression analysis and proteomics have pointed to abnormalities in mitochondrial oxidative phosphorylation and cellular stress in muscle of type 2 diabetic subjects, and recent work suggests that impaired mitochondrial activity is another early defect in the pathogenesis of type 2 diabetes. This review...... will discuss the latest advances in the understanding of the molecular mechanisms underlying insulin resistance in human skeletal muscle in type 2 diabetes with focus on possible links between impaired glycogen synthase activity and mitochondrial dysfunction....

  9. Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

    Energy Technology Data Exchange (ETDEWEB)

    Barends,T.; Domratcheva, T.; Kulik, V.; Blumenstein, L.; Niks, D.; Dunn, M.; Schlichting, I.

    2008-01-01

    Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of tryptophan synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.

  10. Reduced Expression of Lipoic Acid Synthase Accelerates Diabetic Nephropathy

    OpenAIRE

    Yi, Xianwen; Xu, Longquan; Hiller, Sylvia; Kim, Hyung-Suk; Nickeleit, Volker; James, Leighton R; Maeda, Nobuyo

    2011-01-01

    Oxidative stress contributes to the pathogenesis of diabetic nephropathy. In mitochondria, lipoic acid synthase produces α-lipoic acid, an antioxidant and an essential cofactor in α-ketoacid dehydrogenase complexes, which participate in glucose oxidation and ATP generation. Administration of lipoic acid abrogates diabetic nephropathy in animal models, but whether lower production of endogenous lipoic acid promotes diabetic nephropathy is unknown. Here, we crossed mice heterozygous for lipoic ...

  11. The cellulose synthase superfamily in fully sequenced plants and algae

    Directory of Open Access Journals (Sweden)

    Xu Ying

    2009-07-01

    Full Text Available Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl families and one cellulose synthase (CesA family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  12. Congenital erythropoietic porphyria due to a mutation in GATA1: the first trans-acting mutation causative for a human porphyria.

    Science.gov (United States)

    Phillips, John D; Steensma, David P; Pulsipher, Michael A; Spangrude, Gerald J; Kushner, James P

    2007-03-15

    Congenital erythropoietic porphyria (CEP), an autosomal recessive disorder, is due to mutations of uroporphyrinogen III synthase (UROS). Deficiency of UROS results in excess uroporphyrin I, which causes photosensitization. We evaluated a 3-year-old boy with CEP. A hypochromic, microcytic anemia was present from birth, and platelet counts averaged 70 x 10(9)/L (70,000/microL). Erythrocyte UROS activity was 21% of controls. Red cell morphology and globin chain labeling studies were compatible with beta-thalassemia. Hb electrophoresis revealed 36.3% A, 2.4% A(2), 59.5% F, and 1.8% of an unidentified peak. No UROS or alpha- and beta-globin mutations were found in the child or the parents. The molecular basis of the phenotype proved to be a mutation of GATA1, an X-linked transcription factor common to globin genes and heme biosynthetic enzymes in erythrocytes. A mutation at codon 216 in the child and on one allele of his mother changed arginine to tryptophan (R216W). This is the first report of a human porphyria due to a mutation in a trans-acting factor and the first association of CEP with thalassemia and thrombocytopenia. The Hb F level of 59.5% suggests a role for GATA-1 in globin switching. A bone marrow allograft corrected both the porphyria and the thalassemia.

  13. Genomic structure and sequence polymorphism of E,E-alphafarnesene synthase gene in apples (Malus domestica Borkh.)

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Primer pairs were designed to amplify the genomic DNA sequence of the alpha-farnesene synthase (AFS) gene by PCR.The PCR products were sequenced,spliced and compared to Cdna sequences in the GenBank (accession No.AY182241).The genomic sequence and intron-exon organization of the AFS gene were thus obtained.The AFS genomic sequence has been registered in the GenBank (accession No.DQ901739).It has 6 introns and 7 exons,encoding a protein of 576 amino acids.The sizes of the 6 introns were 108 bp,113 bp,>1000 bp,125 bp,220 bp and 88 bp,and their phases were 0,1,2,2,0,0,respectively.The sizes of the deduced amino acids of the 7 exons were 57,89,127,73,48,83 and 99,respectively.The AFS protein contained three motifs:the RR(X8)W motif encoded by a sequence in exon 1,and the RxR motif and DDxxD motif encoded by two sequences in exon 4.After comparing the AFS genomic sequence (accession No.DQ901739) to the Cdna sequence (accession No.AY523409) in the GenBank,it was found that there were 6 single-nucleotide polymorphisms between the two sequences,four of which caused mutations at the amino acid level.Interestingly,one amino acid mutation (291R→G) was found in the RxR motif,and further investigation is needed to determine whether the alpha-farnesene synthesis ability and superficial scald susceptibility of apples are influenced by this amino acid mutation and other mutations.

  14. Rational Design of Small-Molecule Stabilizers of Spermine Synthase Dimer by Virtual Screening and Free Energy-Based Approach

    Science.gov (United States)

    Zhang, Zhe; Martiny, Virginie; Lagorce, David; Ikeguchi, Yoshihiko; Alexov, Emil; Miteva, Maria A.

    2014-01-01

    Snyder-Robinson Syndrome (SRS) is a rare mental retardation disorder which is caused by the malfunctioning of an enzyme, the spermine synthase (SMS), which functions as a homo-dimer. The malfunctioning of SMS in SRS patients is associated with several identified missense mutations that occur away from the active site. This investigation deals with a particular SRS-causing mutation, the G56S mutation, which was shown computationally and experimentally to destabilize the SMS homo-dimer and thus to abolish SMS enzymatic activity. As a proof-of-concept, we explore the possibility to restore the enzymatic activity of the malfunctioning SMS mutant G56S by stabilizing the dimer through small molecule binding at the mutant homo-dimer interface. For this purpose, we designed an in silico protocol that couples virtual screening and a free binding energy-based approach to identify potential small-molecule binders on the destabilized G56S dimer, with the goal to stabilize it and thus to increase SMS G56S mutant activity. The protocol resulted in extensive list of plausible stabilizers, among which we selected and tested 51 compounds experimentally for their capability to increase SMS G56S mutant enzymatic activity. In silico analysis of the experimentally identified stabilizers suggested five distinctive chemical scaffolds. This investigation suggests that druggable pockets exist in the vicinity of the mutation sites at protein-protein interfaces which can be used to alter the disease-causing effects by small molecule binding. The identified chemical scaffolds are drug-like and can serve as original starting points for development of lead molecules to further rescue the disease-causing effects of the Snyder-Robinson syndrome for which no efficient treatment exists up to now. PMID:25340632

  15. Rational design of small-molecule stabilizers of spermine synthase dimer by virtual screening and free energy-based approach.

    Directory of Open Access Journals (Sweden)

    Zhe Zhang

    Full Text Available Snyder-Robinson Syndrome (SRS is a rare mental retardation disorder which is caused by the malfunctioning of an enzyme, the spermine synthase (SMS, which functions as a homo-dimer. The malfunctioning of SMS in SRS patients is associated with several identified missense mutations that occur away from the active site. This investigation deals with a particular SRS-causing mutation, the G56S mutation, which was shown computationally and experimentally to destabilize the SMS homo-dimer and thus to abolish SMS enzymatic activity. As a proof-of-concept, we explore the possibility to restore the enzymatic activity of the malfunctioning SMS mutant G56S by stabilizing the dimer through small molecule binding at the mutant homo-dimer interface. For this purpose, we designed an in silico protocol that couples virtual screening and a free binding energy-based approach to identify potential small-molecule binders on the destabilized G56S dimer, with the goal to stabilize it and thus to increase SMS G56S mutant activity. The protocol resulted in extensive list of plausible stabilizers, among which we selected and tested 51 compounds experimentally for their capability to increase SMS G56S mutant enzymatic activity. In silico analysis of the experimentally identified stabilizers suggested five distinctive chemical scaffolds. This investigation suggests that druggable pockets exist in the vicinity of the mutation sites at protein-protein interfaces which can be used to alter the disease-causing effects by small molecule binding. The identified chemical scaffolds are drug-like and can serve as original starting points for development of lead molecules to further rescue the disease-causing effects of the Snyder-Robinson syndrome for which no efficient treatment exists up to now.

  16. From bacterial to human dihydrouridine synthase: automated structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Fiona, E-mail: fiona.whelan@york.ac.uk; Jenkins, Huw T., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Griffiths, Samuel C. [University of Oxford, Headington, Oxford OX3 7BN (United Kingdom); Byrne, Robert T. [Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich (Germany); Dodson, Eleanor J.; Antson, Alfred A., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom)

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  17. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  18. Rotation and structure of FoF1-ATP synthase.

    Science.gov (United States)

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

  19. The pseudouridine synthases: revisiting a mechanism that seemed settled.

    Science.gov (United States)

    Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2004-10-13

    RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.

  20. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    Science.gov (United States)

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

  1. Phytochelatin synthase activity as a marker of metal pollution

    Energy Technology Data Exchange (ETDEWEB)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Adam, Vojtech [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Zehnalek, Josef; Beklova, Miroslava [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kizek, Rene, E-mail: kizek@sci.muni.cz [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic)

    2011-08-30

    Highlights: {yields} New tool for determination of phytochelatin synthase activity. {yields} The optimization of experimental condition for determination of the enzyme activity. {yields} First evaluation of K{sub m} for the enzyme. {yields} The effects of cadmium (II) not only on the activity of the enzyme but also on K{sub m}. -- Abstract: The synthesis of phytochelatins is catalyzed by {gamma}-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO{sub 3}){sub 2} for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 {sup o}C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K{sub m} for PCS was estimated as 2.3 mM.

  2. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

    Science.gov (United States)

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  3. Multi-substrate terpene synthases: their occurrence and physiological significance

    Directory of Open Access Journals (Sweden)

    Leila Pazouki

    2016-07-01

    Full Text Available Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15, and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5, mono- (C10 and diterpenes (C20. Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles.

  4. Site-directed mutagenesis of bacterial cellulose synthase highlights sulfur–arene interaction as key to catalysis

    OpenAIRE

    Sun, Shi-jing; Horikawa, Yoshiki; Wada, Masahisa; SUGIYAMA, Junji; Imai, Tomoya

    2016-01-01

    Cellulose is one of the most abundant biological polymers on Earth, and is synthesized by the cellulose synthase complex in cell membranes. Although many cellulose synthase genes have been identified over the past 25 years, functional studies of cellulose synthase using recombinant proteins have rarely been conducted. In this study, we conducted a functional analysis of cellulose synthase with site-directed mutagenesis, by using recombinant cellulose synthase reconstituted in living Escherich...

  5. Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues.

    Directory of Open Access Journals (Sweden)

    Hyun Jo Koo

    Full Text Available The essential oils of ginger (Zingiber officinale and turmeric (Curcuma longa contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+-germacrene D synthase and (S-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (--caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+-α-turmerone and (+-β-turmerone, are produced from (--α-zingiberene and (--β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase.

  6. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

    Science.gov (United States)

    Shibuya, M; Zhang, H; Endo, A; Shishikura, K; Kushiro, T; Ebizuka, Y

    1999-11-01

    Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.

  7. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe Brandt; Von Wettstein-Knowles, Penny

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

  8. Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

    Science.gov (United States)

    Chakraborty, Jayashree B; Mahato, Sanjit K; Joshi, Kalpana; Shinde, Vaibhav; Rakshit, Srabanti; Biswas, Nabendu; Choudhury Mukherjee, Indrani; Mandal, Labanya; Ganguly, Dipyaman; Chowdhury, Avik A; Chaudhuri, Jaydeep; Paul, Kausik; Pal, Bikas C; Vinayagam, Jayaraman; Pal, Churala; Manna, Anirban; Jaisankar, Parasuraman; Chaudhuri, Utpal; Konar, Aditya; Roy, Siddhartha; Bandyopadhyay, Santu

    2012-01-01

    Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings.

  9. Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

    Institute of Scientific and Technical Information of China (English)

    Hui LU; Bing Zhu; Xin-Dong Xue

    2006-01-01

    AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue.METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (LPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined.RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury.CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.

  10. Filaggrin mutations and the skin

    Directory of Open Access Journals (Sweden)

    Dipankar De

    2012-01-01

    Full Text Available Filaggrin is very important in the terminal differentiation of the skin and the formation of cornified envelope in the stratum corneum. Several mutations in the filaggrin gene have been identified in the last decade, mostly from the European countries. Loss of function mutations in the filaggrin gene results in reduced production of filaggrin, depending on the type and site of mutation. Such mutations in the filaggrin gene have been shown to be the most significant genetic risk factor for development of atopic dermatitis and undoubtedly has a role in the pathogenesis of ichthyosis vulgaris. Though there is theoretical possibility of association with hand eczema and allergic contact dermatitis; in clinical studies, the strength of these associations was not significantly strong. In this review, we have discussed the structure and function of filaggrin, basic genetics, type of mutations in filaggrin gene, and association of such mutations with different dermatoses.

  11. Muller's ratchet with compensatory mutations

    CERN Document Server

    Pfaffelhuber, Peter; Wakolbinger, Anton

    2011-01-01

    We consider an infinite dimensional system of stochastic differential equations which describes the evolution of type frequencies in a large population. Random reproduction is modeled by a Wright-Fisher noise whose inverse diffusion coefficient $N$ corresponds to the total population size. The type of an individual is the number $k$ of deleterious mutations it carries. We assume that fitness of individuals carrying $k$ mutations is decreased by $\\alpha k$ for some $\\alpha >0$. Along the individual lines of descent, (new) mutations accumulate at rate $\\lambda$ per generation, and each of these mutations has a small probability $\\gamma$ per generation to disappear. While the case $\\gamma =0 $ is known as (the Fleming-Viot version of) {\\em Muller's ratchet}, the case $\\gamma > 0$ is referred to as that of {\\em compensatory mutations} in the biological literature. In the former case ($\\gamma=0$), an ever increasing number of mutations is accumulated over time, while in the latter ($\\gamma > 0$) this is prevented ...

  12. Filaggrin mutations and the skin.

    Science.gov (United States)

    De, Dipankar; Handa, Sanjeev

    2012-01-01

    Filaggrin is very important in the terminal differentiation of the skin and the formation of cornified envelope in the stratum corneum. Several mutations in the filaggrin gene have been identified in the last decade, mostly from the European countries. Loss of function mutations in the filaggrin gene results in reduced production of filaggrin, depending on the type and site of mutation. Such mutations in the filaggrin gene have been shown to be the most significant genetic risk factor for development of atopic dermatitis and undoubtedly has a role in the pathogenesis of ichthyosis vulgaris. Though there is theoretical possibility of association with hand eczema and allergic contact dermatitis; in clinical studies, the strength of these associations was not significantly strong. In this review, we have discussed the structure and function of filaggrin, basic genetics, type of mutations in filaggrin gene, and association of such mutations with different dermatoses.

  13. Mutations induced in plant breeding

    Energy Technology Data Exchange (ETDEWEB)

    Barriga B, P. (Universidad Austral de Chile, Valdivia. Inst. de Produccion y Sanidad Vegetal)

    1984-10-01

    The most significant aspects of the use of ionizing radiations in plant breeding are reviewed. Aspects such as basic principles of mutation, expression and selection in obtention of mutants, methods for using induced mutations and sucess achieved with this methodology in plant breeding are reviewed. Results obtained in a program of induced mutation on wheat for high content of protein and lysine at the Universidad Austral de Chile are presented.

  14. 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

    OpenAIRE

    Bunney, Tom D.; van Walraven, Hendrika S.; de Boer, Albertus H.

    2001-01-01

    Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our und...

  15. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  16. Engineering Giardia lamblia trimethylguanosine synthase (GlaTgs2) to transfer non-natural modifications to the RNA 5'-cap.

    Science.gov (United States)

    Holstein, Josephin Marie; Stummer, Daniela; Rentmeister, Andrea

    2015-06-01

    Trimethylguanosine synthase from Giardia lamblia (GlaTgs2) naturally catalyzes methyl transfer from S-adenosyl-L-methionine (AdoMet) to the exocyclic N(2) atom of the 5'-cap--a hallmark of eukaryotic mRNAs. The wild-type enzyme shows substrate promiscuity and can also use the AdoMet-analog AdoPropen for allyl transfer. Here we report on engineering GlaTgs2 to enhance the activity on AdoPropen. A mutational analysis, involving an alanine scan of 10 residues located around the active site, was performed. Positions V34 and S38 were identified as mutational hot spots and analyzed in greater detail by testing NNK libraries. Kinetic analysis and thermostability measurements revealed V34A as the best variant of GlaTgs2, with a ∼10-fold improved specificity for AdoPropen. Double mutants did not yield additional improvements due to low catalytic efficiencies and thermal destabilization. Homologous Tgs enzymes from Homo sapiens and G. intestinalis were also investigated regarding their catalytic activity on AdoPropen. While neither the human wild-type (WT) enzyme nor any of its variants showed activity on AdoPropen, the homologue from G. intestinalis (GinTgs) was remarkably active on AdoPropen. Introducing the best substitution at the homologous position led to variant T34A with ∼40-fold higher specificity for AdoPropen than the original GlaTgs2 WT.

  17. Pseudouridine synthase 1 deficient mice, a model for Mitochondrial Myopathy with Sideroblastic Anemia, exhibit muscle morphology and physiology alterations

    Science.gov (United States)

    Mangum, Joshua E.; Hardee, Justin P.; Fix, Dennis K.; Puppa, Melissa J.; Elkes, Johnathon; Altomare, Diego; Bykhovskaya, Yelena; Campagna, Dean R.; Schmidt, Paul J.; Sendamarai, Anoop K.; Lidov, Hart G. W.; Barlow, Shayne C.; Fischel-Ghodsian, Nathan; Fleming, Mark D.; Carson, James A.; Patton, Jeffrey R.

    2016-01-01

    Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1−/− animals. Pus1−/− mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1−/− mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1−/− mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1−/− mice. PMID:27197761

  18. Modeling functional changes to Escherichia coli thymidylate synthase upon single residue replacements: a structure-based approach

    Directory of Open Access Journals (Sweden)

    Majid Masso

    2015-01-01

    Full Text Available Escherichia coli thymidylate synthase (TS is an enzyme that is indispensable to DNA synthesis and cell division, as it provides the only de novo source of dTMP by catalyzing the reductive methylation of dUMP, thus making it a key target for chemotherapeutic agents. High resolution X-ray crystallographic structures are available for TS and, owing to its relatively small size, successful experimental mutagenesis studies have been conducted on the enzyme. In this study, an in silico mutagenesis technique is used to investigate the effects of single amino acid substitutions in TS on enzymatic activity, one that employs the TS protein structure as well as a knowledge-based, four-body statistical potential. For every single residue TS variant, this approach yields both a global structural perturbation score and a set of local environmental perturbation scores that characterize the mutated position as well as all structurally neighboring residues. Global scores for the TS variants are capable of uniquely characterizing groups of residue positions in the enzyme according to their physicochemical, functional, or structural properties. Additionally, these global scores elucidate a statistically significant structure–function relationship among a collection of 372 single residue TS variants whose activity levels have been experimentally determined. Predictive models of TS variant activity are subsequently trained on this dataset of experimental mutants, whose respective feature vectors encode information regarding the mutated position as well as its six nearest residue neighbors in the TS structure, including their environmental perturbation scores.

  19. Stabilization and enhanced reactivity of actinorhodin polyketide synthase minimal complex in polymer-nucleotide coacervate droplets.

    Science.gov (United States)

    Crosby, John; Treadwell, Tom; Hammerton, Michelle; Vasilakis, Konstantinos; Crump, Matthew P; Williams, David S; Mann, Stephen

    2012-12-18

    Compartmentalization of the minimal complex of actinorhodin polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt polyketides under conditions of low and high ionic strength.

  20. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  1. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent...... on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing...

  2. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

    2015-02-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. © 2014 Wiley Periodicals, Inc.

  3. Argininosuccinate synthase as a novel biomarker for inflammatory conditions.

    Science.gov (United States)

    Cao, Mengde; George, Thomas J; Prima, Victor; Nelson, David; Svetlov, Stanislav

    2013-05-01

    Argininosuccinate synthase (ASS) plays an important role in regulating metabolic functions in mammals. We previously reported that hepatic ASS is released into circulation at very high concentrations in response to endotoxin and acute liver injury. We propose that ASS may serve as a novel biomarker for various inflammatory conditions. Our data showed that ASS accumulated in serum and urine of septic, obese or tumor mice in a condition-dependent fashion. Moreover, ASS significantly increased in urine within the first week after tumor cell implantation in mice which subsequently develop tumors. These results suggest that ASS is a novel biomarker increased upon diverse inflammatory conditions.

  4. Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase.

    Science.gov (United States)

    Girish, Tavarekere S; Sharma, Eshita; Gopal, B

    2008-08-20

    Lysine biosynthesis is crucial for cell-wall formation in bacteria. Enzymes involved in lysine biosynthesis are thus potential targets for anti-microbial therapeutics. Dihydrodipicolinate synthase (DHDPS) catalyzes the first step of this pathway. Unlike its homologues, Staphylococcus aureus DHDPS is a dimer both in solution and in the crystal and is not feedback inhibited by lysine. The crystal structure of S. aureus DHDPS in the free and substrate bound forms provides a structural rationale for its catalytic mechanism. The structure also reveals unique conformational features of the S. aureus enzyme that could be crucial for the design of specific non-competitive inhibitors.

  5. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Marina eKorotkova

    2011-01-01

    Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

  6. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    Science.gov (United States)

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  7. Nitric Oxide Synthase-3 Promotes Embryonic Development of Atrioventricular Valves

    OpenAIRE

    Yin Liu; Xiangru Lu; Fu-Li Xiang; Man Lu; Qingping Feng

    2013-01-01

    Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3(-/-) mice at postnatal day 0. Our data s...

  8. MPL mutations in myeloproliferative disorders

    DEFF Research Database (Denmark)

    Beer, Philip A.; Campbell, Peter J.; Scott, Linda M.

    2008-01-01

    Activating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet cDNA...

  9. Polymorphism of Plasmodium Falciparum Dihydrofolate Reductase and Dihydropteroate Synthase Genes among Pregnant Women with Falciparum Malaria in Banjar District, South Kalimantan Province, Indonesia

    Directory of Open Access Journals (Sweden)

    Sukmawati Basuki

    2012-12-01

    Full Text Available Pregnant women are highly vulnerable to malaria infection in its endemic areas, particularly infection by Plasmodium falciparum that can cause premature, low birth weight, severe anemia in pregnant women, and death. Sulfadoxine-pyrimethamine (SP for Intermittent Preventive Treatment for pregnant (IPTp is used for malaria control in pregnancy recommended by the World Health Organization that has already been implemented in Africa. The P. falciparum resistance to SP has been reported in several malarial endemic areas, and mutations in the genes of Plasmodium falciparum Dihydrofolate Reductase (Pfdhfr and Dihydropteroate Synthase (Pfdhps are shown to be associated with parasite resistance to SP treatment. Genetic analysis of Pfdhfr and Pfdhps genes in pregnant women infected with P. falciparum has not yet been examined in Indonesia. The cross-sectional study was conducted at two subdistricts, Sungai Pinang and Peramasan, in Banjar district of South Kalimantan Province, where 127 pregnant women were recruited from 2008 to April 2010. Two important mutations in Pfdhfr gene (amino acid positions at N51 and S108 and three in Pfdhps gene (A437, K540 and A581 were analyzed by nested PCR-RFLP method. All of the seven pregnant women samples infected with P. falciparum presented PfDHFR 108N and PfDHPS 437G mutations. One of the samples had the additional mutation at PfDHPS 540, in which Lys is substituted by Glu. These results suggested that P. falciparum might present only some resistance to SP at Sungai Pinang and Peramasan subdistricts, Banjar District, South Kalimantan province, Indonesia. Although there were limited number of samples, this study showed only few mutations of Pfdhfr and Pfdhps genes in P. falciparum at Banjar district, South Kalimantan Province, that suggests SP might be effective for IPTp in this area. Thus, further analysis of the other mutation sites in Pfdhfr and Pfdhps genes and in vivo efficacy study of SP with more sufficient

  10. Declining trend of Plasmodium falciparum dihydrofolate reductase (dhfr and dihydropteroate synthase (dhps mutant alleles after the withdrawal of Sulfadoxine-Pyrimethamine in North Western Ethiopia.

    Directory of Open Access Journals (Sweden)

    Sofonias K Tessema

    Full Text Available Antimalarial drug resistance is one of the major challenges in global efforts of malaria control and elimination. In 1998, chloroquine was abandoned and replaced with sulfadoxine/pyrimethamine, which in turn was replaced with artemether/lumefantrine for the treatment of uncomplicated falciparum malaria in 2004. Sulfadoxine/pyrimethamine resistance is associated with mutations in dihydrofolate reductase (Pfdhfr and dihydropteroate synthase (Pfdhps genes. The prevalence of mutation in Pfdhfr and Pfdhps genes were evaluated and compared for a total of 159 isolates collected in two different time points, 2005 and 2007/08, from Pawe hospital, in North Western Ethiopia. The frequency of triple Pfdhfr mutation decreased significantly from 50.8% (32/63 to 15.9% (10/63 (P<0.001, while Pfdhps double mutation remained high and changed only marginally from 69.2% (45/65 to 55.4% (40/65 (P = 0.08. The combined Pfdhfr/Pfdhps quintuple mutation, which is strongly associated with sulfadoxine/pyrimethamine resistance, was significantly decreased from 40.7% (24/59 to 13.6% (8/59 (P<0.0001. On the whole, significant decline in mutant alleles and re-emergence of wild type alleles were observed. The change in the frequency is explained by the reduction of residual drug-resistant parasites caused by the strong drug pressure imposed when sulfadoxine/pyrimethamine was the first-line drug, followed by lower fitness of these resistant parasites in the absence of drug pressure. Despite the decrease in the frequency of mutant alleles, higher percentages of mutation remain prevalent in the study area in 2007/08 in both Pfdhfr and Pfdhps genes. Therefore, further multi-centered studies in different parts of the country will be required to assess the re-emergence of sulfadoxine/pyrimethamine sensitive parasites and to monitor and prevent the establishment of multi drug resistant parasites in this region.

  11. Role of the Tomato Non-Ripening Mutation in Regulating Fruit Quality Elucidated Using iTRAQ Protein Profile Analysis

    Science.gov (United States)

    Yuan, Xin-Yu; Wang, Rui-Heng; Zhao, Xiao-Dan; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Natural mutants of the Non-ripening (Nor) gene repress the normal ripening of tomato fruit. The molecular mechanism of fruit ripening regulation by the Nor gene is unclear. To elucidate how the Nor gene can affect ripening and fruit quality at the protein level, we used the fruits of Nor mutants and wild-type Ailsa Craig (AC) to perform iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The Nor mutation altered tomato fruit ripening and affected quality in various respects, including ethylene biosynthesis by down-regulating the abundance of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), pigment biosynthesis by repressing phytoene synthase 1 (PSY1), ζ-carotene isomerase (Z-ISO), chalcone synthase 1 (CHS1) and other proteins, enhancing fruit firmness by increasing the abundance of cellulose synthase protein, while reducing those of polygalacturonase 2 (PG2) and pectate lyase (PL), altering biosynthesis of nutrients such as carbohydrates, amino acids, and anthocyanins. Conversely, Nor mutation also enhanced the fruit’s resistance to some pathogens by up-regulating the expression of several genes associated with stress and defense. Therefore, the Nor gene is involved in the regulation of fruit ripening and quality. It is useful in the future as a means to improve fruit quality in tomato. PMID:27732677

  12. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  13. Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.

    Science.gov (United States)

    Xiong, S; Chirala, S S; Wakil, S J

    2000-04-11

    To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.

  14. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    Science.gov (United States)

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. The subunit b dimer of the FOF1-ATP synthase: interaction with F1-ATPase as deduced by site-specific spin-labeling.

    Science.gov (United States)

    Motz, Christian; Hornung, Tassilo; Kersten, Michael; McLachlin, Derek T; Dunn, Stanley D; Wise, John G; Vogel, Pia D

    2004-11-19

    We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F1-ATPase as well as to F1-delta. Subsequent addition of delta to F1-delta.b2 complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region.

  16. Mutational meltdown in laboratory yeast populations

    NARCIS (Netherlands)

    Zeyl, C.; Mizesko, M.; Visser, de J.A.G.M.

    2001-01-01

    In small or repeatedly bottlenecked populations, mutations are expected to accumulate by genetic drift, causing fitness declines. In mutational meltdown models, such fitness declines further reduce population size, thus accelerating additional mutation accumulation and leading to extinction. Because

  17. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  18. Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

    Science.gov (United States)

    Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase.

  19. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

    Science.gov (United States)

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C

    2012-04-27

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  1. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing......CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold...... decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein...

  2. Aldosterone synthase inhibitors in hypertension: current status and future possibilities

    Directory of Open Access Journals (Sweden)

    Milan Hargovan

    2014-02-01

    Full Text Available The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors.

  3. Aldosterone synthase inhibitors in hypertension: current status and future possibilities.

    Science.gov (United States)

    Hargovan, Milan; Ferro, Albert

    2014-01-01

    The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors.

  4. Cryptic Polyketide Synthase Genes in Non-Pathogenic Clostridium SPP

    Science.gov (United States)

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides. PMID:22235310

  5. IPC synthase as a useful target for antifungal drugs.

    Science.gov (United States)

    Sugimoto, Yuichi; Sakoh, Hiroki; Yamada, Koji

    2004-12-01

    Inositol phosphorylceramide (IPC) synthase is a common and essential enzyme in fungi and plants, which catalyzes the transfer of phosphoinositol to the C-1 hydroxy of ceramide to produce IPC. This reaction is a key step in fungal sphingolipid biosynthesis, therefore the enzyme is a potential target for the development of nontoxic therapeutic antifungal agents. Natural products with a desired biological activity, aureobasidin A (AbA), khafrefungin, and galbonolide A, have been reported. AbA, a cyclic depsipeptide containing 8 amino acids and a hydroxyl acid, is a broad spectrum antifungal with strong activity against many pathogenic fungi such as Candida spp., Cryptococcus neoformans, and some Aspergillus spp. Khafrefungin, an aldonic acid ester with a C22 long alkyl chain, has antifungal activity against C. albicans, Cr. Neoformans, and Saccharomyces cerevisiae. Galbonolide A is a 14-membered macrolide with fungicidal activity against clinically important strains, and is especially potent against Cr. neoformans. These classes of natural products are potent and specific antifungal agents. We review current progress in the development of IPC synthase inhibitors with antifungal activities, and present structure-activity relationships (SAR), physicochemical and structural properties, and synthetic methodology for chemical modification.

  6. Eugenol synthase genes in floral scent variation in Gymnadenia species.

    Science.gov (United States)

    Gupta, Alok K; Schauvinhold, Ines; Pichersky, Eran; Schiestl, Florian P

    2014-12-01

    Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of "floral scent genes" and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

  7. Cloning and Identification of Methionine Synthase Gene from Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Lan HUANG; Dong-Yang LI; Shao-Xiao WANG; Shi-Ming ZHANG; Jun-Hui CHEN; Xiang-Fu WU

    2005-01-01

    Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.

  8. Tryptophan synthase of Phaeophyceae originated from the secondary host nucleus

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yalan; CHI Shan; WU Shuangxiu; LIU Cui; YU Jun; WANG Xumin; CHEN Shengping; LIU Tao

    2014-01-01

    Tryptophan synthase (TS, EC 4.2.1.20) catalyzes the last two steps of L-tryptophan biosynthesis. In pro-karyotes, tryptophan synthase is a multi-enzyme complex, and it consists ofαandβsubunit which forms anα-ββ-αcomplex. In fungi and diatoms, TS is a bifunctional enzyme. Because of the limited genomic and transcriptomic data of algae, there are few studies on TS evolution of algae. Here we analyzed the data of the 1000 Plants Project (1KP), and focused on red algae and brown algae. We found out that the TS of Phaeophy-ceae were fusion genes, which probably originated from the secondary host nucleus, and that the TS of Rho-dophyta contained two genes, TSA and TSB, which both display a possible cyanobacterial origin at the time of primary endosymbiosis. In addition, there were two types of TSB genes (TSB1 and TSB2). Through the multiple sequence alignment of TSB proteins, we found several residues conserved in TSB1 but variable in TSB2 which connect withαsubunit. The phenomenon may suggest that the TSB2 sequences of Rhodophyta cannot form stable complex with TSA.

  9. The crystal structure of human GDP-L-fucose synthase.

    Science.gov (United States)

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  10. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  11. Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

    Directory of Open Access Journals (Sweden)

    C F Reboul

    Full Text Available Dihydrodipicolinate synthase (DHDPS is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA. These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

  12. Structural and Dynamic Requirements for Optimal Activity of the Essential Bacterial Enzyme Dihydrodipicolinate Synthase

    Science.gov (United States)

    Reboul, C. F.; Porebski, B. T.; Griffin, M. D. W.; Dobson, R. C. J.; Perugini, M. A.; Gerrard, J. A.; Buckle, A. M.

    2012-01-01

    Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a ‘dimer of dimers’, with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface. PMID:22685390

  13. Identification of a glycogen synthase kinase-3β inhibitor that attenuates hyperactivity in CLOCK mutant mice.

    Science.gov (United States)

    Kozikowski, Alan P; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N; Walter, Richard L; Ketcherside, Ariel; McClung, Colleen A; Mesecar, Andrew D; Caldarone, Barbara

    2011-09-05

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called "mood-stabilizing drugs", such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3β (GSK-3β) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3β. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC₅₀ values in the range of 4 to 680 nM against human GSK-3β. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg⁻¹ resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg⁻¹) and the antipsychotic haloperidol (1 mg kg⁻¹). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3β in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3β as a relevant therapeutic target in the identification of new therapies for bipolar patients. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity.

    Science.gov (United States)

    Ahmad, Shabbir; Ytterberg, A Jimmy; Thulasingam, Madhuranayaki; Tholander, Fredrik; Bergman, Tomas; Zubarev, Roman; Wetterholm, Anders; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z

    2016-08-26

    Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 μmol/min/mg) of the activity of WT enzyme (95 μmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Nasal nitric oxide and nitric oxide synthase expression in primary ciliary dyskinesia.

    Science.gov (United States)

    Pifferi, M; Bush, A; Maggi, F; Michelucci, A; Ricci, V; Conidi, M E; Cangiotti, A M; Bodini, A; Simi, P; Macchia, P; Boner, A L

    2011-03-01

    No study has evaluated the correlation between different expression of nitric oxide synthase (NOS) isoforms in nasal epithelial cells and nasal NO (nNO) level in primary ciliary dyskinesia (PCD). Gene expression of endothelial (NOS3) and inducible NOS (NOS2) and their correlation with nNO level, ciliary function and morphology were studied in patients with PCD or secondary ciliary dyskinesia (SCD). NOS3 gene polymorphisms were studied in blood leukocytes. A total of 212 subjects were studied (48 with PCD, 161 with SCD and three normal subjects). nNO level correlated with mean ciliary beat frequency (p = 0.044; r = 0.174). The lower the nNO level the higher was the percentage of immotile cilia (p<0.001; r = -0.375). A significant positive correlation between NOS2 gene expression and nNO levels was demonstrated in all children (p = 0.001; r = 0.428), and this correlation was confirmed in patients with PCD (p = 0.019; r = 0.484). NOS2 gene expression was lower in PCD than in SCD (p = 0.04). The NOS3 isoform correlated with missing central microtubules (p = 0.048; r = 0.447). nNO levels were higher in PCD subjects with the NOS3 thymidine 894 mutation, and this was associated with a higher ciliary beat frequency (p = 0.045). These results demonstrate a relationship between nNO level, NOS mRNA expression and ciliary beat frequency.

  16. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    Energy Technology Data Exchange (ETDEWEB)

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara (Psychogenics); (Purdue); (UIC); (UTSMC)

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  17. A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan; Jin, Zhongmin; Yu, Xiaolin; Zhao, Gengxiang; Haskins, Nantaporn; Allewell, Norma M.; Tuchman, Mendel (Maryland); (GWU); (Georgia)

    2012-05-24

    Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.

  18. Selectivity of the surface binding site (SBS) on barley starch synthase I

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cuesta-Seijo, Jose A.; Palcic, Monica

    2014-01-01

    Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was ...

  19. Insights into the subunit in-teractions of the chloroplast ATP synthase

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  20. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named

  1. KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery

    NARCIS (Netherlands)

    Mansoori Zangir, N.; Timmers, J.F.P.; Desprez, T.; Lessa Alvim Kamei, C.; Dees, D.C.T.; Vincken, J.P.; Visser, R.G.F.; Höfte, H.; Vernhettes, S.; Trindade, L.M.

    2014-01-01

    Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases.

  2. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPIN

  3. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank;

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer c...

  4. HNPCC: Six new pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Epplen Joerg T

    2004-06-01

    Full Text Available Abstract Background Hereditary non-polyposis colorectal cancer (HNPCC is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR. HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6. Methods Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out. Results We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6 resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702], three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298] and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]. All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H. Conclusions HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.

  5. Radiation mutation breeding

    Energy Technology Data Exchange (ETDEWEB)

    Song, Hi Sup; Kim, Jae Sung; Kim, Jin Kyu; Shin, In Chul; Lim, Young Taek

    1998-04-01

    In order to develop an advanced technical knowledge for the selection of better mutants, some of the crops were irradiated and the mutation rate, the survival rate and the method for selction of a mutant were studied. Furthermore, this study aimed to obtain basic data applicable to the development of genetic resources by evaluation and analysis the specific character for selection of the superior mutant and its plant breeding. 1. selection of the mutant with a superior resistance against environment in the principal crops 1) New varieties of mutant rices such as Wonpyeongbyeo, Wongwangbyeo, Winmibyeo, and heogseon chalbeyeo (sticky forma) were registered in the national variety list and made an application to crop variety protection right. They are under review now. 2) We also keep on studying on the number of a grain of 8 lines of excellent mutant rice for the purpose of improvement of breeding . 3) We selected 3 lines which have a resistance to pod and stem blight in large soybean, 31 lines with small grain size and higher yield, 112 lines of soybean of cooking, 7 lines of low lipoxygenase content, and 12 lines with decreased phytic acid content by 20 % compared to the previous level. 2. Selection of advanced Mugunwha (Rose of Sharon) mutant 1) Bagseul, a new variety of mutant, was developed and 30 plantlets of it are being proliferated. 2) Fifty-three lines of a mutant having a various morphologies were selected.

  6. [Primary hypoaldosteronism and moderate bilateral deafness in a child with a homozygous missense mutation (Thr318Met) in the CYP11B2 gene].

    Science.gov (United States)

    Rubio-Cabezas, O; Regueras, L; Muñoz-Calvo, M T; Bartolomé, M; Pozo, J; Argente, J

    2010-07-01

    Isolated congenital hypoaldosteronism is a rare disorder that presents as chronic salt-wasting syndrome during infancy. Aldosterone synthase deficiency due to mutations in CYP11B2 is the underlying cause in most cases. Apart from the classical electrolyte disturbances (hyponatremia and hyperkalemia), no other extra-adrenal features have been described to date. We report a male child with congenital hypoaldosteronism due to a homozygous missense mutation (Thr318Met) in CYP11B2 who also presented with unexplained sensorineural hearing loss.

  7. Mutations induced by ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Pfeifer, Gerd P. [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)]. E-mail: gpfeifer@coh.org; You, Young-Hyun [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Besaratinia, Ahmad [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)

    2005-04-01

    The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms

  8. Triplet repeat mutation length gains correlate with cell-type specific vulnerability in Huntington disease brain.

    Science.gov (United States)

    Shelbourne, Peggy F; Keller-McGandy, Christine; Bi, Wenya Linda; Yoon, Song-Ro; Dubeau, Louis; Veitch, Nicola J; Vonsattel, Jean Paul; Wexler, Nancy S; Arnheim, Norman; Augood, Sarah J

    2007-05-15

    Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Here, we provide evidence supporting the hypothesis that somatic increases of mutation length play a role in the progressive nature and cell-selective aspects of HD pathogenesis. Results from micro-dissected tissue and individual laser-dissected cells obtained from human HD cases and knock-in HD mice indicate that the CAG repeat is unstable in all cell types tested although neurons tend to have longer mutation length gains than glia. Mutation length gains occur early in the disease process and continue to accumulate as the disease progresses. In keeping with observed patterns of cell loss, neuronal mutation length gains tend to be more prominent in the striatum than in the cortex of low-grade human HD cases, less so in more advanced cases. Interestingly, neuronal sub-populations of HD mice appear to have different propensities for mutation length gains; in particular, smaller mutation length gains occur in nitric oxide synthase-positive striatal interneurons (a relatively spared cell type in HD) compared with the pan-striatal neuronal population. More generally, the data demonstrate that neuronal changes in HD repeat length can be at least as great, if not greater, than those observed in the germline. The fact that significant CAG repeat length gains occur in non-replicating cells also argues that processes such as inappropriate mismatch repair rather than DNA replication are involved in generating mutation instability in HD brain tissue.

  9. T-786c Polymorphism in nitric oxide synthase 3 gene and Nitrit Oxide Level of Diabetic Retinopathy in Javanese Population

    Directory of Open Access Journals (Sweden)

    Putri Widelia Welkriana

    2015-11-01

    Full Text Available AbstractComplication of retinopathy in type 2 DM is caused of lower level of NO. Nitric oxide level is synthesizedfrom L-arginin in reaction that catalyze Nitric oxide synthase (NOS 3. The T-786C mutation in NOS 3 genedecreases the expression of nitric oxide synthase (NOS 3 so decreases NO synthesis. To investigate theassociation between T-786C polymorphism in NOS 3 gene with NO level of diabetic retinopathy patients. Thisstudy was a case control study, consist of 40 patient of type 2 diabetic with DR (case group and 40 patient oftype 2 diabetic without DR (control group of Javanese ethnic. The genotyping of T-786C polymorphism wasperformed by PCR-RLFP. Level of NO was measured by spectrophotometry. Chi square test and odd ratiowere used to analyze the association of the T-786C polymorphism in NOS 3 gene with DR. Differences ofNO level between TT and TC genotypes were analyzed using independent t test. The distribution of T-786Cpolymorphism in NOS 3 gene of DR subjects showed that frequency of TT genotype was 22.5% and TC genotypewas 77.5%. Non DR subjects showed the frequency of TT genotype was 50% and TC genotype was 50%, (p=0.011. Frequency of T allele in DR group was 61.25% and C allele was 38.75%, and frequency of T allele in nonDR group was 75% and C allele was 25%, (p= 0.62. Odd ratio of TC genotype was 3.444(CI; 95% : 0.964-3.735and C allele was 1.898 (CI; 95% : 1.310-9.058. The NO level of TC genotype was 1.43+0.126 and TT genotypewas 11.27+5.87 (p=0.000. Level of NO between RD and non RD showed not different significantly (p=0.160for retinopathy. The T-786C polymorphism of NOS 3 gene is risk factor for retinopathy in type 2 DiabetesMellitus. Individual with TC genotype of NOS 3 gene has lower level of NO than TT genotype.Keywords : Diabetic Retinopathy, Polymorphism, Nitric Oxide, Nitric Oxide Synthase.

  10. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Science.gov (United States)

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata.

  11. Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos.

    Science.gov (United States)

    Maruyama, T; Ito, M; Honda, G

    2001-10-01

    We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

  12. Mutational profiling reveals PIK3CA mutations in gallbladder carcinoma

    Directory of Open Access Journals (Sweden)

    Bardeesy Nabeel

    2011-02-01

    Full Text Available Abstract Background The genetics of advanced biliary tract cancers (BTC, which encompass intra- and extra-hepatic cholangiocarcinomas as well as gallbladder carcinomas, are heterogeneous and remain to be fully defined. Methods To better characterize mutations in established known oncogenes and tumor suppressor genes we tested a mass spectrometric based platform to interrogate common cancer associated mutations across a panel of 77 formalin fixed paraffin embedded archived BTC cases. Results Mutations among three genes, KRAS, NRAS and PIK3CA were confirmed in this cohort. Activating mutations in PIK3CA were identified exclusively in GBC (4/32, 12.5%. KRAS mutations were identified in 3 (13% intra-hepatic cholangiocarcinomas and 1 (33% perihillar cholangiocarcinoma but were not identified in gallbladder carcinomas and extra-hepatic cholangiocarcinoma. Conclusions The presence of activating mutations in PIK3CA specifically in GBC has clinical implications in both the diagnosis of this cancer type, as well as the potential utility of targeted therapies such as PI3 kinase inhibitors.

  13. Feline congenital erythropoietic porphyria: two homozygous UROS missense mutations cause the enzyme deficiency and porphyrin accumulation.

    Science.gov (United States)

    Clavero, Sonia; Bishop, David F; Giger, Urs; Haskins, Mark E; Desnick, Robert J

    2010-01-01

    The first feline model of human congenital erythropoietic porphyria (CEP) due to deficient uroporphyrinogen III synthase (URO-synthase) activity was identified by its characteristic clinical phenotype, and confirmed by biochemical and molecular genetic studies. The proband, an adult domestic shorthair cat, had dark-red urine and brownish discolored teeth with red fluorescence under ultraviolet light. Biochemical studies demonstrated markedly increased uroporphyrinogen I in urine and plasma (2,650- and 10,700-fold greater than wild type, respectively), whereas urinary 5-aminolevulinic acid and porphobilinogen were lower than normal. Erythrocytic URO-synthase activity was UROS gene revealed two missense mutations, c.140C>T (p.S47F) in exon 3 and c.331G>A (p.G111S) in exon 6, both of which were homozygous, presumably owing to parental consanguinity. Neither was present in 100 normal cat alleles. Prokaryotic expression and thermostability studies of the purified monomeric wild-type, p.S47F, p.G111S, and p.S47F/G111S enzymes showed that the p.S47F enzyme had 100% of wild-type specific activity but ~50% decreased thermostability, whereas the p.G111S and p.S47F/G111S enzymes had about 60% and 20% of wild-type specific activity, respectively, and both were markedly thermolabile. Molecular modeling results indicated that the less active/less stable p.G111S enzyme was further functionally impaired by a structural interaction induced by the presence of the S47F substitution. Thus, the synergistic interaction of two rare amino acid substitutions in the URO-synthase polypeptide caused the feline model of human CEP.

  14. Protons, the thylakoid membrane, and the chloroplast ATP synthase.

    Science.gov (United States)

    Junge, W

    1989-01-01

    According to the chemiosmotic theory, proton pumps and ATP synthases are coupled by lateral proton flow through aqueous phases. Three long-standing challenges to this concept, all of which have been loosely subsumed under 'localized coupling' in the literature, were examined in the light of experiments carried out with thylakoids: (1) Nearest neighbor interaction between pumps and ATP synthases. Considering the large distances between photosystem II and CFoCF1, in stacked thylakoids this is a priori absent. (2) Enhanced proton diffusion along the surface of the membrane. This could not be substantiated for the outer side of the thylakoid membrane. Even for the interface between pure lipid and water, two laboratories have reported the absence of enhanced diffusion. (3) Localized proton ducts in the membrane. Intramembrane domains that can transiently trap protons do exist in thylakoid membranes, but because of their limited storage capacity for protons, they probably do not matter for photophosphorylation under continuous light. Seemingly in favor of localized proton ducts is the failure of a supposedly permeant buffer to enhance the onset lag of photophosphorylation. However, it was found that failure of some buffers and the ability of others in this respect were correlated with their failure/ability to quench pH transients in the thylakoid lumen, as predicted by the chemiosmotic theory. It was shown that the chemiosmotic concept is a fair approximation, even for narrow aqueous phases, as in stacked thylakoids. These are approximately isopotential, and protons are taken in by the ATP synthase straight from the lumen. The molecular mechanism by which F0F1 ATPases couple proton flow to ATP synthesis is still unknown. The threefold structural symmetry of the headpiece that, probably, finds a corollary in the channel portion of these enzymes appeals to the common wisdom that structural symmetry causes functional symmetry. "Rotation catalysis" has been proposed. It is

  15. Markov models for accumulating mutations

    CERN Document Server

    Beerenwinkel, Niko

    2007-01-01

    We introduce and analyze a waiting time model for the accumulation of genetic changes. The continuous time conjunctive Bayesian network is defined by a partially ordered set of mutations and by the rate of fixation of each mutation. The partial order encodes constraints on the order in which mutations can fixate in the population, shedding light on the mutational pathways underlying the evolutionary process. We study a censored version of the model and derive equations for an EM algorithm to perform maximum likelihood estimation of the model parameters. We also show how to select the maximum likelihood poset. The model is applied to genetic data from different cancers and from drug resistant HIV samples, indicating implications for diagnosis and treatment.

  16. Dynamical Mutation of Dark Energy

    CERN Document Server

    Abramo, L R; Liberato, L; Rosenfeld, R

    2007-01-01

    We discuss the intriguing possibility that dark energy may change its equation of state in situations where large dark energy fluctuations are present. We show indications of this dynamical mutation in some generic models of dark energy.

  17. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  18. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  19. The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

    NARCIS (Netherlands)

    Müller, Patrick; Ewers, C.; Bertsche, U.; Anstett, M.; Kallis, T.; Breukink, E.J.; Fraipont, Claudine; Terrak, Mohammed; Nguyen-Distèche, Martine; Vollmer, W.

    2007-01-01

    Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP

  20. PPARγ mutations, lipodystrophy and diabetes.

    Science.gov (United States)

    Astapova, Olga; Leff, Todd

    2014-11-01

    The focus of this review is the lipodystrophy syndrome caused by mutation in the PPARγ nuclear receptor - partial familial lipodystrophy FPLD3. To provide a broader context for how these mutations act to generate the clinical features of partial lipodystrophy we will review the basic biology of PPARγ and also survey the set PPARγ genetic variants that do not cause lipodystrophy, but are nonetheless associated with clinically related syndromes, specifically type 2 diabetes.

  1. Gene mutations in hepatocellular adenomas

    DEFF Research Database (Denmark)

    Raft, Marie B; Jørgensen, Ernö N; Vainer, Ben

    2015-01-01

    is associated with bi-allelic mutations in the TCF1 gene and morphologically has marked steatosis. β-catenin activating HCA has increased activity of the Wnt/β-catenin pathway and is associated with possible malignant transformation. Inflammatory HCA is characterized by an oncogene-induced inflammation due....... This review offers an overview of the reported gene mutations associated with hepatocellular adenomas together with a discussion of the diagnostic and prognostic value....

  2. Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance1

    Science.gov (United States)

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R. Douglas; Powles, Stephen B.

    2015-01-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. PMID:25717039

  3. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.

    Science.gov (United States)

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R Douglas; Powles, Stephen B

    2015-04-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I+P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.

  4. Mutations causative of familial hypercholesterolaemia

    DEFF Research Database (Denmark)

    Benn, Marianne; Watts, Gerald F; Tybjærg-Hansen, Anne

    2016-01-01

    AIMS: Ideally, familial hypercholesterolaemia (FH) is diagnosed by testing for mutations that decrease the catabolism of low-density lipoprotein (LDL) cholesterol; however, genetic testing is not universally available. The aim of the present study was to assess the frequency and predictors of FH....... The prevalence of the four FH mutations was 0.18% (1:565), suggesting a total prevalence of FH mutations of 0.46% (1:217). Using the Dutch Lipid Clinic Network (DLCN) criteria, odds ratios for an FH mutation were 439 (95% CI: 170-1 138) for definite FH, 90 (53-152) for probable FH, and 18 (13-25) for possible FH......-cholesterol concentration to discriminate between mutation carriers and non-carriers was 4.4 mmol/L. CONCLUSION: Familial hypercholesterolaemia-causing mutations are estimated to occur in 1:217 in the general population and are best identified by a definite or probable phenotypic diagnosis of FH based on the DLCN criteria...

  5. Hyperactivity: glycogen synthase kinase-3 as a therapeutic target.

    Science.gov (United States)

    Mines, Marjelo A

    2013-05-15

    The diagnosis of hyperactivity-associated disorders has increased within the past few years. The prevalence of hyperactivity-associated disorders is indicative of the need to more fully understand the underlying causes and to develop improved therapeutic interventions. There is increasing evidence that glycogen synthase kinase-3 (GSK3) mediates locomotor hyperactivity in a number of animal models, and therefore may be a potential target for therapeutic intervention in hyperactivity-associated behaviors. In this review, we discuss 1) the effect of manipulations of GSK3 in the absence of drugs and disorders on locomotor activity, 2) the role of GSK3 in drug-induced hyperactivity in rodents, and 3) regulation of locomotor activity by GSK3 in transgenic mouse models related to specific disorders. These studies link GSK3 regulation and activity to hyperactivity-associated behaviors and disease pathologies. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Alterations in Nitric Oxide Synthase in the Aged CNS

    Directory of Open Access Journals (Sweden)

    Junyang Jung

    2012-01-01

    Full Text Available Aging is associated with neuronal loss, gross weight reduction of the brain, and glial proliferation in the cortex, all of which lead to functional changes in the brain. It is known that oxidative stress is a critical factor in the pathogenesis of aging; additionally, growing evidence suggests that excessive nitric oxide (NO production contributes to the aging process. However, it is still unclear how NO plays a role in the aging process. This paper describes age-related changes in the activity of NADPH-diaphorase (NADPH-d, a marker for neurons containing nitric oxide synthase (NOS, in many CNS regions. Understanding these changes may provide a novel perspective in identifying the aging mechanism.

  7. Recurrent encephalopathy: NAGS (N-acetylglutamate synthase) deficiency in adults.

    Science.gov (United States)

    Cartagena, A; Prasad, A N; Rupar, C A; Strong, M; Tuchman, M; Ah Mew, N; Prasad, C

    2013-01-01

    N-acetyl-glutamate synthase (NAGS) deficiency is a rare autosomal recessive urea cycle disorder (UCD) that uncommonly presents in adulthood. Adult presentations of UCDs include; confusional episodes, neuropsychiatric symptoms and encephalopathy. To date, there have been no detailed neurological descriptions of an adult onset presentation of NAGS deficiency. In this review we examine the clinical presentation and management of UCDs with an emphasis on NAGS deficiency. An illustrative case is provided. Plasma ammonia levels should be measured in all adult patients with unexplained encephalopathy, as treatment can be potentially life-saving. Availability of N-carbamylglutamate (NCG; carglumic acid) has made protein restriction largely unnecessary in treatment regimens currently employed. Genetic counselling remains an essential component of management of NAGS.

  8. Amino acids conferring herbicide resistance in tobacco acetohydroxyacid synthase.

    Science.gov (United States)

    Le, Dung Tien; Choi, Jung-Do; Tran, Lam-Son Phan

    2010-01-01

    Acetohydroxyacid synthase (AHAS) (EC 4.1.3.18) is a target of commercially available herbicides such as sulfonylurea, imidazolinone, and triazolopyrimidine. In plants and microorganisms, AHAS catalyzes the first common reaction in the biosynthesis pathways leading to leucine, isoleucine and valine. Intensive studies using different approaches - including site-directed mutagenesis, molecular modeling and structural analysis - on plant AHAS-s have contributed to the understanding of the herbicide-AHAS interaction. Knowledge of the critical roles of amino acid residues of plant AHAS in conferring herbicide resistance will enable the creation of new herbicide-tolerant AHAS which could be used to develop herbicide-resistant transgenic plants. Moreover, such information will also elucidate design strategies for more efficient herbicides that could also kill weeds resistant to previously used AHAS-inhibiting herbicides. In this review, we summarize the results of intensive searches for amino acid residues and their substitutions that confer herbicide resistance in tobacco AHAS.

  9. A new motif for inhibitors of geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Foust, Benjamin J; Allen, Cheryl; Holstein, Sarah A; Wiemer, David F

    2016-08-15

    The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

  10. Identification of sucrose synthase as an actin-binding protein

    Science.gov (United States)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  11. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering.

    Science.gov (United States)

    Zerbe, Philipp; Bohlmann, Jörg

    2015-07-01

    Plants produce thousands of diterpenoid natural products; some of which are of significant industrial value as biobased pharmaceuticals (taxol), fragrances (sclareol), food additives (steviosides), and commodity chemicals (diterpene resin acids). In nature, diterpene synthase (diTPS) enzymes are essential for generating diverse diterpene hydrocarbon scaffolds. While some diTPSs also form oxygenated compounds, more commonly, oxygenation is achieved by cytochrome P450-dependent mono-oxygenases. Recent genome-, transcriptome-, and metabolome-guided gene discovery and enzyme characterization identified novel diTPS functions that form the core of complex modular pathway systems. Insights into diterpene metabolism may translate into the development of new bioengineered microbial and plant-based production systems.

  12. A novel mutation in CLCN1 associated with feline myotonia congenita.

    Directory of Open Access Journals (Sweden)

    Barbara Gandolfi

    Full Text Available Myotonia congenita (MC is a skeletal muscle channelopathy characterized by inability of the muscle to relax following voluntary contraction. Worldwide population prevalence in humans is 1:100,000. Studies in mice, dogs, humans and goats confirmed myotonia associated with functional defects in chloride channels and mutations in a skeletal muscle chloride channel (CLCN1. CLCN1 encodes for the most abundant chloride channel in the skeletal muscle cell membrane. Five random bred cats from Winnipeg, Canada with MC were examined. All cats had a protruding tongue, limited range of jaw motion and drooling with prominent neck and proximal limb musculature. All cats had blepharospasm upon palpebral reflex testing and a short-strided gait. Electromyograms demonstrated myotonic discharges at a mean frequency of 300 Hz resembling the sound of a 'swarm of bees'. Muscle histopathology showed hypertrophy of all fiber types. Direct sequencing of CLCN1 revealed a mutation disrupting a donor splice site downstream of exon 16 in only the affected cats. In vitro translation of the mutated protein predicted a premature truncation and partial lack of the highly conserved CBS1 (cystathionine β-synthase domain critical for ion transport activity and one dimerization domain pivotal in channel formation. Genetic screening of the Winnipeg random bred population of the cats' origin identified carriers of the mutation. A genetic test for population screening is now available and carrier cats from the feral population can be identified.

  13. Mutations in COX7B cause microphthalmia with linear skin lesions, an unconventional mitochondrial disease.

    Science.gov (United States)

    Indrieri, Alessia; van Rahden, Vanessa Alexandra; Tiranti, Valeria; Morleo, Manuela; Iaconis, Daniela; Tammaro, Roberta; D'Amato, Ilaria; Conte, Ivan; Maystadt, Isabelle; Demuth, Stephanie; Zvulunov, Alex; Kutsche, Kerstin; Zeviani, Massimo; Franco, Brunella

    2012-11-02

    Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possible candidates, we analyzed the X-linked COX7B and found deleterious de novo mutations in two simplex cases and a nonsense mutation, which segregates with the disease, in a familial case. COX7B encodes a poorly characterized structural subunit of cytochrome c oxidase (COX), the MRC complex IV. We demonstrated that COX7B is indispensable for COX assembly, COX activity, and mitochondrial respiration. Downregulation of the COX7B ortholog (cox7B) in medaka (Oryzias latipes) resulted in microcephaly and microphthalmia that recapitulated the MLS phenotype and demonstrated an essential function of complex IV activity in vertebrate CNS development. Our results indicate an evolutionary conserved role of the MRC complexes III and IV for the proper development of the CNS in vertebrates and uncover a group of mitochondrial diseases hallmarked by a developmental phenotype.

  14. X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation.

    Science.gov (United States)

    Sankaran, Vijay G; Ulirsch, Jacob C; Tchaikovskii, Vassili; Ludwig, Leif S; Wakabayashi, Aoi; Kadirvel, Senkottuvelan; Lindsley, R Coleman; Bejar, Rafael; Shi, Jiahai; Lovitch, Scott B; Bishop, David F; Steensma, David P

    2015-04-01

    Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5'-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5'-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.

  15. In vivo mutational analysis of the mupirocin gene cluster reveals labile points in the biosynthetic pathway: the "leaky hosepipe" mechanism.

    Science.gov (United States)

    Wu, Ji'en; Hothersall, Joanne; Mazzetti, Carlo; O'Connell, Yvonne; Shields, Jennifer A; Rahman, Ayesha S; Cox, Russell J; Crosby, John; Simpson, Thomas J; Thomas, Christopher M; Willis, Christine L

    2008-06-16

    A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.

  16. CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations

    Science.gov (United States)

    Scott, John W.; Hawley, Simon A.; Green, Kevin A.; Anis, Miliea; Stewart, Greg; Scullion, Gillian A.; Norman, David G.; Hardie, D. Grahame

    2004-01-01

    CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (γ2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine β-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine β-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives. PMID:14722619

  17. The Cer-cqu gene cluster determines three key players in a β-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes

    Science.gov (United States)

    Schneider, Lizette M; Adamski, Nikolai M; Christensen, Caspar Elo; Stuart, David B; Vautrin, Sonia; Hansson, Mats; Uauy, Cristobal; von Wettstein-Knowles, Penny

    2016-01-01

    Aliphatic compounds on plant surfaces, called epicuticular waxes, are the first line of defense against pathogens and pests, contribute to reducing water loss and determine other important phenotypes. Aliphatics can form crystals affecting light refraction, resulting in a color change and allowing identification of mutants in their synthesis or transport. The present study discloses three such Eceriferum (cer) genes in barley – Cer-c, Cer-q and Cer-u – known to be tightly linked and functioning in a biochemical pathway forming dominating amounts of β-diketone and hydroxy-β-diketones plus some esterified alkan-2-ols. These aliphatics are present in many Triticeae as well as dicotyledons such as Eucalyptus and Dianthus. Recently developed genomic resources and mapping populations in barley defined these genes to a small region on chromosome arm 2HS. Exploiting Cer-c and -u potential functions pinpointed five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase and Cer-u is a P450 enzyme. All were highly expressed in pertinent leaf sheath tissue of wild type. A physical map revealed the order Cer-c, Cer-u, Cer-q with the flanking genes 101kb apart, confirming they are a gene cluster, Cer-cqu. Homology-based modeling suggests that many of the mutant alleles affect overall protein structure or specific active site residues. The rich diversity of identified mutations will facilitate future studies of three key enzymes involved in synthesis of plant apoplast waxes. PMID:26962211

  18. The Cer-cqu gene cluster determines three key players in a β-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes.

    Science.gov (United States)

    Schneider, Lizette M; Adamski, Nikolai M; Christensen, Caspar Elo; Stuart, David B; Vautrin, Sonia; Hansson, Mats; Uauy, Cristobal; von Wettstein-Knowles, Penny

    2016-03-09

    Aliphatic compounds on plant surfaces, called epicuticular waxes, are the first line of defense against pathogens and pests, contribute to reducing water loss and determine other important phenotypes. Aliphatics can form crystals affecting light refraction, resulting in a color change and allowing identification of mutants in their synthesis or transport. The present study discloses three such Eceriferum (cer) genes in barley - Cer-c, Cer-q and Cer-u - known to be tightly linked and functioning in a biochemical pathway forming dominating amounts of β-diketone and hydroxy-β-diketones plus some esterified alkan-2-ols. These aliphatics are present in many Triticeae as well as dicotyledons such as Eucalyptus and Dianthus. Recently developed genomic resources and mapping populations in barley defined these genes to a small region on chromosome arm 2HS. Exploiting Cer-c and -u potential functions pinpointed five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase and Cer-u is a P450 enzyme. All were highly expressed in pertinent leaf sheath tissue of wild type. A physical map revealed the order Cer-c, Cer-u, Cer-q with the flanking genes 101kb apart, confirming they are a gene cluster, Cer-cqu. Homology-based modeling suggests that many of the mutant alleles affect overall protein structure or specific active site residues. The rich diversity of identified mutations will facilitate future studies of three key enzymes involved in synthesis of plant apoplast waxes.

  19. Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase.

    Science.gov (United States)

    Cong, Weitao; Zhao, Ting; Zhu, Zhongxin; Huang, Binbin; Ma, Weide; Wang, Yuehui; Tan, Yi; Chakrabarti, Subrata; Li, Xiaokun; Jin, Litai; Cai, Lu

    2014-04-01

    Mitochondrial ATP production is the main energy source for the cell. Diabetes reduces the efficient generation of ATP, possibly due to the inactivation of ATP synthase. However, the exact mechanism by which diabetes induces inactivation of ATP synthase remains unknown, as well as whether such inactivation has a role in the development of pathological abnormalities of the diabetic heart. To address these issues, we used cardiac metallothionein-transgenic (MT-TG) and wild-type (WT) mice with streptozotocin-induced diabetes, since we have demonstrated previously that diabetes-induced cardiac damage and remodeling were found in WT diabetic mice, but not in MT-TG diabetic mice. Immunohistochemical and biochemical assays were used to compare pathological and biochemical changes of the heart between MT-TG and WT diabetic mice, and a proteomic assay to evaluate ATP synthase expression and tyrosine nitration, with its activity. LC/MS analysis revealed that diabetes increased tyrosine nitration of the ATP synthase α subunit at Tyr(271), Tyr(311), and Tyr(476), and the β subunit at Tyr(269) and Tyr(508), and also significantly reduced ATP synthase activity by ~32%. These changes were not observed in MT-TG diabetic mice. Furthermore, parallel experiments with induced expression of cardiac MT by zinc supplementation in diabetic mice produced similar effects. These results suggest that MT can preserve ATP synthase activity in streptozotocin-induced diabetes, probably through the inhibition of ATP synthase nitration. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Viggiano, J M; Pérez Martínez, S; de Gimeno, M F

    1997-01-01

    In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitation in vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 microM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reaction in vitro and that nitric oxide induces this event.

  1. Cloning and Characterization of Cheilanthifoline and Stylopine Synthase Genes from Chelidonium majus.

    Science.gov (United States)

    Yahyazadeh, Mahdi; Ratmoyo, Purwanto; Bittner, Florian; Sato, Fumihiko; Selmar, Dirk

    2017-06-19

    The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristical benzophenanthridine skeleton. Up to now, any information about the enzymes responsible for its biosynthesis in C. majus and the related genes respectively, is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming cytochrome P450-enzymes involved in the isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast.The activity of the heterologously expressed cytochrome P450-enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that the cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo - under the acidic conditions in the vacuole - this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming cytochrome P450-enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine.Quantitative RT-PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both, in roots and leaves from C. majus, although the alkaloid accumulation pattern in these organs are quite different. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Production and characterisation of monoclonal antibodies to phytoene synthase of Lycopersicon esculentum.

    Science.gov (United States)

    Fraser, P D; Misawa, N; Sandmann, G; Johnson, J; Schuch, W; Bramley, P M

    1998-10-01

    Monoclonal antibodies have been prepared against the tomato (Lycopersicon esculentum Mill.) fruit ripening-enhanced phytoene synthase (PSY1). The antigen was prepared as a beta-galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of E. coli. The fusion protein, induced by IPTG, was purified by preparative SDS-PAGE and used to elicit an immune response. The cell lines were screened for cross-reactivity against beta-galactosidase-phytoene synthase fusion protein in E. coli extracts using western blotting and ELISA detection procedures. Positive clones were further screened for their ability to cross-react with the mature phytoene synthase protein on western blots as well as their ability to inhibit enzyme activity. Eleven monoclonal lines were obtained. Nine of these, all of the IgM isotype, exhibited strong responses to phytoene synthase of ripe tomato fruit on western blots, but did not inhibit enzyme activity effectively. The other two lines (IgG/la 2 isotypes) inhibited phytoene synthase activity in ripe tomato stroma, but produced a poor response to the protein on western blots. The monoclonals identified a ripe fruit phytoene synthase of 38 kDa, exclusively located in the chromoplast. In contrast, antibodies were unable to detect microbial phytoene synthases, nor phytoene synthase of maize leaf, tomato chloroplast or mango fruit extracts, either on western blots or from inhibition of phytoene synthase activity. However, they did cross-react with a 44 kDa protein from carrot leaf stroma and with three different proteins (44, 41, and 37 kDa) in carrot root. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.

  3. The role of 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase gene family in citrus carotenoid accumulation.

    Science.gov (United States)

    Peng, Gang; Wang, Chunyan; Song, Song; Fu, Xiumin; Azam, Muhammad; Grierson, Don; Xu, Changjie

    2013-10-01

    Three 1-deoxy-D-xylulose-5-phosphate synthases (DXS) and three phytoene synthases (PSY) were identified in citrus, from Affymetrix GeneChip Citrus Genome Array, GenBank and public orange genome databases. Tissue-specific expression analysis of these genes was carried out on fruit peel and flesh, flower and leaf of Satsuma mandarin (Citrus unshiu Marc.) in order to determine their roles in carotenoid accumulation in different tissues. Expression of CitDXS1 and CitPSY1 was highest in all test tissues, while that of CitDXS2 and CitPSY2 was lower, and that of CitDXS3 and CitPSY3 undetectable. The transcript profiles of CitDXS1 and CitPSY1 paralleled carotenoid accumulation in flesh of Satsuma mandarin and orange (Citrus sinensis Osbeck) during fruit development, and CitPSY1 expression was also associated with carotenoid accumulation in peel, while the CitDXS1 transcript level was only weakly correlated with carotenoid accumulation in peel. Similar results were obtained following correlation analysis between expression of CitDXS1 and CitPSY1 and carotenoid accumulation in peel and flesh of 16 citrus cultivars. These findings identify CitPSY1 and CitDXS1 as the main gene members controlling carotenoid biosynthesis in citrus fruit. Furthermore, chromoplasts were extracted from flesh tissue of these citrus, and chromoplasts of different shape (spindle or globular), different size, and color depth were observed in different cultivars, indicating chromoplast abundance, number per gram tissue, size and color depth were closely correlated with carotenoid content in most cultivars. The relationship between carotenoid biosynthesis and chromoplast development was discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  4. Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury

    Institute of Scientific and Technical Information of China (English)

    Guang-Jin Yuan; Xiao-Rong Zhou; Zuo-Jiong Gong; Pin Zhang; Xiao-Mei Sun; Shi-Hua Zheng

    2006-01-01

    AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-KB (NF-кB) and tumor necrosis factor-α (TNF-α)expression in the liver.METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT)activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-KB p65, iNOS, eNOS and TNF-αprotein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR).RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-кB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-кB, and TNF-α mRNA expression.CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-KB and TNF-αexpression. eNOS activity is reduced, but its mRNA expression is not affected.

  5. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms...... competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure....

  6. Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Lindebjerg, Jan; Crüger, Dorthe G.;

    2008-01-01

    The possible associations between microsatellite instability, homocysteine and thymidylate synthase were investigated in tumors and plasma from 130 patients with colorectal cancer. Other analyses included thymidylate synthase and 5,10-methylene-tetrahydrofolate reductase gene polymorphisms......, carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p ... factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors....

  7. Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold.

    Science.gov (United States)

    Kaya, Yusuf; Del Campo, Mark; Ofengand, James; Malhotra, Arun

    2004-04-30

    TruD, a recently discovered novel pseudouridine synthase in Escherichia coli, is responsible for modifying uridine13 in tRNA(Glu) to pseudouridine. It has little sequence homology with the other 10 pseudouridine synthases in E. coli which themselves have been grouped into four related protein families. Crystal structure determination of TruD revealed a two domain structure consisting of a catalytic domain that differs in sequence but is structurally very similar to the catalytic domain of other pseudouridine synthases and a second large domain (149 amino acids, 43% of total) with a novel alpha/beta fold that up to now has not been found in any other protein.

  8. ESR-spektroskopische Untersuchungen der F0F1-ATP-Synthase aus Escherichia coli

    OpenAIRE

    Motz, Christian

    1999-01-01

    Die FoF1-ATP-Synthase katalysiert die Synthese von ATP aus ADP und Pi bei der oxidativen bzw. Photophosphorylierung. Der ATP-Synthase-Komplex läßt sich in zwei funktionelle Einheiten unterteilen: Fo ist ein integraler Membranproteinkomplex, der den Protonenkanal bildet. F1 hingegen ist ein wasserlöslicher Proteinkomplex, der die Nukleotidbindungsstellen trägt. Die ATP-Synthase aus Escherichia coli hat die Zusammensetzung alpha3beta3gamma delta epsilon für die F1 und ab2c9-12 für den Fo-Teil. ...

  9. Identification of Mutations in TMEM5 and ISPD as a Cause of Severe Cobblestone Lissencephaly

    Science.gov (United States)

    Vuillaumier-Barrot, Sandrine; Bouchet-Séraphin, Céline; Chelbi, Malika; Devisme, Louise; Quentin, Samuel; Gazal, Steven; Laquerrière, Annie; Fallet-Bianco, Catherine; Loget, Philippe; Odent, Sylvie; Carles, Dominique; Bazin, Anne; Aziza, Jacqueline; Clemenson, Alix; Guimiot, Fabien; Bonnière, Maryse; Monnot, Sophie; Bole-Feysot, Christine; Bernard, Jean-Pierre; Loeuillet, Laurence; Gonzales, Marie; Socha, Koryna; Grandchamp, Bernard; Attié-Bitach, Tania; Encha-Razavi, Férechté; Seta, Nathalie

    2012-01-01

    Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a “cobblestone” brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies. PMID:23217329

  10. Three-factor reciprocal cross mapping of a gene that causes expression of feedback-resistant acetohydroxy acid synthase in Escherichia coli K-12.

    Science.gov (United States)

    Jackson, J H; Davis, E J; Madu, A C; Braxter, S E

    1981-01-01

    The ilv-662 allele was previously identified as a mutation that caused acetohydroxy acid synthase activity to be resistant to feedback inhibition by valine (Davis et al. 1977). This allele was mapped between thr and leu by cotransduction analysis and labeled ilvJ. This report describes the mapping of ilvJ relative to genes that lie between thr and leu (ara, carA and pdxA) by three factor reciprocal cross analyses. We find that the probable gene order is thr-carA-pdxA-ilvJ-ara-leu. Although the phenotypic properties of ilvJ662 appear to be quite distinct from brnS, a gene reported to involve branched chain amino acid transport (Guardiola et al. 1974), we do not rule out possible allelism because of the uncertainty of the map position of brnS.

  11. Common Β- Thalassaemia Mutations in

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    P Azarfam

    2005-01-01

    Full Text Available Introduction: β –Thalassaemia was first explained by Thomas Cooly as Cooly’s anaemia in 1925. The β- thalassaemias are hereditary autosomal disorders with decreased or absent β-globin chain synthesis. The most common genetic defects in β-thalassaemias are caused by point mutations, micro deletions or insertions within the β-globin gene. Material and Methods: In this research , 142 blood samples (64 from childrens hospital of Tabriz , 15 samples from Shahid Gazi hospital of Tabriz , 18 from Urumia and 45 samples from Aliasghar hospital of Ardebil were taken from thalassaemic patients (who were previously diagnosed .Then 117 non-familial samples were selected . The DNA of the lymphocytes of blood samples was extracted by boiling and Proteinase K- SDS procedure, and mutations were detected by ARMS-PCR methods. Results: From the results obtained, eleven most common mutations,most of which were Mediterranean mutations were detected as follows; IVS-I-110(G-A, IVS-I-1(G-A ،IVS-I-5(G-C ,Frameshift Codon 44 (-C,( codon5(-CT,IVS-1-6(T-C, IVS-I-25(-25bp del ,Frameshift 8.9 (+G ,IVS-II-1(G-A ,Codon 39(C-T, Codon 30(G-C the mutations of the samples were defined. The results showed that Frameshift 8.9 (+G, IVS-I-110 (G-A ,IVS-II-I(G-A, IVS-I-5(G-C, IVS-I-1(G-A , Frameshift Codon 44(-C , codon5(-CT , IVS-1-6(T-C , IVS-I-25(-25bp del with a frequency of 29.9%, 25.47%,17.83%, 7.00%, 6.36% , 6.63% , 3.8% , 2.5% , 0.63% represented the most common mutations in North - west Iran. No mutations in Codon 39(C-T and Codon 30(G-C were detected. Cunclusion: The frequency of the same mutations in patients from North - West of Iran seems to be different as compared to other regions like Turkey, Pakistan, Lebanon and Fars province of Iran. The pattern of mutations in this region is more or less the same as in the Mediterranean region, but different from South west Asia and East Asia.

  12. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

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    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  13. [Founder mutation in Lynch syndrome].

    Science.gov (United States)

    Cajal, Andrea R; Piñero, Tamara A; Verzura, Alicia; Santino, Juan Pablo; Solano, Angela R; Kalfayan, Pablo G; Ferro, Alejandra; Vaccaro, Carlos

    2016-01-01

    Lynch syndrome is the most frequent syndrome in hereditary colorectal cancer, a family-specific deleterious mutations in genes encoding DNA reparation proteins: MLH1 (mutL homolog 1), MSH2, MSH6 (mutS homolog 2 y 6, respectively), PMS2 (PMS1 homolog 2, mismatch repair system component) y MUTYH (mutY DNA glycosylase). The c.2252_2253delAA, p.Lys751Serfs*3 mutation in MLH1 gene segregates with a haplotype reported in the northern region of Italy and whose origin was attributed to a founder effect. This mutation co-segregates with typical characteristics of Lynch syndrome, including early age at onset and multiple primary tumors in the same individual, a high frequency of pancreatic cancer, high microsatellite instability and lack of PMS2 expression. This report describes a mutation in an Argentinian patient with endometrioid adenocarcinoma of uterus. Her first-degree relatives had a history of colon cancer diagnosed before 50 years, fulfilling the Amsterdam Criteria I and Lynch syndrome II. The high pathogenicity associated to this mutation makes necessary the study of all members from families with hereditary cancer, allowing pre-symptomatic genetic diagnosis, early assessment and the instauration of preventive treatments.

  14. SQSTM1 Mutations and Glaucoma.

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    Todd E Scheetz

    Full Text Available Glaucoma is the most common cause of irreversible blindness worldwide. One subset of glaucoma, normal tension glaucoma (NTG occurs in the absence of high intraocular pressure. Mutations in two genes, optineurin (OPTN and TANK binding kinase 1 (TBK1, cause familial NTG and have known roles in the catabolic cellular process autophagy. TKB1 encodes a kinase that phosphorylates OPTN, an autophagy receptor, which ultimately activates autophagy. The sequestosome (SQSTM1 gene also encodes an autophagy receptor and also is a target of TBK1 phosphorylation. Consequently, we hypothesized that mutations in SQSTM1 may also cause NTG. We tested this hypothesis by searching for glaucoma-causing mutations in a cohort of NTG patients (n = 308 and matched controls (n = 157 using Sanger sequencing. An additional 1098 population control samples were also analyzed using whole exome sequencing. A total of 17 non-synonymous mutations were detected which were not significantly skewed between cases and controls when analyzed separately, or as a group (p > 0.05. These data suggest that SQSTM1 mutations are not a common cause of NTG.

  15. Driven by Mutations: The Predictive Value of Mutation Subtype in EGFR-Mutated Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Castellanos, Emily; Feld, Emily; Horn, Leora

    2016-12-23

    EGFR-mutated NSCLC is a genetically heterogeneous disease that includes more than 200 distinct mutations. The implications of mutational subtype for both prognostic and predictive value are being increasingly understood. Although the most common EGFR mutations-exon 19 deletions or L858R mutations-predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs), it is now being recognized that outcomes may be improved in patients with exon 19 deletions. Additionally, 10% of patients will have an uncommon EGFR mutation, and response to EGFR TKI therapy is highly variable depending on the mutation. Given the growing recognition of the genetic and clinical variation seen in this disease, the development of comprehensive bioinformatics-driven tools to both analyze response in uncommon mutation subtypes and inform clinical decision making will be increasingly important. Clinical trials of novel EGFR TKIs should prospectively account for the presence of uncommon mutation subtypes in study design.

  16. Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase.

    Science.gov (United States)

    Hannibal, Luciana; Somasundaram, Ramasamy; Tejero, Jesús; Wilson, Adjele; Stuehr, Dennis J

    2011-11-11

    Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B. subtilis NOS to investigate how heme-thiolate electronic properties control enzyme catalysis. The mutations had opposite effects on heme midpoint potential (-302, -361, and -427 mV for W66H, wild-type (WT), and W66F, respectively). These changes were associated with rank order (W66H < WT < W66F) changes in the rates of oxygen activation and product formation in Arg hydroxylation and N-hydroxyarginine (NOHA) oxidation single turnover reactions, and in the O(2) reactivity of the ferrous heme-NO product complex. However, enzyme ferrous heme-O(2) autoxidation showed an opposite rank order. Tetrahydrofolate supported NO synthesis by WT and the mutant NOS. All three proteins showed similar extents of product formation (L-Arg → NOHA or NOHA → citrulline) in single turnover studies, but the W66F mutant showed a 2.5 times lower activity when the reactions were supported by flavoproteins and NADPH. We conclude that Trp(66) controls several catalytic parameters by tuning the electron density of the heme-thiolate bond. A greater electron density (as in W66F) improves oxygen activation and reactivity toward substrate, but decreases heme-dioxy stability and lowers the driving force for heme reduction. In the WT enzyme the Trp(66) residue balances these opposing effects for optimal catalysis.

  17. Rescue of cystathionine beta-synthase (CBS) mutants with chemical chaperones: purification and characterization of eight CBS mutant enzymes.

    Science.gov (United States)

    Majtan, Tomas; Liu, Lu; Carpenter, John F; Kraus, Jan P

    2010-05-21

    Missense mutations represent the most common cause of many genetic diseases including cystathionine beta-synthase (CBS) deficiency. Many of these mutations result in misfolded proteins, which lack biological function. The presence of chemical chaperones can sometimes alleviate or even restore protein folding and activity of mutant proteins. We present the purification and characterization of eight CBS mutants expressed in the presence of chemical chaperones such as ethanol, dimethyl sulfoxide, or trimethylamine-N-oxide. Preliminary screening in Escherichia coli crude extracts showed that their presence during protein expression had a significant impact on the amount of recovered CBS protein, formation of tetramers, and catalytic activity. Subsequently, we purified eight CBS mutants to homogeneity (P49L, P78R, A114V, R125Q, E176K, P422L, I435T, and S466L). The tetrameric mutant enzymes fully saturated with heme had the same or higher specific activities than wild type CBS. Thermal stability measurements demonstrated that the purified mutants are equally or more thermostable than wild type CBS. The response to S-adenosyl-L-methionine stimulation or thermal activation varied. The lack of response of R125Q and E176K to both stimuli indicated that their specific conformations were unable to reach the activated state. Increased levels of molecular chaperones in crude extracts, particularly DnaJ, indicated a rather indirect effect of the chemical chaperones on folding of CBS mutants. In conclusion, the chemical chaperones present in the expression medium were able to fully restore the activity of eight CBS mutants by improving their protein folding. This finding could have direct implications for the development of a therapeutical approach to pyridoxine unresponsive homocystinuria.

  18. Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

    Science.gov (United States)

    Chevreux, Guillaume; Atmanene, Cédric; Lopez, Philippe; Ouazzani, Jamal; Van Dorsselaer, Alain; Badet, Bernard; Badet-Denisot, Marie-Ange; Sanglier-Cianférani, Sarah

    2011-03-01

    Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using 14N versus 15N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.

  19. Probing the Mechanism of the Mycobacterium tuberculosis [beta]-Ketoacyl-Acyl Carrier Protein Synthase III mtFabH: Factors Influencing Catalysis and Substrate Specificity

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    Brown, Alistair K.; Sridharan, Sudharsan; Kremer, Laurent; Lindenberg, Sandra; Dover, Lynn G.; Sacchettini, James C.; Besra, Gurdyal S. (TAM); (Birmingham); (CNRS)

    2010-11-30

    Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These {alpha}-alkyl, {beta}-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C{sub 24}-C{sub 26} fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The {beta}-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg{sup 46} revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg{sup 161} {yields} Ala substitution. Our structural studies suggested that His{sup 258}, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys{sup 122}.

  20. Rapid generation of hypomorphic mutations

    Science.gov (United States)

    Arthur, Laura L.; Chung, Joyce J.; Jankirama, Preetam; Keefer, Kathryn M.; Kolotilin, Igor; Pavlovic-Djuranovic, Slavica; Chalker, Douglas L.; Grbic, Vojislava; Green, Rachel; Menassa, Rima; True, Heather L.; Skeath, James B.; Djuranovic, Sergej

    2017-01-01

    Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels. PMID:28106166

  1. Neutral Evolution of Mutational Robustness

    CERN Document Server

    Van Nimwegen, E; Huynen, M; Nimwegen, Erik van; Crutchfield, James P.; Huynen, Martijn

    1999-01-01

    We introduce and analyze a general model of a population evolving over a network of selectively neutral genotypes. We show that the population's limit distribution on the neutral network is solely determined by the network topology and given by the principal eigenvector of the network's adjacency matrix. Moreover, the average number of neutral mutant neighbors per individual is given by the matrix spectral radius. This quantifies the extent to which populations evolve mutational robustness: the insensitivity of the phenotype to mutations. Since the average neutrality is independent of evolutionary parameters---such as, mutation rate, population size, and selective advantage---one can infer global statistics of neutral network topology using simple population data available from {\\it in vitro} or {\\it in vivo} evolution. Populations evolving on neutral networks of RNA secondary structures show excellent agreement with our theoretical predictions.

  2. Mutations in ANKH cause chondrocalcinosis.

    Science.gov (United States)

    Pendleton, Adrian; Johnson, Michelle D; Hughes, Anne; Gurley, Kyle A; Ho, Andrew M; Doherty, Michael; Dixey, Josh; Gillet, Pierre; Loeuille, Damien; McGrath, Rodney; Reginato, Antonio; Shiang, Rita; Wright, Gary; Netter, Patrick; Williams, Charlene; Kingsley, David M

    2002-10-01

    Chondrocalcinosis (CC) is a common cause of joint pain and arthritis that is caused by the deposition of calcium-containing crystals within articular cartilage. Although most cases are sporadic, rare familial forms have been linked to human chromosomes 8 (CCAL1) or 5p (CCAL2) (Baldwin et al. 1995; Hughes et al. 1995; Andrew et al. 1999). Here, we show that two previously described families with CCAL2 have mutations in the human homolog of the mouse progressive ankylosis gene (ANKH). One of the human mutations results in the substitution of a highly conserved amino acid residue within a predicted transmembrane segment. The other creates a new ATG start site that adds four additional residues to the ANKH protein. Both mutations segregate completely with disease status and are not found in control subjects. In addition, 1 of 95 U.K. patients with sporadic CC showed a deletion of a single codon in the ANKH gene. The same change was found in a sister who had bilateral knee replacement for osteoarthritis. Each of the three human mutations was reconstructed in a full-length ANK expression construct previously shown to regulate pyrophosphate levels in cultured cells in vitro. All three of the human mutations showed significantly more activity than a previously described nonsense mutation that causes severe hydroxyapatite mineral deposition and widespread joint ankylosis in mice. These results suggest that small sequence changes in ANKH are one cause of CC and joint disease in humans. Increased ANK activity may explain the different types of crystals commonly deposited in human CCAL2 families and mutant mice and may provide a useful pharmacological target for treating some forms of human CC.

  3. Solanapyrone synthase, a possible Diels-Alderase and iterative type I polyketide synthase encoded in a biosynthetic gene cluster from Alternaria solani.

    Science.gov (United States)

    Kasahara, Ken; Miyamoto, Takanori; Fujimoto, Takashi; Oguri, Hiroki; Tokiwano, Tetsuo; Oikawa, Hideaki; Ebizuka, Yutaka; Fujii, Isao

    2010-06-14

    The solanapyrone biosynthetic gene cluster was cloned from Alternaria solani. It consists of six genes-sol1-6-coding for a polyketide synthase, an O-methyltransferase, a dehydrogenase, a transcription factor, a flavin-dependent oxidase, and cytochrome P450. The prosolanapyrone synthase (PSS) encoded by sol1 was expressed in Aspergillus oryzae and its product was identified as desmethylprosolanapyrone I (8). Although PSS is closely related to the PKSs/Diels-Alderases LovB and MlcA of lovastatin and compactin biosynthesis, it did not catalyze cycloaddition. Sol5, encoding a flavin-dependent oxidase (solanapyrone synthase, SPS), was expressed in Pichia pastoris and purified. The purified recombinant SPS showed activity for the formation of (-)-solanapyrone A (1) from achiral prosolanapyrone II (2), establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels-Alder enzyme.

  4. Mutations determining mitomycin resistance in Bacillus subtilis.

    Science.gov (United States)

    Iyer, V N

    1966-12-01

    Iyer, V. N. (Microbiology Research Institute, Canada Department of Agriculture, Ottawa, Canada). Mutations determining mitomycin resistance in Bacillus subtilis. J. Bacteriol. 92:1663-1669. 1966.-The pattern of development of genetic resistance in Bacillus subtilis to mitomycin C was studied, and spontaneous single and multistep mutants were obtained. The transmission and expression of these mutations in sensitive strains proved possible by means of genetic transformation. The mutations were genetically studied in relation to a chromosomal mutation, mac-1, which confers resistance to the macrolide antibiotic erythromycin and which has been previously localized in the early-replicating segment of the B. subtilis chromosome. The results indicate that all of three primary mutations studied in this manner, as well as a secondary and tertiary mutation derived from one of the primary mutations, are clustered in this early-replicating segment. It appears that the secondary and tertiary mutations enhance the resistance conferred by the primary mutation, apparently without themselves conferring any resistance.

  5. Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content1[OPEN

    Science.gov (United States)

    2017-01-01

    Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production. PMID:27940492

  6. Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

    Science.gov (United States)

    Newton, Derek C; Bevan, Sian C; Choi, Stephen; Robb, G Brett; Millar, Adam; Wang, Yang; Marsden, Philip A

    2003-01-03

    Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

  7. Hydrogen sulfide generation in mammals: the molecular biology of cystathionine-β- synthase (CBS) and cystathionine-γ-lyase (CSE).

    Science.gov (United States)

    Renga, Barbara

    2011-04-01

    Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) are two key enzymes involved in the synthesis of hydrogen sulphide (H(2)S). CBS catalyzes the pyridoxal 5'-phosphate (PLP)-dependent conversion of homocysteine in Cystathionine whilst CSE the pyridoxal 5'-phosphate (PLP)-dependent synthesis of L-cysteine from Cystathionine. In mammals, CBS gene transcription is poorly investigated and the activity of the enzyme is highly regulated. In fact, the CBS enzyme contains a heme cofactor that functions as a redox sensor and utilizes S-adenosylmethionine (SAM) as an allosteric activator. Impaired CBS activity causes hyperhomocystinuria and hyperhomocysteinemia, both risk factors for cardiovascular diseases. Murine CSE gene regulation is well characterized but little is known about the human counterpart and there is no information regarding the enzyme activity regulation. Recently it has been demonstrated that CSE transcription is regulated by the nuclear receptor Farnesoid X Receptor (FXR). Mutations that decrease the activity of CSE cause cystathioninuria, hypercystathioninemia and increase the risk of developing atherosclerosis and bladder cancer. This review focuses on the recent aspects of the molecular regulation of both CBS and CSE and highlights the possibility that members of the nuclear receptors superfamily might be involved in the regulation of hydrogen sulphide metabolism.

  8. Crystal structures of human HMG-CoA synthase isoforms provide insights into inherited ketogenesis disorders and inhibitor design.

    Science.gov (United States)

    Shafqat, Naeem; Turnbull, Andrew; Zschocke, Johannes; Oppermann, Udo; Yue, Wyatt W

    2010-05-14

    3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.

  9. Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-β precursor protein.

    Science.gov (United States)

    Parr, Callum; Carzaniga, Raffaela; Gentleman, Steve M; Van Leuven, Fred; Walter, Jochen; Sastre, Magdalena

    2012-11-01

    Alzheimer's disease (AD) has been associated with altered activity of glycogen synthase kinase 3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. However, the molecular basis by which GSK3 affects the formation of Aβ remains unknown. Our aim was to identify the underlying mechanisms of GSK3-dependent effects on the processing of amyloid precursor protein (APP). For this purpose, N2a cells stably expressing APP carrying the Swedish mutation were treated with specific GSK3 inhibitors or transfected with GSK3α/β short interfering RNA. We show that inhibition of GSK3 leads to decreased expression of APP by enhancing its degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Our data indicate that GSK3 inhibition reduces Aβ through an increase of the degradation of APP and its carboxy-terminal fragment (CTF) by activation of the lysosomal/autophagy pathway. These results suggest that an increased propensity toward autophagic/lysosomal alterations in AD patients could have consequences for neuronal function.

  10. Less is more: Independent loss-of-function OCIMENE SYNTHASE alleles parallel pollination syndrome diversification in monkeyflowers (Mimulus).

    Science.gov (United States)

    Peng, Foen; Byers, Kelsey J R P; Bradshaw, Harvey D

    2017-07-19

    Pollinator-mediated selection on flower phenotypes (e.g., shape, color, scent) is key to understanding the adaptive radiation of angiosperms, many of which have evolved specialized relationships with a particular guild of animal pollinators (e.g., birds, bats, moths, bees). E-β-Ocimene, a monoterpene produced by OCIMENE SYNTHASE (OS) in Mimulus lewisii, is a floral scent important in attracting the species' bumblebee pollinators. The taxa closely related to M. lewisii have evolved several different pollination syndromes, including hummingbird pollination and self pollination (autogamy). We are interested in how floral scent variation contributed to species diversification in this clade. We analyzed variation in E-β-ocimene emission within this Mimulus clade and explored its molecular basis through a combination of DNA sequencing, reverse transcriptase PCR, and enzyme functional analysis in vitro. We found that none of the taxa, other than M. lewisii, emitted E-β-ocimene from flowers. But the molecular basis underlying loss of E-β-ocimene emission is unique in each taxon, including deletion, missense, or frameshift mutations in the OS gene, and potential posttranscriptional downregulation. The molecular evidence suggests that parallel loss-of-function in OS is the best explanation for the observed pattern of E-β-ocimene emission, likely as the result of natural selection. © 2017 Botanical Society of America.

  11. Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.

    Science.gov (United States)

    Shevchuk, Olga; Pägelow, Dennis; Rasch, Janine; Döhrmann, Simon; Günther, Gabriele; Hoppe, Julia; Ünal, Can Murat; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel; Steinert, Michael

    2014-11-01

    L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.

  12. Regulation of expression of citrate synthase by the retinoic acid receptor-related orphan receptor α (RORα.

    Directory of Open Access Journals (Sweden)

    Christine Crumbley

    Full Text Available The retinoic acid receptor-related orphan receptor α (RORα is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression.

  13. Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

    2001-01-01

    beta -Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamy-beta -alanine as the sole nitrogen source and exhibits diminished beta -alanine synthase...... no pyrimidine catabolic pathway, it enabled growth on N-carbamyl- beta -alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta -alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial...... N- carbamyl amidohydrolases. All three beta -alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta...

  14. Lack of inhibition of endothelial nitric oxide synthase in the isolated rat aorta by doxorubicin.

    NARCIS (Netherlands)

    Hartog, den GJ; Boots, AW; Haenen, GR; Vijgh, van der W.J.F.

    2003-01-01

    Besides inducing cardiotoxicity, doxorubicin also affects the vasculature. Recent observations in cultured endothelial cells indicated that the endothelial form of nitric oxide synthase might be inhibited by doxorubicin thereby seriously interfering with vascular function. We have investigated the

  15. Prostaglandin-endoperoxide H synthase-2 expression and activity increases with term labor in human chorion

    National Research Council Canada - National Science Library

    Mijovic, J E; Zakar, T; Nairn, T K; Olson, D M

    1997-01-01

    We investigated the changes in prostaglandin-endoperoxide H synthase (PGHS) specific activity and the levels and distribution of PGHS-1 and PGHS-2 mRNA in chorion collected at term before the onset of labor (CS...

  16. The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng; Garavito, R. Michael (MSU); (NWU)

    2014-10-02

    Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.

  17. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

    Science.gov (United States)

    Schrepfer, Patrick; Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-02-23

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

  18. SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

    Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

  19. Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

    NARCIS (Netherlands)

    Douma, Anneke C.; Veenhuis, Marten; de Koning, Willem; Evers, Melchior; Harder, Willem

    1985-01-01

    The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared

  20. EPSP合成酶的纯化与制备%Purification and Preparation of EPSP Synthase

    Institute of Scientific and Technical Information of China (English)

    向文胜; 王相晶; 覃兆海; 任天瑞; 张雅莉; 张文吉; 苏少泉

    2000-01-01

    The rapid purification(less than 1.5 h) of EPSP synthase from bean seedling by S ephadex G-50 and Mono-Q chromtography was reported. Specific activity of E PSP synthase obtained by the method was 175.2 nmol.min-1.mg-1.Conc entrated enzyme solution after adjusting to 50% glycerol(V/V) and 1mg.mL -1BSA, was stored at -20℃. EPSP synthase activity was stable at least f or 150 days.The activity of EPSP synthase was inhibited approximately 50% by 6.3 μmol.L-1 glyphosate. It showed that the purified EPSP synth ase as herbicide screening model is possible. This purified method has been used to study enzyme mechanism of the glyphosate resistant bean.

  1. Transcriptional Modulation of Squalene Synthase Genes in Barley Treated with PGPR

    OpenAIRE

    Anam eYousaf; Abdul eQadir; Tehmina eAnjum; Aqeel eAhmad

    2015-01-01

    Phytosterol contents and food quality of plant produce is directly associated with transcription of gene Squalene Synthase (SS). In current study, barley plants were treated with different rhizobacterial strains under semi controlled (27±3°C) greenhouse conditions in order to modulate expression of SS gene. Plant samples were analysed through semi-quantitative PCR to evaluate effect of rhizobacterial application on transcriptional status of squalene synthase. Results revealed that among four ...

  2. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

  3. Synthèse bibliographique: la divinyl éther synthase de plantes

    OpenAIRE

    Fauconnier M.L.; Hoyaux P.; Delcarte J.; Marlier M.; du Jardin P.

    2001-01-01

    Divinyl ether synthase in plants: a review. Divinyl ether synthase, an enzyme of the lipoxygenase pathway transforms, in potato tubers, 9-hydroperoxides of fatty acids into colneleic and colnelenic acid, two divinyl ethers of fatty acids. The enzyme has been described in a limited number of quite different plants. The enzyme has also been detected in tomato roots, garlic bulbs, tobacco plants and in marine algae. The enzyme is bound to membranes and is located in the microsomal fraction. The ...

  4. The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

    Science.gov (United States)

    Riga, Maria; Bajda, Sabina; Themistokleous, Christos; Papadaki, Stavrini; Palzewicz, Maria; Dermauw, Wannes; Vontas, John; Leeuwen, Thomas Van

    2017-08-23

    The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

  5. Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, Phytophthora infestans.

    Science.gov (United States)

    Blum, Mathias; Boehler, Martine; Randall, Eva; Young, Vanessa; Csukai, Michael; Kraus, Sabrina; Moulin, Florence; Scalliet, Gabriel; Avrova, Anna O; Whisson, Stephen C; Fonne-Pfister, Raymonde

    2010-03-01

    Oomycete plant pathogens cause a wide variety of economically and environmentally important plant diseases. Mandipropamid (MPD) is a carboxylic acid amide (CAA) effective against downy mildews, such as Plasmopara viticola on grapes and potato late blight caused by Phytophthora infestans. Historically, the identification of the mode of action of oomycete-specific control agents has been problematic. Here, we describe how a combination of biochemical and genetic techniques has been utilized to identify the molecular target of MPD in P. infestans. Phytophthora infestans germinating cysts treated with MPD produced swelling symptoms typical of cell wall synthesis inhibitors, and these effects were reversible after washing with H(2)O. Uptake studies with (14)C-labelled MPD showed that this oomycete control agent acts on the cell wall and does not enter the cell. Furthermore, (14)C glucose incorporation into cellulose was perturbed in the presence of MPD which, taken together, suggests that the inhibition of cellulose synthesis is the primary effect of MPD. Laboratory mutants, insensitive to MPD, were raised by ethyl methane sulphonate (EMS) mutagenesis, and gene sequence analysis of cellulose synthase genes in these mutants revealed two point mutations in the PiCesA3 gene, known to be involved in cellulose synthesis. Both mutations in the PiCesA3 gene result in a change to the same amino acid (glycine-1105) in the protein. The transformation and expression of a mutated PiCesA3 allele was carried out in a sensitive wild-type isolate to demonstrate that the mutations in PiCesA3 were responsible for the MPD insensitivity phenotype.

  6. Molecular dynamics analysis of the structural and dynamic properties of the functionally enhanced hepta-variant of mouse 5-aminolevulinate synthase.

    Science.gov (United States)

    Na, Insung; DeForte, Shelly; Stojanovski, Bosko M; Ferreira, Gloria C; Uversky, Vladimir N

    2017-01-09

    Heme biosynthesis, a complex, multistage, and tightly controlled process, starts with 5-aminolevulinate (ALA) production, which, in metazoa and certain bacteria, is a reaction catalyzed by 5-aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme. Functional aberrations in ALAS are associated with several human diseases. ALAS can adopt open and closed conformations, with segmental rearrangements of a C-terminal, 16-amino acid loop and an α-helix regulating accessibility to the ALAS active site. Of the murine erythroid ALAS (mALAS2) forms previously engineered to assess the role of the flexible C-terminal loop versus mALAS2 function one stood out due to its impressive gain in catalytic power. To elucidate how the simultaneously introduced seven mutations of this activity-enhanced variant affected structural and dynamic properties of mALAS2, we conducted extensive molecular dynamics simulation analysis of the dimeric forms of wild-type mALAS2, hepta-variant and Rhodobacter capsulatus ALAS (aka R. capsulatus HemA). This analysis revealed that the seven simultaneous mutations in the C-terminal loop, which extends over the active site of the enzyme, caused the bacterial and murine proteins to adopt different conformations. Specifically, a new β-strand in the mutated 'loop' led to interaction with two preexisting β-strands and formation of an anti-parallel three-stranded β-sheet, which likely endowed the murine hepta-variant a more 'stable' open conformation than that of wild-type mALAS2, consistent with a kinetic mechanism involving a faster closed-to-open conformation transition and product release for the mutated than wild-type enzyme. Further, the dynamic behavior of the mALAS2 protomers was strikingly different in the two dimeric forms.

  7. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva;

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and....... The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases....

  8. Loss of ceramide synthase 3 causes lethal skin barrier disruption.

    Science.gov (United States)

    Jennemann, Richard; Rabionet, Mariona; Gorgas, Karin; Epstein, Sharon; Dalpke, Alexander; Rothermel, Ulrike; Bayerle, Aline; van der Hoeven, Franciscus; Imgrund, Silke; Kirsch, Joachim; Nickel, Walter; Willecke, Klaus; Riezman, Howard; Gröne, Hermann-Josef; Sandhoff, Roger

    2012-02-01

    The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1-6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.

  9. Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

    Directory of Open Access Journals (Sweden)

    Kao Ming-Ching

    2011-02-01

    Full Text Available Abstract Background Hyperbaric oxygen therapy (HBOT is a known adjuvant for treating ischemia-related inner ear diseases. Controversies still exist in the role of HBOT in cochlear diseases. Few studies to date have investigated the cellular changes that occur in inner ears after HBOT. Nitric oxide, which is synthesized by nitric oxide synthase (NOS, is an important signaling molecule in cochlear physiology and pathology. Here we investigated the effects of hyperbaric oxygen on eardrum morphology, cochlear function and expression of NOS isoforms in cochlear substructures after repetitive HBOT in guinea pigs. Results Minor changes in the eardrum were observed after repetitive HBOT, which did not result in a significant hearing threshold shift by tone burst auditory brainstem responses. A differential effect of HBOT on the expression of NOS isoforms was identified. Upregulation of constitutive NOS (nNOS and eNOS was found in the substructures of the cochlea after HBOT, but inducible NOS was not found in normal or HBOT animals, as shown by immunohistochemistry. There was no obvious DNA fragmentation present in this HBOT animal model. Conclusions The present evidence indicates that the customary HBOT protocol may increase constitutive NOS expression but such upregulation did not cause cell death in the treated cochlea. The cochlear morphology and auditory function are consequently not changed through the protocol.

  10. Adenovirus-mediated nitric oxide synthase gene transfer.

    Science.gov (United States)

    Raman, Kathleen G; Shapiro, Richard A; Tzeng, Edith; Kibbe, Melina R

    2004-01-01

    The varied biological effects of nitric oxide (NO) have led to intense research into its diverse physiologic and pathophysiologic roles in multiple disease processes. It has been implicated in the development of altered vasomotor tone, intimal hyperplasia, atherosclerosis, impotence, host defense, and wound healing. Using the modern technologies of recombinant DNA and gene transfer using adenoviral vectors, the effects of NO derived from various NO synthase (NOS) enzymes can be studied in a variety of tissues and the therapeutic applications of NOS is possible. Such uses of NOS gene transfer have been investigated extensively in the vasculature where NO is critical to regulating vascular homeostasis. NOS gene therapy has the theoretical advantage of allowing NO delivery to be localized, thereby limiting potential adverse effects of NO. The benefits of adenoviral vectors in gene transfer include relatively high transduction efficiencies, both replicating and nonreplicating cells may be infected, and the high titers of adenovirus that can be produced. The methods described in this chapter include the cloning of the iNOS cDNA into a recombinant adenoviral vector, large-scale production of that vector AdiNOS preparation, and the use of the vector to transduce tissue in vitro and in vivo.

  11. ASMPKS: an analysis system for modular polyketide synthases

    Directory of Open Access Journals (Sweden)

    Kong Eun-Bae

    2007-09-01

    Full Text Available Abstract Background Polyketides are secondary metabolites of microorganisms with diverse biological activities, including pharmacological functions such as antibiotic, antitumor and agrochemical properties. Polyketides are synthesized by serialized reactions of a set of enzymes called polyketide synthase(PKSs, which coordinate the elongation of carbon skeletons by the stepwise condensation of short carbon precursors. Due to their importance as drugs, the volume of data on polyketides is rapidly increasing and creating a need for computational analysis methods for efficient polyketide research. Moreover, the increasing use of genetic engineering to research new kinds of polyketides requires genome wide analysis. Results We describe a system named ASMPKS (Analysis System for Modular Polyketide Synthesis for computational analysis of PKSs against genome sequences. It also provides overall management of information on modular PKS, including polyketide database construction, new PKS assembly, and chain visualization. ASMPKS operates on a web interface to construct the database and to analyze PKSs, allowing polyketide researchers to add their data to this database and to use it easily. In addition, the ASMPKS can predict functional modules for a protein sequence submitted by users, estimate the chemical composition of a polyketide synthesized from the modules, and display the carbon chain structure on the web interface. Conclusion ASMPKS has powerful computation features to aid modular PKS research. As various factors, such as starter units and post-processing, are related to polyketide biosynthesis, ASMPKS will be improved through further development for study of the factors.

  12. Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

    Directory of Open Access Journals (Sweden)

    Vinciane Régnier

    Full Text Available BACKGROUND: The cystathionine β-synthase (CBS gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA metabolism, a pathway important for several brain physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1 expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line. CONCLUSION/SIGNIFICANCE: We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

  13. SUCROSE SYNTHASE: ELUCIDATION OF COMPLEX POST-TRANSLATIONAL REGULATORY MECHANISMS

    Energy Technology Data Exchange (ETDEWEB)

    Steven C. Huber

    2009-05-12

    Studies have focused on the enzyme sucrose synthase, which plays an important role in the metabolism of sucrose in seeds and tubers. There are three isoforms of SUS in maize, referred to as SUS1, SUS-SH1, and SUS2. SUS is generally considered to be tetrameric protein but recent evidence suggests that SUS can also occur as a dimeric protein. The formation of tetrameric SUS is regulated by sucrose concentration in vitro and this could also be an important factor in the cellular localization of the protein. We found that high sucrose concentrations, which promote tetramer formation, also inhibit the binding of SUS1 to actin filaments in vitro. Previously, high sucrose concentrations were shown to promote SUS association with the plasma membrane. The specific regions of the SUS molecule involved in oligomerization are not known, but we identified a region of the SUS1 moelcule by bioinformatic analysis that was predicted to form a coiled coil. We demonstrated that this sequence could, in fact, self-associate as predicted for a coiled coil, but truncation analysis with the full-length recombinant protein suggested that it was not responsible for formation of dimers or tetramers. However, the coiled coil may function in binding of other proteins to SUS1. Overall, sugar availability may differentially influence the binding of SUS to cellular structures, and these effects may be mediated by changes in the oligomeric nature of the enzyme.

  14. Mechanics of Cellulose Synthase Complexes in Living Plant Cells

    Science.gov (United States)

    Zehfroosh, Nina; Liu, Derui; Ramos, Kieran P.; Yang, Xiaoli; Goldner, Lori S.; Baskin, Tobias I.

    The polymer cellulose is one of the major components of the world's biomass with unique and fascinating characteristics such as its high tensile strength, renewability, biodegradability, and biocompatibility. Because of these distinctive aspects, cellulose has been the subject of enormous scientific and industrial interest, yet there are still fundamental open questions about cellulose biosynthesis. Cellulose is synthesized by a complex of transmembrane proteins called ``Cellulose Synthase A'' (CESA) in the plasma membrane. Studying the dynamics and kinematics of the CESA complex will help reveal the mechanism of cellulose synthesis and permit the development and validation of models of CESA motility. To understand what drives these complexes through the cell membrane, we used total internal reflection fluorescence microscopy (TIRFM) and variable angle epi-fluorescence microscopy to track individual, fluorescently-labeled CESA complexes as they move in the hypocotyl and root of living plants. A mean square displacement analysis will be applied to distinguish ballistic, diffusional, and other forms of motion. We report on the results of these tracking experiments. This work was funded by NSF/PHY-1205989.

  15. Radical mechanism of cyanophage phycoerythrobilin synthase (PebS).

    Science.gov (United States)

    Busch, Andrea W U; Reijerse, Edward J; Lubitz, Wolfgang; Hofmann, Eckhard; Frankenberg-Dinkel, Nicole

    2011-02-01

    PEB (phycoerythrobilin) is a pink-coloured open-chain tetrapyrrole molecule found in the cyanobacterial light-harvesting phycobilisome. Within the phycobilisome, PEB is covalently bound via thioether bonds to conserved cysteine residues of the phycobiliprotein subunits. In cyanobacteria, biosynthesis of PEB proceeds via two subsequent two-electron reductions catalysed by the FDBRs (ferredoxin-dependent bilin reductases) PebA and PebB starting from the open-chain tetrapyrrole biliverdin IXα. A new member of the FDBR family has been identified in the genome of a marine cyanophage. In contrast with the cyanobacterial enzymes, PebS (PEB synthase) from cyanophages combines both two-electron reductions for PEB synthesis. In the present study we show that PebS acts via a substrate radical mechanism and that two conserved aspartate residues at position 105 and 206 are critical for stereospecific substrate protonation and conversion. On the basis of the crystal structures of both PebS mutants and presented biochemical and biophysical data, a mechanism for biliverdin IXα conversion to PEB is postulated and discussed with respect to other FDBR family members.

  16. Nitric oxide synthase is induced in sporulation of Physarum polycephalum

    Science.gov (United States)

    Golderer, Georg; Werner, Ernst R.; Leitner, Stefan; Gröbner, Peter; Werner-Felmayer, Gabriele

    2001-01-01

    The myxomycete Physarum polycephalum expresses a calcium-independent nitric oxide (NO) synthase (NOS) resembling the inducible NOS isoenzyme in mammals. We have now cloned and sequenced this, the first nonanimal NOS to be identified, showing that it shares Physarum macroplasmodia during the 5-day starvation period needed to induce sporulation competence. Induction of both NOS and sporulation competence were inhibited by glucose, a growth signal and known repressor of sporulation, and by l-N6–(1-iminoethyl)-lysine (NIL), an inhibitor of inducible NOS. Sporulation, which is triggered after the starvation period by light exposure, was also prevented by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-sensitive guanylate cyclase. In addition, also expression of lig1, a sporulation-specific gene, was strongly attenuated by NIL or ODQ. 8-Bromo-cGMP, added 2 h before the light exposure, restored the capacity of NIL-treated macroplasmodia to express lig1 and to sporulate. This indicates that the second messenger used for NO signaling in sporulation of Physarum is cGMP and links this signaling pathway to expression of lig1. PMID:11358872

  17. Role of lupeol synthase in Lotus japonicus nodule formation.

    Science.gov (United States)

    Delis, Costas; Krokida, Afrodite; Georgiou, Sofia; Peña-Rodríguez, Luis M; Kavroulakis, Nektarios; Ioannou, Efstathia; Roussis, Vassilios; Osbourn, Anne E; Papadopoulou, Kalliope K

    2011-01-01

    • Triterpenes are plant secondary metabolites, derived from the cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs). Here, we investigated the role of lupeol synthase, encoded by OSC3, and its product, lupeol, in developing roots and nodules of the model legume Lotus japonicus. • The expression patterns of OSC3 in different developmental stages of uninfected roots and in roots infected with Mesorhizobium loti were determined. The tissue specificity of OSC3 expression was analysed by in situ hybridization. Functional analysis, in which transgenic L. japonicus roots silenced for OSC3 were generated, was performed. The absence of lupeol in the silenced plant lines was determined by GC-MS. • The expression of ENOD40, a marker gene for nodule primordia initiation, was increased significantly in the OSC3-silenced plant lines, suggesting that lupeol influences nodule formation. Silenced plants also showed a more rapid nodulation phenotype, consistent with this. Exogenous application of lupeol to M. loti-infected wild-type plants provided further evidence for a negative regulatory effect of lupeol on the expression of ENOD40. • The synthesis of lupeol in L. japonicus roots and nodules can be solely attributed to OSC3. Taken together, our data suggest a role for lupeol biosynthesis in nodule formation through the regulation of ENOD40 gene expression.

  18. Human leucocytes in asthenozoospermic patients: endothelial nitric oxide synthase expression.

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