Targeting the GPI biosynthetic pathway.

Science.gov (United States)

Yadav, Usha; Khan, Mohd Ashraf

2018-02-27

The GPI (Glycosylphosphatidylinositol) biosynthetic pathway is a multistep conserved pathway in eukaryotes that culminates in the generation of GPI glycolipid which in turn anchors many proteins (GPI-APs) to the cell surface. In spite of the overall conservation of the pathway, there still exist subtle differences in the GPI pathway of mammals and other eukaryotes which holds a great promise so far as the development of drugs/inhibitors against specific targets in the GPI pathway of pathogens is concerned. Many of the GPI structures and their anchored proteins in pathogenic protozoans and fungi act as pathogenicity factors. Notable examples include GPI-anchored variant surface glycoprotein (VSG) in Trypanosoma brucei, GPI-anchored merozoite surface protein 1 (MSP1) and MSP2 in Plasmodium falciparum, protein-free GPI related molecules like lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) in Leishmania spp., GPI-anchored Gal/GalNAc lectin and proteophosphoglycans in Entamoeba histolytica or the GPI-anchored mannoproteins in pathogenic fungi like Candida albicans. Research in this active area has already yielded encouraging results in Trypanosoma brucei by the development of parasite-specific inhibitors of GlcNCONH 2 -β-PI, GlcNCONH 2 -(2-O-octyl)-PI and salicylic hydroxamic acid (SHAM) targeting trypanosomal GlcNAc-PI de-N-acetylase as well as the development of antifungal inhibitors like BIQ/E1210/gepinacin/G365/G884 and YW3548/M743/M720 targeting the GPI specific fungal inositol acyltransferase (Gwt1) and the phosphoethanolamine transferase-I (Mcd4), respectively. These confirm the fact that the GPI pathway continues to be the focus of researchers, given its implications for the betterment of human life.

Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

DEFF Research Database (Denmark)

Liu, Qing; Manzano, David; Tanić, Nikola

2014-01-01

Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew tha...

Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli.

Science.gov (United States)

Stahlhut, Steen G; Siedler, Solvej; Malla, Sailesh; Harrison, Scott J; Maury, Jérôme; Neves, Ana Rute; Forster, Jochen

2015-09-01

Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer's disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

A kinetic model for the penicillin biosynthetic pathway in

DEFF Research Database (Denmark)

Nielsen, Jens; Jørgensen, Henrik

1996-01-01

A kinetic model for the first two steps in the penicillin biosynthetic pathway, i.e. the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS) is proposed. The model is based on Michaelis-Menten type kinetics with non-competitive inhibition of the ACVS by ACV, and competitive inhibition...... of the IPNS by glutathione. The model predicted flux through the pathway corresponds well with the measured rate of penicillin biosynthesis. From the kinetic model the elasticity coefficients and the flux control coefficients are calculated throughout a fed-batch cultivation, and it is found...

Consortium analysis of gene and gene–folate interactions in purine and pyrimidine metabolism pathways with ovarian carcinoma risk

DEFF Research Database (Denmark)

Kelemen, Linda E; Terry, Kathryn L; Goodman, Marc T

2014-01-01

SCOPE: We reevaluated previously reported associations between variants in pathways of one-carbon (1-C) (folate) transfer genes and ovarian carcinoma (OC) risk, and in related pathways of purine and pyrimidine metabolism, and assessed interactions with folate intake. METHODS AND RESULTS: Odds rat...

Linking metabolic QTLs with network and cis-eQTLs controlling biosynthetic pathways.

Directory of Open Access Journals (Sweden)

Adam M Wentzell

2007-09-01

Full Text Available Phenotypic variation between individuals of a species is often under quantitative genetic control. Genomic analysis of gene expression polymorphisms between individuals is rapidly gaining popularity as a way to query the underlying mechanistic causes of variation between individuals. However, there is little direct evidence of a linkage between global gene expression polymorphisms and phenotypic consequences. In this report, we have mapped quantitative trait loci (QTLs-controlling glucosinolate content in a population of 403 Arabidopsis Bay x Sha recombinant inbred lines, 211 of which were previously used to identify expression QTLs controlling the transcript levels of biosynthetic genes. In a comparative study, we have directly tested two plant biosynthetic pathways for association between polymorphisms controlling biosynthetic gene transcripts and the resulting metabolites within the Arabidopsis Bay x Sha recombinant inbred line population. In this analysis, all loci controlling expression variation also affected the accumulation of the resulting metabolites. In addition, epistasis was detected more frequently for metabolic traits compared to transcript traits, even when both traits showed similar distributions. An analysis of candidate genes for QTL-controlling networks of transcripts and metabolites suggested that the controlling factors are a mix of enzymes and regulatory factors. This analysis showed that regulatory connections can feedback from metabolism to transcripts. Surprisingly, the most likely major regulator of both transcript level for nearly the entire pathway and aliphatic glucosinolate accumulation is variation in the last enzyme in the biosynthetic pathway, AOP2. This suggests that natural variation in transcripts may significantly impact phenotypic variation, but that natural variation in metabolites or their enzymatic loci can feed back to affect the transcripts.

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic and related bacteria

International Nuclear Information System (INIS)

Avissar, Y.J.; Beale, S.I.; Ormerod, J.G.

1989-01-01

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, δ-aminolevulinic acid (ALA): condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO 2 , and conversion of the intact carbon skeleton of glutamate to ALA in a process requiring tRNA Glu , ATP, Mg 2+ , NADPH, and pyridoxal phosphate. The distribution of the two ALA biosynthetic pathways among various bacterial genera was determined, using cell-free extracts obtained from representative organisms. Evidence for the operation of the glutamate pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA using 3,4-[ 3 H]glutamate and 1-[ 14 C]glutamate as substrate. The glycine pathway was indicated by RNase-insensitive incorporation of level from 2-[ 14 C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously phylogenetic relationships and clearly indicates that the glutamate pathway is the more ancient process, whereas the glycine pathway probably evolved much later. The glutamate pathway is the more widely utilized one among bacteria, while the glycine pathway is apparently limited to the α subgroup of purple bacteria (including Rhodobacter, Rhodospirillum, and Rhizobium). E. coli was found ALA via the glutamate pathway. The ALA-requiring hemA mutant of E. coli was determined to lack the dehydrogenase activity that utilizes glutamyl-tRNA as a substrate

Associations between purine metabolites and clinical symptoms in schizophrenia.

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Jeffrey K Yao

Full Text Available The antioxidant defense system, which is known to be dysregulated in schizophrenia, is closely linked to the dynamics of purine pathway. Thus, alterations in the homeostatic balance in the purine pathway may be involved in the pathophysiology of schizophrenia.Breakdown products in purine pathway were measured using high-pressure liquid chromatography coupled with a coulometric multi-electrode array system for 25 first-episode neuroleptic-naïve patients with schizophrenia at baseline and at 4-weeks following initiation of treatment with antipsychotic medication. Associations between these metabolites and clinical and neurological symptoms were examined at both time points. The ratio of uric acid and guanine measured at baseline predicted clinical improvement following four weeks of treatment with antipsychotic medication. Baseline levels of purine metabolites also predicted clinical and neurological symtpoms recorded at baseline; level of guanosine was associated with degree of clinical thought disturbance, and the ratio of xanthosine to guanosine at baseline predicted degree of impairment in the repetition and sequencing of actions.Findings suggest an association between optimal levels of purine byproducts and dynamics in clinical symptoms and adjustment, as well as in the integrity of sensory and motor processing. Taken together, alterations in purine catabolism may have clinical relevance in schizophrenia pathology.

Identification of sugarcane genes involved in the purine synthesis pathway

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Mario A. Jancso

2001-12-01

Full Text Available Nucleotide synthesis is of central importance to all cells. In most organisms, the purine nucleotides are synthesized de novo from non-nucleotide precursors such as amino acids, ammonia and carbon dioxide. An understanding of the enzymes involved in sugarcane purine synthesis opens the possibility of using these enzymes as targets for chemicals which may be effective in combating phytopathogen. Such an approach has already been applied to several parasites and types of cancer. The strategy described in this paper was applied to identify sugarcane clusters for each step of the de novo purine synthesis pathway. Representative sequences of this pathway were chosen from the National Center for Biotechnology Information (NCBI database and used to search the translated sugarcane expressed sequence tag (SUCEST database using the available basic local alignment search tool (BLAST facility. Retrieved clusters were further tested for the statistical significance of the alignment by an implementation (PRSS3 of the Monte Carlo shuffling algorithm calibrated using known protein sequences of divergent taxa along the phylogenetic tree. The sequences were compared to each other and to the sugarcane clusters selected using BLAST analysis, with the resulting table of p-values indicating the degree of divergence of each enzyme within different taxa and in relation to the sugarcane clusters. The results obtained by this strategy allowed us to identify the sugarcane proteins participating in the purine synthesis pathway.A via de síntese de purino nucleotídeos é considerada uma via de central importância para todas as células. Na maioria dos organismos, os purino nucleotídeos são sintetizados ''de novo'' a partir de precursores não-nucleotídicos como amino ácidos, amônia e dióxido de carbono. O conhecimento das enzimas envolvidas na via de síntese de purinas da cana-de-açúcar vai abrir a possibilidade do uso dessas enzimas como alvos no desenho

Enhancement of cordyceps polysaccharide production via biosynthetic pathway analysis in Hirsutella sinensis.

Science.gov (United States)

Lin, Shan; Liu, Zhi-Qiang; Baker, Peter James; Yi, Ming; Wu, Hui; Xu, Feng; Teng, Yi; Zheng, Yu-Guo

2016-11-01

The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2mM of manganese sulfate on 0day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcriptional repressor role of PocR on the 1,3-propanediol biosynthetic pathway by Lactobacillus panis PM1.

Science.gov (United States)

Kang, Tae Sun; Korber, Darren R; Tanaka, Takuji

2014-06-01

The regulatory role of a transcriptional regulator (PocR) in the 1,3-propanediol biosynthetic pathway of Lactobacillus panis PM1 contributes to the optimization of 1,3-propanediol production by this strain, which potentially will lead to 1,3-propanediol manufacturing efficiencies. Lactobacillus panis PM1 can utilize a 1,3-propanediol (1,3-PDO) biosynthetic pathway, consisting of diol dehydratase (PduCDE) and 1,3-PDO dehydrogenase, as a NADH recycling system, to survive under various environmental conditions. In this study, we identified a key transcriptional repressor (PocR) which was annotated as a transcriptional factor of AraC family as part of the 1,3-PDO biosynthetic pathway of L. panis PM1. The over-expression of the PocR gene resulted in the significant repression (81 %) of pduC (PduCDE large subunit) transcription, and subsequently, the decreased activity of PduCDE by 22 %. As a result of the regulation of PduCDE, production of both 3-hydroxypropionaldehyde and 1,3-PDO in the PocR over-expressing strain were significantly decreased by 40 % relative to the control strain. These results clearly demonstrate the transcriptional repressor role of PocR in the 1,3-PDO biosynthetic pathway.

Construction of a controllable β-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae.

Science.gov (United States)

Xie, Wenping; Liu, Min; Lv, Xiaomei; Lu, Wenqiang; Gu, Jiali; Yu, Hongwei

2014-01-01

Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi-gene pathways by employing the GAL regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof-of-principle, a controllable β-carotene biosynthesis pathway (∼16 kb) was reconstructed and optimized by repeatedly using GAL10-GAL1 bidirectional promoters with high efficiency (80-100%). By controling the switch time of the pathway, production of 11 mg/g DCW of total carotenoids (72.57 mg/L) and 7.41 mg/g DCW of β-carotene was achieved in shake-flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture. The results demonstrated a controllable and genetically stable biosynthetic pathway capable of increasing the yield of target products. Furthermore, the strategy presented in this study could be extended to construct other pathways in S. cerevisisae. © 2013 Wiley Periodicals, Inc.

Unravelling Protein-Protein Interaction Networks Linked to Aliphatic and Indole Glucosinolate Biosynthetic Pathways in Arabidopsis

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Sebastian J. Nintemann

2017-11-01

Full Text Available Within the cell, biosynthetic pathways are embedded in protein-protein interaction networks. In Arabidopsis, the biosynthetic pathways of aliphatic and indole glucosinolate defense compounds are well-characterized. However, little is known about the spatial orchestration of these enzymes and their interplay with the cellular environment. To address these aspects, we applied two complementary, untargeted approaches—split-ubiquitin yeast 2-hybrid and co-immunoprecipitation screens—to identify proteins interacting with CYP83A1 and CYP83B1, two homologous enzymes specific for aliphatic and indole glucosinolate biosynthesis, respectively. Our analyses reveal distinct functional networks with substantial interconnection among the identified interactors for both pathway-specific markers, and add to our knowledge about how biochemical pathways are connected to cellular processes. Specifically, a group of protein interactors involved in cell death and the hypersensitive response provides a potential link between the glucosinolate defense compounds and defense against biotrophic pathogens, mediated by protein-protein interactions.

Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

OpenAIRE

Cirillo, J. D.; Weisbrod, T. R.; Banerjee, A.; Bloom, B. R.; Jacobs, W. R.

1994-01-01

The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the constru...

Molecular evolution of the lysine biosynthetic pathways.

Science.gov (United States)

Velasco, A M; Leguina, J I; Lazcano, A

2002-10-01

Among the different biosynthetic pathways found in extant organisms, lysine biosynthesis is peculiar because it has two different anabolic routes. One is the diaminopimelic acid pathway (DAP), and the other over the a-aminoadipic acid route (AAA). A variant of the AAA route that includes some enzymes involved in arginine and leucine biosyntheses has been recently reported in Thermus thermophilus (Nishida et al. 1999). Here we describe the results of a detailed genomic analysis of each of the sequences involved in the two lysine anabolic routes, as well as of genes from other routes related to them. No evidence was found of an evolutionary relationship between the DAP and AAA enzymes. Our results suggest that the DAP pathway is related to arginine metabolism, since the lysC, asd, dapC, dapE, and lysA genes from lysine biosynthesis are related to the argB, argC, argD, argE, and speAC genes, respectively, whose products catalyze different steps in arginine metabolism. This work supports previous reports on the relationship between AAA gene products and some enzymes involved in leucine biosynthesis and the tricarboxylic acid cycle (Irvin and Bhattacharjee 1998; Miyazaki et al. 2001). Here we discuss the significance of the recent finding that several genes involved in the arginine (Arg) and leucine (Leu) biosynthesis participate in a new alternative route of the AAA pathway (Miyazaki et al. 2001). Our results demonstrate a clear relationship between the DAP and Arg routes, and between the AAA and Leu pathways.

Neurosteroid biosynthetic pathway changes in substantia nigra and caudate nucleus in Parkinson's disease

NARCIS (Netherlands)

Luchetti, Sabina; Bossers, Koen; Frajese, Giovanni Vanni; Swaab, Dick F.

2010-01-01

There is emerging evidence from animal studies for a neuroprotective role of sex steroids in neurodegenerative diseases, but studies in human brain are lacking. We have carried out an extensive study of the neurosteroid biosynthetic pathways in substantia nigra (SN), caudate nucleus (CN) and putamen

Molecular analysis of "de novo" purine biosynthesis in solanaceous species and in Arabidopsis thaliana

DEFF Research Database (Denmark)

van der Graaff, Eric; Hooykaas, Paul; Lein, Wolfgang

2004-01-01

Purine nucleotides are essential components to sustain plant growth and development. In plants they are either synthesized "de novo" during the process of purine biosynthesis or are recycled from purine bases and purine nucleosides throughout the salvage pathway. Comparison between animals...... biosynthesis pathway in plants, and the in planta functional analysis of PRPP (5-phosphoribosyl-1-pyrophoshate) amidotransferase (ATase), catalyzing the first committed step of the "de novo" purine biosynthesis. The cloning of the genes involved in the purine biosynthesis pathway was attained by a screening...... strategy with heterologous cDNA probes and by using S. cerevisiae mutants for complementation. Southern hybridization showed a complex genomic organization for these genes in solanaceous species and their organ- and developmental specific expression was analyzed by Northern hybridization. The specific role...

Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator

DEFF Research Database (Denmark)

Schultz, Anna Charlotte; Nygaard, P.; Saxild, Hans Henrik

2001-01-01

The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system...... for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires......ABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control....

Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Rugbjerg, Peter; Naesby, Michael; Mortensen, Uffe Hasbro

2013-01-01

production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways. RESULTS: In this study, we established...

Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

Science.gov (United States)

Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

2008-06-01

Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

Pediatric neurological syndromes and inborn errors of purine metabolism.

Science.gov (United States)

Camici, Marcella; Micheli, Vanna; Ipata, Piero Luigi; Tozzi, Maria Grazia

2010-02-01

This review is devised to gather the presently known inborn errors of purine metabolism that manifest neurological pediatric syndromes. The aim is to draw a comprehensive picture of these rare diseases, characterized by unexpected and often devastating neurological symptoms. Although investigated for many years, most purine metabolism disorders associated to psychomotor dysfunctions still hide the molecular link between the metabolic derangement and the neurological manifestations. This basically indicates that many of the actual functions of nucleosides and nucleotides in the development and function of several organs, in particular central nervous system, are still unknown. Both superactivity and deficiency of phosphoribosylpyrophosphate synthetase cause hereditary disorders characterized, in most cases, by neurological impairments. The deficiency of adenylosuccinate lyase and 5-amino-4-imidazolecarboxamide ribotide transformylase/IMP cyclohydrolase, both belonging to the de novo purine synthesis pathway, is also associated to severe neurological manifestations. Among catabolic enzymes, hyperactivity of ectosolic 5'-nucleotidase, as well as deficiency of purine nucleoside phosphorylase and adenosine deaminase also lead to syndromes affecting the central nervous system. The most severe pathologies are associated to the deficiency of the salvage pathway enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase: the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to a clear impairment of mitochondrial functions. The assessment of hypo- or hyperuricemic conditions is suggestive of purine enzyme dysfunctions, but most disorders of purine metabolism may escape the clinical investigation because they are not associated to these metabolic derangements. This review may represent a starting point stimulating both scientists and physicians involved in the study of

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

Science.gov (United States)

Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

2008-01-01

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

Elucidation of the biosynthetic pathway for the production of the pigment chrysogine by Penicillium chrysogenum

NARCIS (Netherlands)

Viggiano, Annarita; Salo, Oleksandr; Ali, Hazrat; Szymanski, Wiktor; Lankhorst, Peter P; Nygård, Yvonne; Bovenberg, Roel A L; Driessen, Arnold J M

Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although it was first isolated in 1973, the biosynthetic pathway has so far not been resolved. Here, we show that the deletion of the highly expressed non-ribosomal peptide synthetase (NRPS) gene

Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

Energy Technology Data Exchange (ETDEWEB)

Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika; Alfaro, Joshua F.; Yang, Feng; Burnum-Johnson, Kristin E.; Petyuk, Vladislav A.; Weitz, Karl K.; Camp, David G.; Smith, Richard D.; Wilmarth, Phillip A.; David, Larry L.; Ramasamy, Gowthaman; Myler, Peter J.; Carter, Nicola S.

2014-02-27

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.

Science.gov (United States)

Ishida, Mariko; Kitao, Naoko; Mizuno, Kouichi; Tanikawa, Natsu; Kato, Misako

2009-02-01

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-(14)C]adenine and [8-(14)C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue.

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

Directory of Open Access Journals (Sweden)

Sandra Fresquet-Corrales

Full Text Available Proanthocyanidins (PAs, or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (- catechin/g FW and 228.5 nmol (- epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloat-safe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass are discussed.

Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

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Mattanovich Diethard

2008-07-01

Full Text Available Abstract Background High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. Results Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1 is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP increases the riboflavin accumulation significantly. Conclusion The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.

Functional Characterization of a Novel R2R3-MYB Transcription Factor Modulating the Flavonoid Biosynthetic Pathway from Epimedium sagittatum

Directory of Open Access Journals (Sweden)

Wenjun Huang

2017-07-01

Full Text Available Epimedium species have been widely used both as traditional Chinese medicinal plants and ornamental perennials. Both flavonols, acting as the major bioactive components (BCs and anthocyanins, predominantly contributing to the color diversity of Epimedium flowers belong to different classes of flavonoids. It is well-acknowledged that flavonoid biosynthetic pathway is predominantly regulated by R2R3-MYB transcription factor (TF as well as bHLH TF and WD40 protein at the transcriptional level. MYB TFs specifically regulating anthocyanin or flavonol biosynthetic pathway have been already isolated and functionally characterized from Epimedium sagittatum, but a R2R3-MYB TF involved in regulating both these two pathways has not been functionally characterized to date in Epimedium plants. In this study, we report the functional characterization of EsMYB9, a R2R3-MYB TF previously isolated from E. sagittatum. The previous study indicated that EsMYB9 belongs to a small subfamily of R2R3-MYB TFs containing grape VvMYB5a and VvMYB5b TFs, which regulate flavonoid biosynthetic pathway. The present studies show that overexpression of EsMYB9 in tobacco leads to increased transcript levels of flavonoid pathway genes and increased contents of anthocyanins and flavonols. Yeast two-hybrid assay indicates that the C-terminal region of EsMYB9 contributes to the autoactivation activity, and EsMYB9 interacts with EsTT8 or AtTT8 bHLH regulator. Transient reporter assay shows that EsMYB9 slightly activates the expression of EsCHS (chalcone synthase promoter in transiently transformed leaves of Nicotiana benthamiana, but the addition of AtTT8 or EsTT8 bHLH regulator strongly enhances the transcriptional activation of EsMYB9 against five promoters of the flavonoid pathway genes except EsFLS (flavonol synthase. In addition, co-transformation of EsMYB9 and EsTT8 in transiently transfected tobacco leaves strongly induces the expressions of flavonoid biosynthetic genes. The

Purine biosynthesis is the bottleneck in trimethoprim-treated Bacillus subtilis.

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Stepanek, Jennifer Janina; Schäkermann, Sina; Wenzel, Michaela; Prochnow, Pascal; Bandow, Julia Elisabeth

2016-10-01

Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preliminary crystallographic studies of purine nucleoside phosphorylase from the cariogenic pathogen Streptococcus mutans

International Nuclear Information System (INIS)

Hou, Qiao-Ming; Liu, Xiang; Brostromer, Erik; Li, Lan-Fen; Su, Xiao-Dong

2009-01-01

Purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, has been expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique. The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and α-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6 Å resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1 Å

Synthesis of C-Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies-Based Biosynthetic Pathway.

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Andersen-Ranberg, Johan; Kongstad, Kenneth Thermann; Nafisi, Majse; Staerk, Dan; Okkels, Finn Thyge; Mortensen, Uffe Hasbro; Lindberg Møller, Birger; Frandsen, Rasmus John Normand; Kannangara, Rubini

2017-10-05

Carminic acid is a C-glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane-bound enzymes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling Method for Increased Precision and Scope of Directly Measurable Fluxes at a Genome-Scale

DEFF Research Database (Denmark)

McCloskey, Douglas; Young, Jamey D.; Xu, Sibei

2016-01-01

pathways of traditional MFA models and also covers the additional pathways of purine, pyrimidine, isoprenoid, methionine, riboflavin, coenzyme A, and folate, as well as other biosynthetic pathways. When evaluating the iDM2014 using a set of measured intracellular intermediate and cofactor mass isotopomer...

Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP-l-galactose mutase.

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Zhu, Qinghua; Chen, Qi; Song, Yongxiang; Huang, Hongbo; Li, Jun; Ma, Junying; Li, Qinglian; Ju, Jianhua

2017-05-09

Galactose, a monosaccharide capable of assuming two possible configurational isomers (d-/l-), can exist as a six-membered ring, galactopyranose (Gal p ), or as a five-membered ring, galactofuranose (Gal f ). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for d-Gal f Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive l-Gal f production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-l-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: ( i ) decipher the unique enzyme, GDP-l-galactose mutase associated with production of two unique d-mannose-derived sugars, and ( ii ) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of l-Gal f -containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.

TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] ameliorates diet induced obesity and insulin resistance via inhibition of the IP6K1 pathway.

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Ghoshal, Sarbani; Zhu, Qingzhang; Asteian, Alice; Lin, Hua; Xu, Haifei; Ernst, Glen; Barrow, James C; Xu, Baoji; Cameron, Michael D; Kamenecka, Theodore M; Chakraborty, Anutosh

2016-10-01

Obesity and type 2 diabetes (T2D) lead to various life-threatening diseases such as coronary heart disease, stroke, osteoarthritis, asthma, and neurodegeneration. Therefore, extensive research is ongoing to identify novel pathways that can be targeted in obesity/T2D. Deletion of the inositol pyrophosphate (5-IP7) biosynthetic enzyme, inositol hexakisphosphate kinase-1 (IP6K1), protects mice from high fat diet (HFD) induced obesity (DIO) and insulin resistance. Yet, whether this pathway is a valid pharmacologic target in obesity/T2D is not known. Here, we demonstrate that TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine], a pan-IP6K inhibitor, has strong anti-obesity and anti-diabetic effects in DIO mice. Q-NMR, GTT, ITT, food intake, energy expenditure, QRT-PCR, ELISA, histology, and immunoblot studies were conducted in short (2.5-week)- and long (10-week)-term TNP treated DIO C57/BL6 WT and IP6K1-KO mice, under various diet and temperature conditions. TNP, when injected at the onset of HFD-feeding, decelerates initiation of DIO and insulin resistance. Moreover, TNP facilitates weight loss and restores metabolic parameters, when given to DIO mice. However, TNP does not reduce weight gain in HFD-fed IP6K1-KO mice. TNP specifically enhances insulin sensitivity in DIO mice via Akt activation. TNP decelerates weight gain primarily by enhancing thermogenic energy expenditure in the adipose tissue. Accordingly, TNP's effect on body weight is partly abolished whereas its impact on glucose homeostasis is preserved at thermoneutral temperature. Pharmacologic inhibition of the inositol pyrophosphate pathway has strong therapeutic potential in obesity, T2D, and other metabolic diseases.

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.

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Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

2016-01-01

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a

Metabolic engineering of biosynthetic pathway for production of renewable biofuels.

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Singh, Vijai; Mani, Indra; Chaudhary, Dharmendra Kumar; Dhar, Pawan Kumar

2014-02-01

Metabolic engineering is an important area of research that involves editing genetic networks to overproduce a certain substance by the cells. Using a combination of genetic, metabolic, and modeling methods, useful substances have been synthesized in the past at industrial scale and in a cost-effective manner. Currently, metabolic engineering is being used to produce sufficient, economical, and eco-friendly biofuels. In the recent past, a number of efforts have been made towards engineering biosynthetic pathways for large scale and efficient production of biofuels from biomass. Given the adoption of metabolic engineering approaches by the biofuel industry, this paper reviews various approaches towards the production and enhancement of renewable biofuels such as ethanol, butanol, isopropanol, hydrogen, and biodiesel. We have also identified specific areas where more work needs to be done in the future.

Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways.

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Janevska, Slavica; Tudzynski, Bettina

2018-01-01

The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

Purine biosynthesis de novo by lymphocytes in gout

International Nuclear Information System (INIS)

Kamoun, P.; Chanard, J.; Brami, M.; Funck-Brentano, J.L.

1978-01-01

A method of measurement in vitro of purine biosynthesis de novo in human circulating blood lymphocytes is proposed. The rate of early reactions of purine biosynthesis de novo was determined by the incorporation of [ 14 C]formate into N-formyl glycinamide ribonucleotide when the subsequent reactions of the metabolic pathway were completely inhibited by the antibiotic azaserine. Synthesis of 14 C-labelled N-formyl glycinamide ribonucleotide by lymphocytes was measured in healthy control subjects and patients with primary gout or hyperuricaemia secondary to renal failure, with or without allopurinol therapy. The average synthesis was higher in gouty patients without therapy than in control subjects, but the values contained overlap the normal range. In secondary hyperuricaemia the synthesis was at same value as in control subjects. These results are in agreement with the inconstant acceleration of purine biosynthesis de novo in gouty patients as seen by others with measurement of [ 14 C]glycine incorporation into urinary uric acid. (author)

Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

Science.gov (United States)

Kuraoka, Isao; Bender, Christina; Romieu, Anthony; Cadet, Jean; Wood, Richard D.; Lindahl, Tomas

2000-01-01

Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5′ position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5′R or 5′S form of 5′,8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals. PMID:10759556

The Stringent Response Induced by Phosphate Limitation Promotes Purine Salvage in Agrobacterium fabrum.

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Sivapragasam, Smitha; Deochand, Dinesh K; Meariman, Jacob K; Grove, Anne

2017-10-31

Agrobacterium fabrum induces tumor growth in susceptible plant species. The upregulation of virulence genes that occurs when the bacterium senses plant-derived compounds is enhanced by acidic pH and limiting inorganic phosphate. Nutrient starvation may also trigger the stringent response, and purine salvage is among the pathways expected to be favored under such conditions. We show here that phosphate limitation induces the stringent response, as evidenced by production of (p)ppGpp, and that the xdhCSML operon encoding the purine salvage enzyme xanthine dehydrogenase is upregulated ∼15-fold. The xdhCSML operon is under control of the TetR family transcription factor XdhR; direct binding of ppGpp to XdhR attenuates DNA binding, and the enhanced xdhCSML expression correlates with increased cellular levels of (p)ppGpp. Xanthine dehydrogenase may also divert purines away from salvage pathways to form urate, the ligand for the transcription factor PecS, which in the plant pathogen Dickeya dadantii is a key regulator of virulence gene expression. However, urate levels remain low under conditions that produce increased levels of xdhCSML expression, and neither acidic pH nor limiting phosphate results in induction of genes under control of PecS. Instead, expression of such genes is induced only by externally supplemented urate. Taken together, our data indicate that purine salvage is favored during the stringent response induced by phosphate starvation, suggesting that control of this pathway may constitute a novel approach to modulating virulence. Because bacterial purine catabolism appears to be unaffected, as evidenced by the absence of urate accumulation, we further propose that the PecS regulon is induced by only host-derived urate.

The flavonoid biosynthetic pathway in plants: function and evolution

International Nuclear Information System (INIS)

Koes, R.E.; Quattrocchio, F.; Mol, J.N.M.

1994-01-01

Flavonoids are a class of low molecular weight phenolic compounds that is widely distributed in the plant kingdom. They exhibit a diverse spectrum of biological functions and play an important role in the interaction between plants and their environment. Flavonoids not only protect the plant from the harmful effects of UV irradiation but also play a crucial role in the sexual reproduction process. A special class of flavonoid polymers, the tannins, plays a structural role in the plant. Yet other classes of flavonoids, flavonols and anthocyanins, have been implicated in the attraction of pollinators. Certain flavonoids participate in the interaction between plants and other organisms such as symbiotic bacteria and parasites. This raises the intriguing question as to how these different compounds arose and evolved. Based on taxonomy and molecular analysis of gene expression patterns it is possible to deduce a putative sequence of acquisition of the different branches of the biosynthetic pathway and their regulators. (author)

The flavonoid biosynthetic pathway in plants: function and evolution

Energy Technology Data Exchange (ETDEWEB)

Koes, R. E.; Quattrocchio, F.; Mol, J. N.M. [Department of Genetics, Institute for Molecular Biological Sciences, Vrije Universiteit, BioCentrum Amsterdam, De Boelelaan 1087, 1081HV, Amsterdam (Netherlands)

1994-07-01

Flavonoids are a class of low molecular weight phenolic compounds that is widely distributed in the plant kingdom. They exhibit a diverse spectrum of biological functions and play an important role in the interaction between plants and their environment. Flavonoids not only protect the plant from the harmful effects of UV irradiation but also play a crucial role in the sexual reproduction process. A special class of flavonoid polymers, the tannins, plays a structural role in the plant. Yet other classes of flavonoids, flavonols and anthocyanins, have been implicated in the attraction of pollinators. Certain flavonoids participate in the interaction between plants and other organisms such as symbiotic bacteria and parasites. This raises the intriguing question as to how these different compounds arose and evolved. Based on taxonomy and molecular analysis of gene expression patterns it is possible to deduce a putative sequence of acquisition of the different branches of the biosynthetic pathway and their regulators. (author)

Expression of carotenoid biosynthetic pathway genes and changes in carotenoids during ripening in tomato (Lycopersicon esculentum).

Science.gov (United States)

Namitha, Kanakapura Krishnamurthy; Archana, Surya Narayana; Negi, Pradeep Singh

2011-04-01

To study the expression pattern of carotenoid biosynthetic pathway genes, changes in their expression at different stages of maturity in tomato fruit (cv. Arka Ahuti) were investigated. The genes regulating carotenoid production were quantified by a dot blot method using a DIG (dioxigenin) labelling and detection kit. The results revealed that there was an increase in the levels of upstream genes of the carotenoid biosynthetic pathway such as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase (Lyt B), phytoene synthase (PSY), phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) by 2-4 fold at the breaker stage as compared to leaf. The lycopene and β-carotene content was analyzed by HPLC at different stages of maturity. The lycopene (15.33 ± 0.24 mg per 100 g) and β-carotene (10.37 ± 0.46 mg per 100 g) content were found to be highest at 5 days post-breaker and 10 days post-breaker stage, respectively. The lycopene accumulation pattern also coincided with the color values at different stages of maturity. These studies may provide insight into devising gene-based strategies for enhancing carotenoid accumulation in tomato fruits.

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin-Fatty Acid Biosynthetic Pathway.

Science.gov (United States)

Haushalter, Robert W; Phelan, Ryan M; Hoh, Kristina M; Su, Cindy; Wang, George; Baidoo, Edward E K; Keasling, Jay D

2017-04-05

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

The Cremeomycin Biosynthetic Gene Cluster Encodes a Pathway for Diazo Formation.

Science.gov (United States)

Waldman, Abraham J; Pechersky, Yakov; Wang, Peng; Wang, Jennifer X; Balskus, Emily P

2015-10-12

Diazo groups are found in a range of natural products that possess potent biological activities. Despite longstanding interest in these metabolites, diazo group biosynthesis is not well understood, in part because of difficulties in identifying specific genes linked to diazo formation. Here we describe the discovery of the gene cluster that produces the o-diazoquinone natural product cremeomycin and its heterologous expression in Streptomyces lividans. We used stable isotope feeding experiments and in vitro characterization of biosynthetic enzymes to decipher the order of events in this pathway and establish that diazo construction involves late-stage N-N bond formation. This work represents the first successful production of a diazo-containing metabolite in a heterologous host, experimentally linking a set of genes with diazo formation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera

DEFF Research Database (Denmark)

Ono, Hajime; Rewitz, Kim; Shinoda, Tetsu

2006-01-01

is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both...... Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development....

Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

Science.gov (United States)

Dangoudoubiyam, Sriveny; Zhang, Zijing; Howe, Daniel K

2014-09-01

Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite.

Perturbations in the Photosynthetic Pigment Status Result in Photooxidation-Induced Crosstalk between Carotenoid and Porphyrin Biosynthetic Pathways

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Joon-Heum Park

2017-11-01

Full Text Available Possible crosstalk between the carotenoid and porphyrin biosynthetic pathways under photooxidative conditions was investigated by using their biosynthetic inhibitors, norflurazon (NF and oxyfluorfen (OF. High levels of protoporphyrin IX (Proto IX accumulated in rice plants treated with OF, whereas Proto IX decreased in plants treated with NF. Both NF and OF treatments resulted in greater decreases in MgProto IX, MgProto IX methyl ester, and protochlorophyllide. Activities and transcript levels of most porphyrin biosynthetic enzymes, particularly in the Mg-porphyrin branch, were greatly down-regulated in NF and OF plants. In contrast, the transcript levels of GSA, PPO1, and CHLD as well as FC2 and HO2 were up-regulated in NF-treated plants, while only moderate increases in FC2 and HO2 were observed in the early stage of OF treatment. Phytoene, antheraxanthin, and zeaxanthin showed high accumulation in NF-treated plants, whereas other carotenoid intermediates greatly decreased. Transcript levels of carotenoid biosynthetic genes, PSY1 and PDS, decreased in response to NF and OF, whereas plants in the later stage of NF treatment exhibited up-regulation of BCH and VDE as well as recovery of PDS. However, perturbed porphyrin biosynthesis by OF did not noticeably influence levels of carotenoid metabolites, regardless of the strong down-regulation of carotenoid biosynthetic genes. Both NF and OF plants appeared to provide enhanced protection against photooxidative damage, not only by scavenging of Mg-porphyrins, but also by up-regulating FC2, HO2, and Fe-chelatase, particularly with increased levels of zeaxanthin via up-regulation of BCH and VDE in NF plants. On the other hand, the up-regulation of GSA, PPO1, and CHLD under inhibition of carotenogenic flux may be derived from the necessity to recover impaired chloroplast biogenesis during photooxidative stress. Our study demonstrates that perturbations in carotenoid and porphyrin biosynthesis coordinate

Perturbations in the Photosynthetic Pigment Status Result in Photooxidation-Induced Crosstalk between Carotenoid and Porphyrin Biosynthetic Pathways.

Science.gov (United States)

Park, Joon-Heum; Tran, Lien H; Jung, Sunyo

2017-01-01

Possible crosstalk between the carotenoid and porphyrin biosynthetic pathways under photooxidative conditions was investigated by using their biosynthetic inhibitors, norflurazon (NF) and oxyfluorfen (OF). High levels of protoporphyrin IX (Proto IX) accumulated in rice plants treated with OF, whereas Proto IX decreased in plants treated with NF. Both NF and OF treatments resulted in greater decreases in MgProto IX, MgProto IX methyl ester, and protochlorophyllide. Activities and transcript levels of most porphyrin biosynthetic enzymes, particularly in the Mg-porphyrin branch, were greatly down-regulated in NF and OF plants. In contrast, the transcript levels of GSA, PPO1 , and CHLD as well as FC2 and HO2 were up-regulated in NF-treated plants, while only moderate increases in FC2 and HO2 were observed in the early stage of OF treatment. Phytoene, antheraxanthin, and zeaxanthin showed high accumulation in NF-treated plants, whereas other carotenoid intermediates greatly decreased. Transcript levels of carotenoid biosynthetic genes, PSY1 and PDS , decreased in response to NF and OF, whereas plants in the later stage of NF treatment exhibited up-regulation of BCH and VDE as well as recovery of PDS . However, perturbed porphyrin biosynthesis by OF did not noticeably influence levels of carotenoid metabolites, regardless of the strong down-regulation of carotenoid biosynthetic genes. Both NF and OF plants appeared to provide enhanced protection against photooxidative damage, not only by scavenging of Mg - porphyrins, but also by up-regulating FC2, HO2 , and Fe-chelatase, particularly with increased levels of zeaxanthin via up-regulation of BCH and VDE in NF plants. On the other hand, the up-regulation of GSA, PPO1 , and CHLD under inhibition of carotenogenic flux may be derived from the necessity to recover impaired chloroplast biogenesis during photooxidative stress. Our study demonstrates that perturbations in carotenoid and porphyrin biosynthesis coordinate the

Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

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Xu Guoqiang

2012-02-01

Full Text Available Abstract Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH and fumarase (RoFUM1 were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2 was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1 than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner.

Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors.

Science.gov (United States)

Suzuki, Hiroyoshi; Yokokura, Junpei; Ito, Tsukasa; Arai, Ryoma; Yokoyama, Chiaki; Toshima, Hiroaki; Nagata, Shinji; Asami, Tadao; Suzuki, Yoshihito

2014-10-01

Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.

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Michael T Guarnieri

Full Text Available Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga.

Purine content of some Philippine foods

International Nuclear Information System (INIS)

Lontoc, A.V.; Ladines, E.O.; Jalandoon, S.A.

1993-01-01

To facilitate dietary advice for prevention and management of hyperuricemia, the content of individual purines, namely: adenine, hypoxanthine, guanine and xanthine was determined in market samples of 161 legumes and dried beans, 41 organ meats, and 25 finfishes and shellfishes using Waters HPLC Model ALC/GPC 244. Analysis for moisture, protein and purines were conducted on the slurries or dried forms of the edible portions of the food samples. Food samples were subsequently classified into three categories adapted from Williams' dietary guide for purines, expressed in decreasing levels of total purines per 100 g serving. Category 1 foods are those that contain very large amount (150 to 1000 mg) of total purines; category 2 foods are those that contain a large amount (75-<150 mg) of total purines; and category 3 foods are those that contain a moderate amount (up to 75 mg) of total purines. (auth.). 17 refs.; 8 tabs

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

Science.gov (United States)

Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

2012-03-01

Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

Decoding Biosynthetic Pathways in Plants by Pulse-Chase Strategies Using 13CO2 as a Universal Tracer

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Adelbert Bacher

2016-07-01

Full Text Available 13CO2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectrometry can provide 13C-isotopologue compositions in biosynthetic products. Experiments with a variety of plant species have documented that the isotopologue profiles generated with 13CO2 pulse-chase labeling are directly comparable to those that can be generated by the application of [U-13C6]glucose to aseptically growing plants. However, the application of the 13CO2 labeling technology is not subject to the experimental limitations that one has to take into account for experiments with [U-13C6]glucose and can be applied to plants growing under physiological conditions, even in the field. In practical terms, the results of biosynthetic studies with 13CO2 consist of the detection of pairs, triples and occasionally quadruples of 13C atoms that have been jointly contributed to the target metabolite, at an abundance that is well above the stochastic occurrence of such multiples. Notably, the connectivities of jointly transferred 13C multiples can have undergone modification by skeletal rearrangements that can be diagnosed from the isotopologue data. As shown by the examples presented in this review article, the approach turns out to be powerful in decoding the carbon topology of even complex biosynthetic pathways.

Minimum Information about a Biosynthetic Gene cluster : commentary

NARCIS (Netherlands)

Medema, Marnix H; Kottmann, Renzo; Yilmaz, Pelin; Cummings, Matthew; Biggins, John B; Blin, Kai; de Bruijn, Irene; Chooi, Yit Heng; Claesen, Jan; Coates, R Cameron; Cruz-Morales, Pablo; Duddela, Srikanth; Dusterhus, Stephanie; Edwards, Daniel J; Fewer, David P; Garg, Neha; Geiger, Christoph; Gomez-Escribano, Juan Pablo; Greule, Anja; Hadjithomas, Michalis; Haines, Anthony S; Helfrich, Eric J N; Hillwig, Matthew L; Ishida, Keishi; Jones, Adam C; Jones, Carla S; Jungmann, Katrin; Kegler, Carsten; Kim, Hyun Uk; Kotter, Peter; Krug, Daniel; Masschelein, Joleen; Melnik, Alexey V; Mantovani, Simone M; Monroe, Emily A; Moore, Marcus; Moss, Nathan; Nutzmann, Hans-Wilhelm; Pan, Guohui; Pati, Amrita; Petras, Daniel; Reen, F Jerry; Rosconi, Federico; Rui, Zhe; Tian, Zhenhua; Tobias, Nicholas J; Tsunematsu, Yuta; Wiemann, Philipp; Wyckoff, Elizabeth; Yan, Xiaohui; Yim, Grace; Yu, Fengan; Xie, Yunchang; Aigle, Bertrand; Apel, Alexander K; Balibar, Carl J; Balskus, Emily P; Barona-Gomez, Francisco; Bechthold, Andreas; Bode, Helge B; Borriss, Rainer; Brady, Sean F; Brakhage, Axel A; Caffrey, Patrick; Cheng, Yi-Qiang; Clardy, Jon; Cox, Russell J; De Mot, Rene; Donadio, Stefano; Donia, Mohamed S; van der Donk, Wilfred A; Dorrestein, Pieter C; Doyle, Sean; Driessen, Arnold J M; Ehling-Schulz, Monika; Entian, Karl-Dieter; Fischbach, Michael A; Gerwick, Lena; Gerwick, William H; Gross, Harald; Gust, Bertolt; Hertweck, Christian; Hofte, Monica; Jensen, Susan E; Ju, Jianhua; Katz, Leonard; Kaysser, Leonard; Klassen, Jonathan L; Keller, Nancy P; Kormanec, Jan; Kuipers, Oscar P; Kuzuyama, Tomohisa; Kyrpides, Nikos C; Kwon, Hyung-Jin; Lautru, Sylvie; Lavigne, Rob; Lee, Chia Y; Linquan, Bai; Liu, Xinyu; Liu, Wen; Luzhetskyy, Andriy; Mahmud, Taifo; Mast, Yvonne; Mendez, Carmen; Metsa-Ketela, Mikko; Micklefield, Jason; Mitchell, Douglas A; Moore, Bradley S; Moreira, Leonilde M; Muller, Rolf; Neilan, Brett A; Nett, Markus; Nielsen, Jens; O'Gara, Fergal; Oikawa, Hideaki; Osbourn, Anne; Osburne, Marcia S; Ostash, Bohdan; Payne, Shelley M; Pernodet, Jean-Luc; Petricek, Miroslav; Piel, Jorn; Ploux, Olivier; Raaijmakers, Jos M; Salas, Jose A; Schmitt, Esther K; Scott, Barry; Seipke, Ryan F; Shen, Ben; Sherman, David H; Sivonen, Kaarina; Smanski, Michael J; Sosio, Margherita; Stegmann, Evi; Sussmuth, Roderich D; Tahlan, Kapil; Thomas, Christopher M; Tang, Yi; Truman, Andrew W; Viaud, Muriel; Walton, Jonathan D; Walsh, Christopher T; Weber, Tilmann; van Wezel, Gilles P; Wilkinson, Barrie; Willey, Joanne M; Wohlleben, Wolfgang; Wright, Gerard D; Ziemert, Nadine; Zhang, Changsheng; Zotchev, Sergey B; Breitling, Rainer; Takano, Eriko; Glockner, Frank Oliver

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit.

[Construction of Corynebacterium crenatum AS 1.542 δ argR and analysis of transcriptional levels of the related genes of arginine biosynthetic pathway].

Science.gov (United States)

Chen, Xuelan; Tang, Li; Jiao, Haitao; Xu, Feng; Xiong, Yonghua

2013-01-04

ArgR, coded by the argR gene from Corynebacterium crenatum AS 1.542, acts as a negative regulator in arginine biosynthetic pathway. However, the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported. Here, we constructed a deletion mutant of argR gene: C. crenatum AS 1.542 Delta argR using marker-less knockout technology, and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain. We used marker-less knockout technology to construct C. crenatum AS 1.542 Delta argR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR. C. crenatum AS 1.542 Delta argR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162.1 folds. The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR. However, the deletion of this regulator does not result in a clear change in arginine production in the bacteria.

Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels.

Science.gov (United States)

Zargar, Amin; Bailey, Constance B; Haushalter, Robert W; Eiben, Christopher B; Katz, Leonard; Keasling, Jay D

2017-06-01

Advances in retooling microorganisms have enabled bioproduction of 'drop-in' biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gas-turbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), 'drop-in' biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel. Copyright © 2017 Elsevier Ltd. All rights reserved.

Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

OpenAIRE

Vasudevan, Gayatri; Ullman, Buddy; Landfear, Scott M.

2001-01-01

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. do...

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin–Fatty Acid Biosynthetic Pathway

Energy Technology Data Exchange (ETDEWEB)

Haushalter, Robert W. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Phelan, Ryan M. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Hoh, Kristina M. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Su, Cindy [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Baidoo, Edward E. K. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Keasling, Jay D. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division

2017-03-14

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here in this paper we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

Gene set analysis of purine and pyrimidine antimetabolites cancer therapies.

Science.gov (United States)

Fridley, Brooke L; Batzler, Anthony; Li, Liang; Li, Fang; Matimba, Alice; Jenkins, Gregory D; Ji, Yuan; Wang, Liewei; Weinshilboum, Richard M

2011-11-01

Responses to therapies, either with regard to toxicities or efficacy, are expected to involve complex relationships of gene products within the same molecular pathway or functional gene set. Therefore, pathways or gene sets, as opposed to single genes, may better reflect the true underlying biology and may be more appropriate units for analysis of pharmacogenomic studies. Application of such methods to pharmacogenomic studies may enable the detection of more subtle effects of multiple genes in the same pathway that may be missed by assessing each gene individually. A gene set analysis of 3821 gene sets is presented assessing the association between basal messenger RNA expression and drug cytotoxicity using ethnically defined human lymphoblastoid cell lines for two classes of drugs: pyrimidines [gemcitabine (dFdC) and arabinoside] and purines [6-thioguanine and 6-mercaptopurine]. The gene set nucleoside-diphosphatase activity was found to be significantly associated with both dFdC and arabinoside, whereas gene set γ-aminobutyric acid catabolic process was associated with dFdC and 6-thioguanine. These gene sets were significantly associated with the phenotype even after adjusting for multiple testing. In addition, five associated gene sets were found in common between the pyrimidines and two gene sets for the purines (3',5'-cyclic-AMP phosphodiesterase activity and γ-aminobutyric acid catabolic process) with a P value of less than 0.0001. Functional validation was attempted with four genes each in gene sets for thiopurine and pyrimidine antimetabolites. All four genes selected from the pyrimidine gene sets (PSME3, CANT1, ENTPD6, ADRM1) were validated, but only one (PDE4D) was validated for the thiopurine gene sets. In summary, results from the gene set analysis of pyrimidine and purine therapies, used often in the treatment of various cancers, provide novel insight into the relationship between genomic variation and drug response.

Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes.

Science.gov (United States)

Honda, Kohsuke; Maya, Shohei; Omasa, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Ohtake, Hisao

2010-08-02

Six thermophilic enzymes from Thermus thermophilus were used to construct an 'artificial bio-synthetic pathway' for the production of 2-deoxyribose 5-phosphate from fructose. By a simple operation using six recombinant Escherichia coli strains producing the thermophilic enzymes, respectively, fructose was converted to 2-deoxyribose 5-phosphate with a molar yield of 55%. Copyright 2010 Elsevier B.V. All rights reserved.

tRNA-dependent cysteine biosynthetic pathway represents a strategy to increase cysteine contents by preventing it from thermal degradation: thermal adaptation of methanogenic archaea ancestor.

Science.gov (United States)

Qu, Ge; Wang, Wei; Chen, Ling-Ling; Qian, Shao-Song; Zhang, Hong-Yu

2009-10-01

Although cysteine (Cys) is beneficial to stabilize protein structures, it is not prevalent in thermophiles. For instance, the Cys contents in most thermophilic archaea are only around 0.7%. However, methanogenic archaea, no matter thermophilic or not, contain relatively abundant Cys, which remains elusive for a long time. Recently, Klipcan et al. correlated this intriguing property of methanogenic archaea with their unique tRNA-dependent Cys biosynthetic pathway. But, the deep reasons underlying the correlation are ambiguous. Considering the facts that free Cys is thermally labile and the tRNA-dependent Cys biosynthesis avoids the use of free Cys, we speculate that the unique Cys biosynthetic pathway represents a strategy to increase Cys contents by preventing it from thermal degradation, which may be relevant to the thermal adaptation of methanogenic archaea ancestor.

In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

Science.gov (United States)

Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

2015-09-01

miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

International Nuclear Information System (INIS)

Pilch, D.S.; Shafer, R.H.; Levenson, C.

1991-01-01

The authors have investigated the structure and physical chemistry of the d(C 3 T 4 C 3 )·2[d(G 3 A 4 G 3 )] triple helix by polyacrylamide gel electrophoresis (PAGE), 1 H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl 2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur·pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the puring strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 C, depending on the DNA concentration. This marked enhancement in stability, coupled with the lack of an acidic pH requirement, suggests that pur-pur-pyr triplexes are appealing choices for use in applications involving oligonucleotide targeting of duplex DNA in vitro and in vivo

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms.

Directory of Open Access Journals (Sweden)

Sacha Coesel

Full Text Available Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE and zeaxanthin epoxidase (ZEP enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms.

Science.gov (United States)

Coesel, Sacha; Oborník, Miroslav; Varela, Joao; Falciatore, Angela; Bowler, Chris

2008-08-06

Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP) enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several components of the

Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

DEFF Research Database (Denmark)

Nygaard, P.; Bested, S. M.; Andersen, K. A. K.

2000-01-01

amino acid polypeptide and was preceded by a promoter sequence that is recognized by the A form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular...

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

Science.gov (United States)

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling

International Nuclear Information System (INIS)

Park, Joon-Heum; Jung, Sunyo

2017-01-01

In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F v /F m . NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. - Highlights: • Two modes of photooxidation by carotenoid and tetrapyrrole biosynthetic inhibitors. • We examine differential alterations in chloroplast function and plastid signaling. • NF and OF cause differential alterations in chloroplast ultrastructure and function. • Photooxidation coordinates photosynthetic gene expression from nucleus and plastid.

A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant, Epimedium sagittatum

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Wenjun Huang

2016-07-01

Full Text Available Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from E. sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase and EsFLS (flavonol synthase, but not the promoters of EsDFRs (dihydroflavonol 4-reductase and EsANS (anthocyanidin synthase in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase, NtCHI (chalcone isomerase, NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived bioactive components in E

Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

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Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

2013-07-01

Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans.

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Kazuhiro Hayashi

Full Text Available Coenzyme Q (CoQ is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3-9 that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.

A Novel Antibiotic Mechanism of l-Cyclopropylalanine Blocking the Biosynthetic Pathway of Essential Amino Acid l-Leucine

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Bingji Ma

2017-12-01

Full Text Available The unusual amino acid l-cyclopropylalanine was isolated from the mushroom Amanita virgineoides after detection in an anti-fungal screening test. l-Cyclopropylalanine was found to exhibit broad-spectrum inhibition against fungi and bacteria. The anti-fungal activity was found to be abolished in the presence of the amino acid l-leucine, but not any other amino acids, indicating that l-cyclopropylalanine may block the biosynthesis of the essential amino acid l-leucine, thereby inhibiting fungal and bacteria growth. Further biochemical studies found l-cyclopropylalanine indeed inhibits α-isopropylmalate synthase (α-IMPS, the enzyme that catalyzes the rate-limiting step in the biosynthetic pathway of l-leucine. Inhibition of essential l-leucine synthesis in fungal and bacteria organisms, a pathway absent in host organisms such as humans, may represent a novel antibiotic mechanism to counter the ever-increasing problem of drug resistance to existing antibiotics.

Sex pheromone biosynthetic pathways are conserved between moths and the butterfly Bicyclus anynana

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Liénard, Marjorie A; Wang, Hong-Lei; Lassance, Jean-Marc; Löfstedt, Christer

2014-01-01

Although phylogenetically nested within the moths, butterflies have diverged extensively in a number of life history traits. Whereas moths rely greatly on chemical signals, visual advertisement is the hallmark of mate finding in butterflies. In the context of courtship, however, male chemical signals are widespread in both groups although they likely have multiple evolutionary origins. Here, we report that in males of the butterfly Bicyclus anynana, courtship scents are produced de novo via biosynthetic pathways shared with females of many moth species. We show that two of the pheromone components that play a major role in mate choice, namely the (Z)-9-tetradecenol and hexadecanal, are produced through the activity of a fatty acyl Δ11-desaturase and two specialized alcohol-forming fatty acyl reductases. Our study provides the first evidence of conservation and sharing of ancestral genetic modules for the production of FA-derived pheromones over a long evolutionary timeframe thereby reconciling mate communication in moths and butterflies. PMID:24862548

Expression profile of genes coding for carotenoid biosynthetic ...

Indian Academy of Sciences (India)

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits. Shuchi Smita, Ravi Rajwanshi, Sangram Keshari Lenka, Amit Katiyar, Viswanathan Chinnusamy and. Kailash Chander Bansal. J. Genet. 92, 363–368. Table 1.

The hedgehog pathway gene shifted functions together with the hmgcr-dependent isoprenoid biosynthetic pathway to orchestrate germ cell migration.

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Girish Deshpande

Full Text Available The Drosophila embryonic gonad is assembled from two distinct cell types, the Primordial Germ Cells (PGCs and the Somatic Gonadal Precursor cells (SGPs. The PGCs form at the posterior of blastoderm stage embryos and are subsequently carried inside the embryo during gastrulation. To reach the SGPs, the PGCs must traverse the midgut wall and then migrate through the mesoderm. A combination of local repulsive cues and attractive signals emanating from the SGPs guide migration. We have investigated the role of the hedgehog (hh pathway gene shifted (shf in directing PGC migration. shf encodes a secreted protein that facilitates the long distance transmission of Hh through the proteoglycan matrix after it is released from basolateral membranes of Hh expressing cells in the wing imaginal disc. shf is expressed in the gonadal mesoderm, and loss- and gain-of-function experiments demonstrate that it is required for PGC migration. Previous studies have established that the hmgcr-dependent isoprenoid biosynthetic pathway plays a pivotal role in generating the PGC attractant both by the SGPs and by other tissues when hmgcr is ectopically expressed. We show that production of this PGC attractant depends upon shf as well as a second hh pathway gene gγ1. Further linking the PGC attractant to Hh, we present evidence indicating that ectopic expression of hmgcr in the nervous system promotes the release/transmission of the Hh ligand from these cells into and through the underlying mesodermal cell layer, where Hh can contact migrating PGCs. Finally, potentiation of Hh by hmgcr appears to depend upon cholesterol modification.

Abiotic synthesis of purines and other heterocyclic compounds by the action of electrical discharges

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Yuasa, S.; Flory, D.; Basile, B.; Oro, J.

1984-01-01

The synthesis of purines and pyrimidines using Oparin-Urey-type primitive earth atmospheres has been demonstrated by reacting methane, ethane, and ammonia in electrical discharges. Adenine, guaine, 4-aminoimidazole-5-carboxamide (AICA), and isocytosine have been identified by UV spectrometry and paper chromatography as the products of the reaction. The total yields of the identified heterocyclic compounds are 0.0023 percent. It is concluded that adenine synthesis occurs at a much lower concentration of hydrogen cyanide than has been shown by earlier studies. Pathways for the synthesis of purines from hydrogen cyanide are discussed, and a comparison of the heterocyclic compounds that have been identified in meteorites and in prebiotic reactions is presented.

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism.

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Fukuuchi, T; Iyama, N; Yamaoka, N; Kaneko, K

2018-04-13

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.

Alterations in tryptophan and purine metabolism in cocaine addiction: a metabolomic study.

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Patkar, Ashwin A; Rozen, Steve; Mannelli, Paolo; Matson, Wayne; Pae, Chi-Un; Krishnan, K Ranga; Kaddurah-Daouk, Rima

2009-10-01

Mapping metabolic "signatures" can provide new insights into addictive mechanisms and potentially identify biomarkers and therapeutic targets. We examined the differences in metabolites related to the tyrosine, tryptophan, purine, and oxidative stress pathways between cocaine-dependent subjects and healthy controls. Several of these metabolites serve as biological indices underlying the mechanisms of reinforcement, toxicity, and oxidative stress. Metabolomic analysis was performed in 18 DSM-IV-diagnosed cocaine-dependent individuals with at least 2 weeks of abstinence and ten drug-free controls. Plasma concentrations of 37 known metabolites were analyzed and compared using a liquid chromatography electrochemical array platform. Multivariate analyses were used to study the relationship between severity of drug use [Addiction Severity Index (ASI) scores] and biological measures. Cocaine subjects showed significantly higher levels of n-methylserotonin (p cocaine and control groups with no overlap. Alterations in the methylation processes in the serotonin pathways and purine metabolism seem to be associated with chronic exposure to cocaine. Given the preliminary nature and cross-sectional design of the study, the findings need to be confirmed in larger samples of cocaine-dependent subjects, preferably in a longitudinal design.

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings.

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Kubasek, WL; Shirley, BW; McKillop, A; Goodman, HM; Briggs, W; Ausubel, FM

1992-01-01

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor. PMID:12297632

Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

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Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

2015-09-30

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling.

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Park, Joon-Heum; Jung, Sunyo

2017-01-22

In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F v /F m . NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity

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Tobias Fromme

2018-02-01

Full Text Available Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation. Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration. Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation. Keywords: Purine nucleotides, Uncoupling protein 1, Brown adipose tissue, Non-shivering thermogenesis, HILIC-MS/MS, Guanosine monophosphate reductase

The purine-utilizing bacterium Clostridium acidurici 9a: a genome-guided metabolic reconsideration.

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Katrin Hartwich

Full Text Available Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists of one chromosome (3,105,335 bp and one small circular plasmid (2,913 bp. The lack of candidate genes encoding glycine reductase indicates that C. acidurici 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of C. acidurici 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as C. acetobutylicum, C. saccharolyticum, and C. beijerinckii. The only amino acid that can be degraded by C. acidurici is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by C. acidurici but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of C. acidurici towards bacitracin, acriflavine and azaleucine was experimentally confirmed.

The purine-utilizing bacterium Clostridium acidurici 9a: a genome-guided metabolic reconsideration.

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Hartwich, Katrin; Poehlein, Anja; Daniel, Rolf

2012-01-01

Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists of one chromosome (3,105,335 bp) and one small circular plasmid (2,913 bp). The lack of candidate genes encoding glycine reductase indicates that C. acidurici 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of C. acidurici 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as C. acetobutylicum, C. saccharolyticum, and C. beijerinckii. The only amino acid that can be degraded by C. acidurici is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by C. acidurici but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of C. acidurici towards bacitracin, acriflavine and azaleucine was experimentally confirmed.

A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes

Energy Technology Data Exchange (ETDEWEB)

Tran, Timothy H. [Cornell University, Ithaca, New York 14853-1301 (United States); Krishnamoorthy, Kalyanaraman; Begley, Tadhg P., E-mail: begley@tamu.edu [Texas A& M University, College Station, TX 77842 (United States); Ealick, Steven E., E-mail: begley@tamu.edu [Cornell University, Ithaca, New York 14853-1301 (United States)

2011-10-01

MetY is the first reported structure of an O-acetylhomoserine sulfhydrylase that utilizes a protein thiocarboxylate intermediate as the sulfur source in a novel methionine-biosynthetic pathway instead of catalyzing a direct sulfhydrylation reaction. O-Acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5′-phosphate (PLP) dependent sulfide-utilizing enzyme in the l-cysteine and l-methionine biosynthetic pathways of various enteric bacteria and fungi. OAHS catalyzes the conversion of O-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. However, the source of the sulfur has not been identified and no structures of OAHS have been reported in the literature. Here, the crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution is reported. MetY crystallized in space group C2 with two monomers in the asymmetric unit. Size-exclusion chromatography, dynamic light scattering and crystal packing indicate that the biological unit is a tetramer in solution. This is further supported by the crystal structure, in which a tetramer is formed using a combination of noncrystallographic and crystallographic twofold axes. A search for structurally homologous proteins revealed that MetY has the same fold as cystathionine γ-lyase and methionine γ-lyase. The active sites of these enzymes, which are also PLP-dependent, share a high degree of structural similarity, suggesting that MetY belongs to the γ-elimination subclass of the Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.

Transcriptomic analysis of genes in the nitrogen recycling pathway of meat-type chickens divergently selected for feed efficiency.

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Aggrey, S E; Lee, J; Karnuah, A B; Rekaya, R

2014-04-01

The understanding of the dynamics of ammonia detoxification and excretion in uricotelic species is lagging behind ureotelic species. The relative expression of genes involved in nitrogen recycling and feed efficiency in chickens is unknown. The objective of this study was to investigate the transcriptomics differences in key genes in the nitrogen (N) metabolism and purine biosynthesis pathway in a chicken population divergently selected for low (LRFI) or high (HRFI) residual feed intake at days 35 and 42 using duodenum, liver, pectoralis major (P. major) and kidney. There was a significant positive correlation between RFI and fecal N. The purine salvage pathway was activated in the LRFI compared with HRFI at days 42. The birds in the LRFI population attained greater feed efficiency by having lower FI, increasing their protein retention and producing adequate glutamine to maintain growth compared with the HRFI line. To maintain growth, excess N is deaminated mostly to generate purine nucleotides. Generating purine nucleotides primarily from the purine biosynthesis pathway is energetically costly, and to preserve energy, they preferentially generate nucleotides from the purine salvage pathway. The LRFI birds need to generate sufficient nucleotides to maintain growth despite reduced FI that then results in reduced fecal N. © 2013 Stichting International Foundation for Animal Genetics.

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants.

Science.gov (United States)

Ruiz-López, Noemi; Sayanova, Olga; Napier, Johnathan A; Haslam, Richard P

2012-04-01

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.

Using purine skews to predict genes in AT-rich poxviruses

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Upton Chris

2005-02-01

Full Text Available Abstract Background Clusters or runs of purines on the mRNA synonymous strand have been found in many different organisms including orthopoxviruses. The purine bias that is exhibited by these clusters can be observed using a purine skew and in the case of poxviruses, these skews can be used to help determine the coding strand of a particular segment of the genome. Combined with previous findings that minor ORFs have lower than average aspartate and glutamate composition and higher than average serine composition, purine content can be used to predict the likelihood of a poxvirus ORF being a "real gene". Results Using purine skews and a "quality" measure designed to incorporate previous findings about minor ORFs, we have found that in our training case (vaccinia virus strain Copenhagen, 59 of 65 minor (small and unlikely to be a real genes ORFs were correctly classified as being minor. Of the 201 major (large and likely to be real genes vaccinia ORFs, 192 were correctly classified as being major. Performing a similar analysis with the entomopoxvirus amsacta moorei (AMEV, it was found that 4 major ORFs were incorrectly classified as minor and 9 minor ORFs were incorrectly classified as major. The purine abundance observed for major ORFs in vaccinia virus was found to stem primarily from the first codon position with both the second and third codon positions containing roughly equal amounts of purines and pyrimidines. Conclusion Purine skews and a "quality" measure can be used to predict functional ORFs and purine skews in particular can be used to determine which of two overlapping ORFs is most likely to be the real gene if neither of the two ORFs has orthologs in other poxviruses.

Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

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Netzer, Roman; Stafsnes, Marit H; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

2010-11-01

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

Catalase increases ethanol oxidation through the purine catabolism in rat liver.

Science.gov (United States)

Villalobos-García, Daniel; Hernández-Muñoz, Rolando

2017-08-01

Hepatic ethanol oxidation increases according to its concentration and is raised to near-saturation levels of alcohol dehydrogenase (ADH); therefore, re-oxidation of NADH becomes rate limiting in ethanol metabolism by the liver. Adenosine is able to increase liver ethanol oxidation in both in vivo and in vitro conditions; the enhancement being related with the capacity of the nucleoside to accelerate the transport of cytoplasmic reducing equivalents to mitochondria, by modifying the subcellular distribution of the malate-aspartate shuttle components. In the present study, we explored the putative effects of adenosine and other purines on liver ethanol oxidation mediated by non-ADH pathways. Using the model of high precision-cut rat liver slices, a pronounced increase of ethanol oxidation was found in liver slices incubated with various intermediates of the purine degradation pathway, from adenosine to uric acid (175-230%, over controls). Of these, urate had the strongest (230%), whereas xanthine had the less pronounced effect (178% over controls). The enhancement was not abolished by 4-methylpyrazole, indicating that the effect was independent of alcohol dehydrogenase. Conversely, aminotriazole, a catalase inhibitor, completely abolished the effect, pointing out that this enhanced ethanol oxidation is mediated by catalase activity. It is concluded that the H 2 O 2 needed for catalase activity is derived from the oxidation of (hypo)xanthine by xanthine oxidase and the oxidation of urate by uricase. The present and previous data led us to propose that, depending on the metabolic conditions, adenosine might be able to stimulate the metabolism of ethanol through different pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition by methotrexate (MTX) polyglutamates (PGS) of folate-dependent biosyntheses in L1210 Leukemia cells

International Nuclear Information System (INIS)

Matherly, L.H.; Barlowe, C.K.; Goldman, I.D.

1986-01-01

The inhibition of folate-dependent pathways by MTX PGS was evaluated in folate-depleted L1210 cells incubated with (6S)5-formyl(CHO)tetrahydrofolate(FH 4 )(5μM). The accumulation of MTX PGS during exposure to MTX (10μM;3h) inhibited cell growth (>70%) under these conditions. In the presence of 5-CHO-FH 4 , carbon transfer from 14 C-formate or 3- 14 C-serine into purines, dTMP, and amino acids was suppressed following MTX-pretreatment, suggesting the formation of only low levels of FH 4 to drive these reactions. In cells treated with MTX (6S)5-CHO-[ 3 H]-FH 4 was metabolized predominantly to 10-CHO-[ 3 H]-FH 4 . While intracellular dihydrofolate (FH 2 ) increased 10-fold, indicating a block at FH 2 reductase by MTX PGS, FH 2 represented only 20% of the total metabolites of 5-CHO-FH 4 . The incorporation of 14 C from 5-[ 14 C]-CHO-FH 4 into serine and methionine was not affected by the presence of intracellular MTX PGS, however, carbon transfer into dTMP and purine nucleotides was reduced (50-60%). These findings demonstrate that MTX pretreatment inhibits de novo nucleotide and amino acid biosynthetic pathways even when high levels of reduced folates are present. The data suggest a suppression of dTMP synthase and the purine transformylase(s) by MTX and/or FH 2 PGS that accumulate in drug-treated cells. Inhibition of the purine biosynthetic steps appears to trap 10-CHO-FH 4 , limiting FH 4 for the synthesis of dTMP, serine, and methionine

Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production.

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Lu Jin

Full Text Available Caffeine (1, 3, 7-trimethylxanthine and theobromine (3, 7-dimethylxanthine are the major purine alkaloids in plants, e.g., tea (Camellia sinensis and coffee (Coffea arabica. Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT and Camellia sinensis caffeine synthase (TCS in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.

Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production.

Science.gov (United States)

Jin, Lu; Bhuiya, Mohammad Wadud; Li, Mengmeng; Liu, XiangQi; Han, Jixiang; Deng, WeiWei; Wang, Min; Yu, Oliver; Zhang, Zhengzhu

2014-01-01

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g., tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.

Glucagon infusion increases rate of purine synthesis de novo in rat liver

International Nuclear Information System (INIS)

Itakura, Mitsuo; Maeda, Noriaki; Tsuchiya, Masami; Yamashita, Kamejiro

1987-01-01

Based on the parallel increases of glucagon, the second peak of hepatic cAMP, and the rate of purine synthesis de novo in the prereplicative period in regenerating rate liver after a 70% hepatectomy, it was hypothesized that glucagon is responsible for the increased rate of purine synthesis de novo. To test this hypothesis, the effect of glucagon or dibutyryl cAMP infusion on the rate of purine synthesis de novo in rat liver was studied. Glucagon infusion but not insulin or glucose infusion increased the rate of purine synthesis de novo, which was assayed by [ 14 C]glycine or [ 14 C]formate incorporation, by 2.7- to 4.3-fold. Glucagon infusion increased cAMP concentrations by 4.9-fold and 5-phosphoribosyl-1-pyrophosphate concentrations by 1.5-fold in liver but did not change the specific activity of amidophosphoribosyltransferase or purine ribonucleotide concentrations. Dibutyryl cAMP infusion also increased the rate of purine synthesis de novo by 2.2- to 4.0-fold. Because glucagon infusion increased the rate of purine synthesis de novo in the presence of unchanged purine ribonucleotide concentrations, it is concluded that glucagon after infusion or in animals after a 70% hepatectomy is playing an anabolic role to increase the rate of purine synthesis de novo by increasing cAMP and 5-phosphoribosyl-1-pyrophosphate concentrations

Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production

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Juwairiah Remali

2017-11-01

Full Text Available Background Streptomyces are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy carbonyl phenazine-1-carboxylic acid (HCPCA extracted from Streptomyces kebangsaanensis, an endophyte isolated from the ethnomedicinal Portulaca oleracea. Methods The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of S. kebangsaanensis in producing other useful secondary metabolites. Results The S. kebangsaanensis genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35% consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis. Discussion The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of S. kebangsaanensis for producing various antibiotics and secondary metabolites.

Stretches of alternating pyrimidine/purines and purines are respectively linked with pathogenicity and growth temperature in prokaryotes

DEFF Research Database (Denmark)

Ussery, David; Bohlin, J; Hardy, SP

2009-01-01

BACKGROUND: The genomic fractions of purine (RR) and alternating pyrimidine/purine (YR) stretches of 10 base pairs or more, have been linked to genomic AT content, the formation of different DNA helices, strand-biased gene distribution, DNA structure, and more. Although some of these factors are ...... phyla. RR stretches are overrepresented in all phyla except for the Actinobacteria and beta-Proteobacteria. In contrast, YR tracts are underrepresented in all phyla except for the beta-Proteobacterial group. YR-stretches are associated with phylum, pathogenicity and habitat, whilst RR...

A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

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Borui Pi

Full Text Available Secondary metabolites (SMs produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

A Genomics Based Discovery of Secondary Metabolite Biosynthetic Gene Clusters in Aspergillus ustus

Science.gov (United States)

Pi, Borui; Yu, Dongliang; Dai, Fangwei; Song, Xiaoming; Zhu, Congyi; Li, Hongye; Yu, Yunsong

2015-01-01

Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic. PMID:25706180

Heterosis and heritability estimates of purine alkaloids and ...

African Journals Online (AJOL)

Dry cocoa beans displayed high content of purine alkaloids (2.1 and 8.8 mg g-1 for caffein and theobromine, respectively), and polyphenols (25 and 2978 μg g-1 for catechin and epicatechin, respectively). Among the five cocoa clones, SNK16 was the highest in purine alkaloid (caffein and theobromin) and flavanol ...

Carotenoid Biosynthetic Pathways Are Regulated by a Network of Multiple Cascades of Alternative Sigma Factors in Azospirillum brasilense Sp7.

Science.gov (United States)

Rai, Ashutosh Kumar; Dubey, Ashutosh Prakash; Kumar, Santosh; Dutta, Debashis; Mishra, Mukti Nath; Singh, Bhupendra Narain; Tripathi, Anil Kumar

2016-11-01

Carotenoids constitute an important component of the defense system against photooxidative stress in bacteria. In Azospirillum brasilense Sp7, a nonphotosynthetic rhizobacterium, carotenoid synthesis is controlled by a pair of extracytoplasmic function sigma factors (RpoEs) and their cognate zinc-binding anti-sigma factors (ChrRs). Its genome harbors two copies of the gene encoding geranylgeranyl pyrophosphate synthase (CrtE), the first critical step in the carotenoid biosynthetic pathway in bacteria. Inactivation of each of two crtE paralogs found in A. brasilense caused reduction in carotenoid content, suggesting their involvement in carotenoid synthesis. However, the effect of crtE1 deletion was more pronounced than that of crtE2 deletion. Out of the five paralogs of rpoH in A. brasilense, overexpression of rpoH1 and rpoH2 enhanced carotenoid synthesis. Promoters of crtE2 and rpoH2 were found to be dependent on RpoH2 and RpoE1, respectively. Using a two-plasmid system in Escherichia coli, we have shown that the crtE2 gene of A. brasilense Sp7 is regulated by two cascades of sigma factors: one consisting of RpoE1and RpoH2 and the other consisting of RpoE2 and RpoH1. In addition, expression of crtE1 was upregulated indirectly by RpoE1 and RpoE2. This study shows, for the first time in any carotenoid-producing bacterium, that the regulation of carotenoid biosynthetic pathway involves a network of multiple cascades of alternative sigma factors. Carotenoids play a very important role in coping with photooxidative stress in prokaryotes and eukaryotes. Although extracytoplasmic function (ECF) sigma factors are known to directly regulate the expression of carotenoid biosynthetic genes in bacteria, regulation of carotenoid biosynthesis by one or multiple cascades of sigma factors had not been reported. This study provides the first evidence of the involvement of multiple cascades of sigma factors in the regulation of carotenoid synthesis in any bacterium by showing the

Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters

DEFF Research Database (Denmark)

Blin, Kai; Kim, Hyun Uk; Medema, Marnix H.

2017-01-01

Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly...... conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses...... are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats...

Differential hexosamine biosynthetic pathway gene expression with type 2 diabetes

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Megan Coomer

2014-01-01

Full Text Available The hexosamine biosynthetic pathway (HBP culminates in the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc onto serine/threonine residues of target proteins. The HBP is regulated by several modulators, i.e. O-linked β-N-acetylglucosaminyl transferase (OGT and β-N-acetylglucosaminidase (OGA catalyze the addition and removal of O-GlcNAc moieties, respectively; while flux is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFPT, transcribed by two genes, GFPT1 and GFPT2. Since increased HBP flux is glucose-responsive and linked to insulin resistance/type 2 diabetes onset, we hypothesized that diabetic individuals exhibit differential expression of HBP regulatory genes. Volunteers (n = 60; n = 20 Mixed Ancestry, n = 40 Caucasian were recruited from Stellenbosch and Paarl (Western Cape, South Africa and classified as control, pre- or diabetic according to fasting plasma glucose and HbA1c levels, respectively. RNA was purified from leukocytes isolated from collected blood samples and OGT, OGA, GFPT1 and GFPT2 expressions determined by quantitative real-time PCR. The data reveal lower OGA expression in diabetic individuals (P < 0.01, while pre- and diabetic subjects displayed attenuated OGT expression vs. controls (P < 0.01 and P < 0.001, respectively. Moreover, GFPT2 expression decreased in pre- and diabetic Caucasians vs. controls (P < 0.05 and P < 0.01, respectively. We also found ethnic differences, i.e. Mixed Ancestry individuals exhibited a 2.4-fold increase in GFPT2 expression vs. Caucasians, despite diagnosis (P < 0.01. Gene expression of HBP regulators differs between diabetic and non-diabetic individuals, together with distinct ethnic-specific gene profiles. Thus differential HBP gene regulation may offer diagnostic utility and provide candidate susceptibility genes for different ethnic groupings.

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

Science.gov (United States)

Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

2014-03-13

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

Energy Technology Data Exchange (ETDEWEB)

Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Han, Li; Huang, Xueshi [Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819 (China); He, Jing, E-mail: hejingjj@mail.hzau.edu.cn [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China)

2016-04-22

Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.

Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

International Nuclear Information System (INIS)

Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan; Han, Li; Huang, Xueshi; He, Jing

2016-01-01

Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.

Characterization and engineering of thermophilic aldolases : synthesizing nitrogen-heterocycles in biosynthetic routes

NARCIS (Netherlands)

Wolterink-van Loo, S.

2009-01-01

Aldolases are enzymes that catalyze reactions in both degradation and biosynthetic pathways in vivo and have been discovered in all domains of life. they. An interesting property of aldolases is that they can synthesize carbon-carbon bonds, generating a new stereogenic centre. As enzymes are

Bias of purine stretches in sequenced chromosomes

DEFF Research Database (Denmark)

Ussery, David; Soumpasis, Dikeos Mario; Brunak, Søren

2002-01-01

/pur tracts was slightly less than expected, with an average of 0.8%. One of the most surprising findings is a clear difference in the length distributions of the regions studied between prokaryotes and eukaryotes. Whereas short-range correlations can explain the length distributions in prokaryotes......, in eukaryotes there is an abundance of long stretches of purines or alternating purine/pyrimidine tracts, which cannot be explained in this way; these sequences are likely to play an important role in eukaryotic chromosome organisation....

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

Science.gov (United States)

Pisithkul, Tippapha; Jacobson, Tyler B; O'Brien, Thomas J; Stevenson, David M; Amador-Noguez, Daniel

2015-09-01

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-labeled sugars and [(15)N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. Copyright © 2015, Pisithkul et al.

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis

Science.gov (United States)

Pisithkul, Tippapha; Jacobson, Tyler B.; O'Brien, Thomas J.; Stevenson, David M.

2015-01-01

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. PMID:26070680

Differential control of the cholesterol biosynthetic pathway in tumor versus liver: evidence for decontrolled tumor cholesterogenesis in a cell-free system

International Nuclear Information System (INIS)

Azrolan, N.

1987-01-01

Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant (PMS) systems prepared from both normal rat liver and Morris hepatoma 3924A. Per cell, the rate of cholesterol synthesis from either 14 C-citrate of 14 -acetate in the hepatoma system was 9-fold greater than that observed in the liver system. Furthermore, the ratio of sterol-to-fatty acid synthesis rates from 14 C-citrate was more than 3-fold greater in the tumor than in the normal liver system. Incubations using radiolabeled acetate and mevalonate have demonstrated the loss of a normally rate-limiting control site within the early portion of the cholesterol biosynthetic pathway in the tumor system. Upon analysis of the steady-state levels of early lipogenic intermediates, the specific site of decontrol in the tumor was identified as the 3-hydroxy-3-methylglutaryl-CoA → mevalonate site of this pathway. In contrast, this reaction appeared to retain its rate-limiting properties in the cell-free system from normal liver

Isotopically labelled pyrimidines and purines

International Nuclear Information System (INIS)

Balaban, A.T.; Bally, I.

1987-01-01

Among the three diazines, pyrimidine is by far the most important one because its derivatives uracil, thymine and cytosine are constituents of the ubiquitous deoxynucleic acids (DNA) and ribonucleic acids (RNA). Other derivatives of pyrimidine without condensed rings include barbiturates, alloxan, orotic acid and thiamine or vitamin B 1 . From the polycyclic derivatives of pyrimidine such as pteridine, alloxazine, and purine, the latter, through its derivatives adenine and guanine complete the list of bases which occur in DNA and RNA: in addition, other purine derivatives such as hypoxanthine, xanthine, theobromine, theophylline, caffeine and uric acid are important natural products with biological activity. The paper presents methods for preparing isotopically labeled pyrimidines as well as purine derivatives. For convenience, the authors describe separately carbon-labeled with radioisotopes 11 C (T 1/2 = 20.3 min) and 14 C (T 1/2 = 5736 years) or the stable isotope 13 C (natural abundance 1.1%) and then hydrogen-labeled systems with the radioisotope 3 H ≡ T (T 1/2 = 12.346 years) or with the stable isotope 2 H ≡ D (natural abundance 0.015%). We do not separate stable from radioactive isotopes because the synthetic methods are identical for the same element; however, the introduction of hydrogen isotopes into organic molecules is often performed by reactions such as isotope exchange which cannot take place in the case of carbon isotopes

T.C.G triplet in an antiparallel purine.purine.pyrimidine DNA triplex. Conformational studies by NMR.

Science.gov (United States)

Dittrich, K; Gu, J; Tinder, R; Hogan, M; Gao, X

1994-04-12

The antiparallel purine.purine.pyrimidine DNA triplex, RRY6, which contains a T.C.G inverted triplet in the center of the sequence, was examined by proton and phosphorous two-dimensional NMR spectroscopy. The local conformation of the T.C.G triplet (T4.C11.G18) and the effect of this triplet on the global helical structure were analyzed in detail. The formation of the T.C.G triplet is confirmed by a set of cross-strand NOEs, including unusual cross-strand NOEs between the third strand and the pyrimidine strand as opposed to the purine strand of the duplex. NMR data suggest that the T.C.G triplet may be present in an equilibrium between a non-hydrogen-bonded form and a T(O4)-C(NH2) hydrogen-bonded form and that there is a distortion of the in-plane alignment of the three bases. The flanking G.G.C base triplets are well-defined on the 5'-side of T4, but somewhat interrupted on the 3'-side of T4. The effect of the third strand binding on the Watson-Crick duplex was probed by an NMR study of the free duplex RY6. NMR parameters are affected mostly around the T.C.G inversion site. The perturbations extend to at least two adjacent base triplets on either side. The binding of the third purine strand and the accommodation of a central T.C.G inversion in RRY6 does not require a readjustment in sugar pucker, which remains in the range of C2'-endo. 31P resonances of RRY6 distribute over a range of 2.2 ppm. The H-P coupling patterns of the third strand differ from those of the duplex. General spectral patterns defined by the marker protons of the RRY and YRY triplexes are compared.

Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin

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Rebecca Yee

2015-12-01

Full Text Available Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus.

A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

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Wenjun Huang

Full Text Available Herba epimedii (Epimedium, a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1 from Epimedium sagittatum (Sieb. Et Zucc. Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR and anthocyanidin synthase (ANS. In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

Science.gov (United States)

Huang, Wenjun; Sun, Wei; Lv, Haiyan; Luo, Ming; Zeng, Shaohua; Pattanaik, Sitakanta; Yuan, Ling; Wang, Ying

2013-01-01

Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

DEFF Research Database (Denmark)

Liu, Chengwei; Tagami, Koichi; Minami, Atsushi

2015-01-01

KULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products....

Estimation of rumen microbial-nitrogen of sheep using urinary excretion of purine derivatives

International Nuclear Information System (INIS)

Liu Dasen; Shan Anshan

2004-01-01

Determination of rumen microbial-nitrogen of sheep using urinary excretion of purine derivative was studied. Uric acid and xanthine + hypoxanthine were not affected by diets, but total purine derivatives for 1 mg borax/kg diet was higher than other diets (p<0.05). Microbial-nitrogen estimated from allantoin was not affected by diets, but that of 1 mg borax/kg diet estimated from total purine derivatives was higher than other diets (p<0.05). Microbial-nitrogen estimated from total purine derivatives was higher than that from allantoin

Characterization of the biosynthetic gene cluster for cryptic phthoxazolin A in Streptomyces avermitilis.

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Dian Anggraini Suroto

Full Text Available Phthoxazolin A, an oxazole-containing polyketide, has a broad spectrum of anti-oomycete activity and herbicidal activity. We recently identified phthoxazolin A as a cryptic metabolite of Streptomyces avermitilis that produces the important anthelmintic agent avermectin. Even though genome data of S. avermitilis is publicly available, no plausible biosynthetic gene cluster for phthoxazolin A is apparent in the sequence data. Here, we identified and characterized the phthoxazolin A (ptx biosynthetic gene cluster through genome sequencing, comparative genomic analysis, and gene disruption. Sequence analysis uncovered that the putative ptx biosynthetic genes are laid on an extra genomic region that is not found in the public database, and 8 open reading frames in the extra genomic region could be assigned roles in the biosynthesis of the oxazole ring, triene polyketide and carbamoyl moieties. Disruption of the ptxA gene encoding a discrete acyltransferase resulted in a complete loss of phthoxazolin A production, confirming that the trans-AT type I PKS system is responsible for the phthoxazolin A biosynthesis. Based on the predicted functional domains in the ptx assembly line, we propose the biosynthetic pathway of phthoxazolin A.

Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro Biosynthetic Pathway in Streptomyces sp. US24 Strain

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Samiha Sioud

2007-01-01

Full Text Available We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro diketopiperazine (DKP. To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS. The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

Effect of purines on calcium-independent acetylcholine release at the mouse neuromuscular junction.

Science.gov (United States)

Veggetti, M; Muchnik, S; Losavio, A

2008-07-17

At the mouse neuromuscular junction, activation of adenosine A(1) and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca(2+) influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca(2+)-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) and the P2Y(12-13) agonist 2-methylthio-adenosine 5'-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A(1) and P2Y(12-13) receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran

Purine derivate content and amino acid profile in larval stages of three edible insects.

Science.gov (United States)

Bednářová, Martina; Borkovcová, Marie; Komprda, Tomáš

2014-01-15

Considering their high content of protein, insects are a valuable alternative protein source. However, no evaluation of their purine content has so far been done. High content of purine derivates may lead to the exclusion of such food from the diet of people with specific diseases. The aim of this study was to analyse the content of selected purine derivates and amino acid profile in the three insect species most often used for entomophagy in Europe and compare them with the purine content in egg white and chicken breast. The content of individual purine derivates and their total content were significantly dependent on insect species. The purine content in all three species was significantly higher (P < 0.05) than in egg white, but some values were significantly lower (P < 0.05) than in chicken breast. The total protein content was 548.9 g kg(-1) dry matter (DM) in mealworm (Tenebrio molitor), 551.6 g kg(-1) DM in superworm (Zophobas atratus) and 564.9 g kg(-1) DM in cricket (Gryllus assimilis). Larvae of mealworm and superworm are protein-rich and purine-low meat alternatives. In contrast, cricket nymphs are protein-rich and purine-rich and cannot be recommended for people with hyperuricemia or gout. © 2013 Society of Chemical Industry.

Biological treatment in clarificated purines pilot plant; Depuracion biologica en plant piloto del clarificado de los purines

Energy Technology Data Exchange (ETDEWEB)

Bosque Hernandez, J. L. del; Martin Sanchez, J. L. [Universidad de Salamanca (Spain)

2004-07-01

With the purpose of eliminating the environmental problem that suppose the residuals, denominated. Purines, it is necessary to treat them so that they can take advantage. for it, in the part I of the work, the treatment that facilitates the operation of separation of the solid in suspension of the raw purin that allows to decrease the matter organic present in the liquid phase, settled down as well as, the increase of the power fertilizer in the solid phase. The part II of this investigation have as purpose the treatment of the phase it liquidates obtained previously to adapt it for their use like water of industrial type and the solid phase as fertilizer. (Author) 5 refs.

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

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Gonzalez, Antonio; Zhao, Mingzhe; Leavitt, John M; Lloyd, Alan M

2008-03-01

In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

Purine Restriction Induces Pronounced Translational Upregulation of the NT1 Adenosine/Pyrimidine Nucleoside Transporter in Leishmania major

OpenAIRE

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A.; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M.

2010-01-01

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gen...

Distinct Purine Distribution in Carbonaceous Chondrites

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Callahan, Michael P.; Smith, Karen E.; Cleaves, Henderson J.; Ruzicka, Josef; Stern, Jennifer C.; Glavin, Daniel P.; House, Christopher H.; Dworkin, Jason P.

2011-01-01

Carbonaceous chondrite meteorites are known to contain a diverse suite of organic compounds, many of which are essential components of biochemistry. Amino acids, which are the monomers of proteins, have been extensively studied in such meteorites (e.g. Botta and Bada 2002; Pizzarello et aI., 2006). The origin of amino acids in meteorites has been firmly established as extraterrestrial based on their detection typically as racemic mixtures of amino acids, the presence of many non-protein amino acids, and non-terrestrial values for compound-specific deuterium, carbon, and nitrogen isotopic measurements. In contrast to amino acids, nucleobases in meteorites have been far less studied. Nucleobases are substituted one-ring (pyrimidine) or two-ring (purine) nitrogen heterocyclic compounds and serve as the information carriers of nucleic acids and in numerous coenzymes. All of the purines (adenine, guanine, hypoxanthine, and xanthine) and pyrimidines (uracil) previously reported in meteorites are biologically common and could be interpreted as the result of terrestrial contamination (e.g. van del' Velden and Schwartz, 1974.) Unlike other meteoritic organics, there have been no observations of stochastic molecular diversity of purines and pyrimidines in meteorites, which has been a criterion for establishing extraterrestrial origin. Maltins et al. (2008) performed compound-specific stable carbon isotope measurements for uracil and xanthine in the Murchison meteorite. They assigned a non-terrestrial origin for these nucleobases; however, the possibility that interfering indigenous molecules (e.g. carboxylic acids) contributed to the 13C-enriched isotope values for these nucleobases cannot be completely ruled out. Thus, the origin of these meteoritic nucleobases has never been established unequivocally. Here we report on our investigation of extracts of II different carbonaceous chondrites covering various petrographic types (Cl, CM, and CR) and degrees of aqueous alteration

The crystal structure of the hexameric purine nucleoside phosphorylase from Bacillus subtilis in complex with adenosine

Energy Technology Data Exchange (ETDEWEB)

Giuseppe, P.O.; Meza, A.N.; Martins, N.H.; Santos, C.R.; Murakami, M.T. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil)

2012-07-01

Full text: Purine nucleoside phosphorylases (PNPs) play a key role in the purine-salvage pathway in both prokaryotes and eukaryotes. Its ribosyltransferase activity is of great biotechnological interest due to potential application in the synthesis of nucleoside analogues used in the treatment of antiviral infections and in anticancer chemotherapy. Trimeric PNPs are found mainly in vertebrates and are specific for 6-oxo-purines whereas hexameric PNPs are prevalent in prokaryotes and exhibit a broad range of substrates including 6-oxo and 6-amino purines. BsPNP233, the hexameric PNP from B. subtilis, is able to catalyze the bioconversion of ribavirin, an anti-viral drug, and is relatively thermostable, being a good target for industrial use. Here we report the crystal structures of BsPNP233 in the apo form and in complex with adenosine solved at 2.65 and 1.91 resolution, respectively. The apo and ligand-bound BsPNP233 subunits superposed with an overall r.m.s. deviation of 0.31 for all C{alpha} atoms, which suggests that no major conformational changes occur upon substrate binding. Based on the crystal structure of BsPNP233 in complex with adenosine we have defined the active site residues implicated in binding the ribose (H4{sup *}, R43{sup *}, M64, R87, E178, M179, E180) and the nitrogenous base (S90, C91, G92, S202, V177, F159). These residues are highly conserved among the bacterial hexameric PNPs, suggesting they share the same mode of interaction with the substrates. This work will probably contribute to a better understanding of the molecular basis for the broad substrate specificity of hexameric PNPs and to projects aiming the rational design of PNPs for industrial purposes. (author)

Experimental and theoretical dipole moments of purines in their ground and lowest excited singlet states

Science.gov (United States)

Aaron, Jean-Jacques; Diabou Gaye, Mame; Párkányi, Cyril; Cho, Nam Sook; Von Szentpály, László

1987-01-01

The ground-state dipole moments of seven biologically important purines (purine, 6-chloropurine, 6-mercaptopurine, hypoxanthine, theobromine, theophylline and caffeine) were determined at 25°C in acetic acid (all the above compounds with the exception of purine) and in ethyl acetate (purine, theophylline and caffeine). Because of its low solubility, it was not possible to measure the dipole moment of uric acid. The first excited singlet-state dipole moments were obtained on the basis of the Bakhshiev and Chamma—Viallet equations using the variation of the Stokes shift with the solvent dielectric constant-refractive index term. The theoretical dipole moments for all the purines listed above and including uric acid were calculated by combining the use of the PPP (π-LCI-SCF-MO) method for the π-contribution to the overall dipole moment with the σ-contribution obtained as a vector sum of the σbond moments and group moments. The experimental and theoretical values were compared with the data available in the literature for some of the purines under study. For several purines, the calculations were carried out for different tautomeric forms. Excited singlet-state dipole moments are smaller than the ground-state values by 0.8 to 2.2 Debye units for all purines under study with the exception of 6-chloropurine. The effects of the structure upon the ground- and excited-state dipole moments of the purines are discussed.

Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

Energy Technology Data Exchange (ETDEWEB)

Taylor,E.; Rinaldo-Matthis, A.; Li, L.; Ghanem, M.; Hazleton, K.; Cassera, M.; Almo, S.; Schramm, V.

2007-01-01

The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

Science.gov (United States)

Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

2016-07-01

Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L. leaves

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Gutha Linga R

2010-08-01

Full Text Available Abstract Background Symptoms of grapevine leafroll disease (GLRD in red-fruited wine grape (Vitis vinifera L. cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. The reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis. Results We examined six putative constitutively expressed genes, Ubiquitin, Actin, GAPDH, EF1-a, SAND and NAD5, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR. Using the geNorm program, a combination of two genes (Actin and NAD5 was identified as the stable set of reference genes for normalization of gene expression data obtained from grapevine leaves. By using gene-specific RT-qPCR in combination with a reliable normalization factor, we compared relative expression of the flavonoid biosynthetic pathway genes between leaves infected with Grapevine leafroll-associated virus 3 (GLRaV-3 and exhibiting GLRD symptoms and virus-free green leaves obtained from a red-fruited wine grape cultivar (cv. Merlot. The expression levels of these different genes ranged from two- to fifty-fold increase in virus-infected leaves. Among them, CHS3, F3'5'H, F3H1, LDOX, LAR1 and MybA1 showed greater than 10-fold increase suggesting that they were expressed at significantly higher levels in virus-infected symptomatic leaves. HPLC profiling of anthocyanins extracted from leaves indicated the presence of cyanidin-3-glucoside and malvidin-3-glucoside only in virus-infected symptomatic leaves. The results also showed 24% higher levels of flavonols in virus-infected symptomatic leaves than in virus-free green leaves, with quercetin followed by myricetin being the predominant compounds. Proanthocyanidins, estimated as total tannins by protein precipitation method, were 36% higher in virus

In situ localization of phenylpropanoid biosynthetic mRNAs and proteins in Parsley (Petroselinum crispum)

International Nuclear Information System (INIS)

Reinold, S.; Hahlbrock, K.

1997-01-01

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together constituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence. (author)

Rational engineering of p-hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway.

Science.gov (United States)

Chen, Zhenya; Shen, Xiaolin; Wang, Jian; Wang, Jia; Yuan, Qipeng; Yan, Yajun

2017-11-01

Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Versatile synthesis and biological evaluation of novel 3’-fluorinated purine nucleosides

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Hang Ren

2015-12-01

Full Text Available A unified synthetic strategy accessing novel 3'-fluorinated purine nucleoside derivatives and their biological evaluation were achieved. Novel 3’-fluorinated analogues were constructed from a common 3’-deoxy-3’-fluororibofuranose intermediate. Employing Suzuki and Stille cross-coupling reactions, fifteen 3’-fluororibose purine nucleosides 1–15 and eight 3’-fluororibose 2-chloro/2-aminopurine nucleosides 16–23 with various substituents at position 6 of the purine ring were efficiently synthesized. Furthermore, 3’-fluorine analogs of natural products nebularine and 6-methylpurine riboside were constructed via our convergent synthetic strategy. Synthesized nucleosides were tested against HT116 (colon cancer and 143B (osteosarcoma cancer tumor cell lines. We have demonstrated 3’-fluorine purine nucleoside analogues display potent tumor cell growth inhibition activity at sub- or low micromolar concentration.

Biosynthetic Studies of 13-Desmethylspirolide C Produced by Alexandrium ostenfeldii (= A. peruvianum): Rationalization of the Biosynthetic Pathway Following Incorporation of (13)C-Labeled Methionine and Application of the Odd-Even Rule of Methylation.

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Anttila, Matthew; Strangman, Wendy; York, Robert; Tomas, Carmelo; Wright, Jeffrey L C

2016-03-25

Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding (13)C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd-even methylation rule, the complete biosynthetic pathway has been established.

Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant.

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Ahmad-Faris Seman-Kamarulzaman

Full Text Available Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate

Elucidating the biosynthetic and regulatory mechanisms of flavonoid-derived bioactive components in Epimedium sagittatum

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Wenjun eHuang

2015-09-01

Full Text Available Herba epimedii (Epimedium, a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. In Epimedium, flavonoids have been demonstrated to be the main bioactive components (BCs. However, the molecular biosynthetic and regulatory mechanisms of flavonoid-derived BCs remain obscure. In this study, we isolated twelve structural genes and two putative transcription factors (TFs in the flavonoid pathway. Phytochemical analysis showed that the total content of four representative BCs (epimedin A, B, C and icariin decreased slightly or dramatically in two lines of E. sagittatum during leaf development. Transcriptional analysis revealed that two R2R3-MYB TFs (EsMYBA1 and EsMYBF1, together with a bHLH TF (EsGL3 and WD40 protein (EsTTG1, were supposed to coordinately regulate the anthocyanin and flavonol-derived BCs biosynthesis in leaves. Overexpression of EsFLS (flavonol synthase in tobacco resulted in increased flavonols content and decreased anthocyanins content in flowers. Moreover, EsMYB12 negatively correlated with the accumulation of the four BCs, and might act as a transcriptional repressor in the flavonoid pathway. Therefore, the anthocyanin pathway may coordinate with the flavonol-derived BCs pathway in Epimedium leaves. A better understanding of the flavonoid biosynthetic and regulatory mechanisms in E. sagittatum will facilitate functional characterization, metabolic engineering and molecular breeding studies of Epimedium species.

Enhanced activity of the purine nucleotide cycle of the exercising muscle in patients with hyperthyroidism.

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Fukui, H; Taniguchi , S; Ueta, Y; Yoshida, A; Ohtahara, A; Hisatome, I; Shigemasa, C

2001-05-01

Myopathy frequently develops in patients with hyperthyroidism, but its precise mechanism is not clearly understood. In this study we focused on the purine nucleotide cycle, which contributes to ATP balance in skeletal muscles. To investigate purine metabolism in muscles, we measured metabolites related to the purine nucleotide cycle using the semiischemic forearm test. We examined the following four groups: patients with untreated thyrotoxic Graves' disease (untreated group), patients with Graves' disease treated with methimazole (treated group), patients in remission (remission group), and healthy volunteers (control group). To trace the glycolytic process, we measured glycolytic metabolites (lactate and pyruvate) as well as purine metabolites (ammonia and hypoxanthine). In the untreated group, the levels of lactate, pyruvate, and ammonia released were remarkably higher than those in the control group. Hypoxanthine release also increased in the untreated group, but the difference among the patient groups was not statistically significant. The accelerated purine catabolism did not improve after 3 months of treatment with methimazole, but it was completely normalized in the remission group. This indicated that long-term maintenance of thyroid function was necessary for purine catabolism to recover. We presume that an unbalanced ATP supply or conversion of muscle fiber type may account for the acceleration of the purine nucleotide cycle under thyrotoxicosis. Such acceleration of the purine nucleotide cycle is thought to be in part a protective mechanism against a rapid collapse of the ATP energy balance in exercising muscles of patients with hyperthyroidism.

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

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Wei Sun

Full Text Available Chalcone synthase (CHS catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1 encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

Purine Bases in Blood Plasma of Patients with Chronic Pulmonary Diseases

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Larissa E. Muravluyova

2012-09-01

Full Text Available The article is focused on the study of purine bases and intermediates of purine catabolism in plasma of patients with chronic obstructive bronchitis and idiopathic interstitial pneumonia. Decrease of adenine and hypoxantine in plasma of patients with idiopathic interstitial pneumonia was registered. Increase of guanine in plasma of patients with chronic obstructive pulmonary disease was established.

Purines and Neuronal Excitability: Links to the Ketogenic Diet

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Masino, SA; Kawamura, M; Ruskin, DN; Geiger, JD; Boison, D

2011-01-01

ATP and adenosine are purines that play dual roles in cell metabolism and neuronal signaling. Acting at the A1 receptor (A1R) subtype, adenosine acts directly on neurons to inhibit excitability and is a powerful endogenous neuroprotective and anticonvulsant molecule. Previous research showed an increase in ATP and other cell energy parameters when an animal is administered a ketogenic diet, an established metabolic therapy to reduce epileptic seizures, but the relationship among purines, neuronal excitability and the ketogenic diet was unclear. Recent work in vivo and in vitro tested the specific hypothesis that adenosine acting at A1Rs is a key mechanism underlying the success of ketogenic diet therapy and yielded direct evidence linking A1Rs to the antiepileptic effects of a ketogenic diet. Specifically, an in vitro mimic of a ketogenic diet revealed an A1R-dependent metabolic autocrine hyperpolarization of hippocampal neurons. In parallel, applying the ketogenic diet in vivo to transgenic mouse models with spontaneous electrographic seizures revealed that intact A1Rs are necessary for the seizure-suppressing effects of the diet. This is the first direct in vivo evidence linking A1Rs to the antiepileptic effects of a ketogenic diet. Other predictions of the relationship between purines and the ketogenic diet are discussed. Taken together, recent research on the role of purines may offer new opportunities for metabolic therapy and insight into its underlying mechanisms. PMID:21880467

Quantitative evaluation of the biosynthetic pathways leading to δ-aminolevulinic acid from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus by analysis of 13C-labeled coproporphyrinogen III biosynthesized from [2-13C]glycine, [1-13C]acetate, and [2-13C]acetate using 13C NMR spectroscopy

International Nuclear Information System (INIS)

Katsumi Iida

2013-01-01

The biosynthetic pathways leading to δ-aminolevulinic acid (ALA) from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus were quantitatively evaluated by means of feeding experiments with [2- 13 C]glycine, sodium [1- 13 C]acetate, and sodium [2- 13 C]acetate, followed by analysis of the labeling patterns of coproporphyrinogen III (Copro'gen III) (biosynthesized from ALA) using 13 C NMR spectroscopy. Two biosynthetic pathways leading to ALA from glycine via the C5 pathway were identified: i.e., transformation of glycine to l-serine catalyzed by glycine hydroxymethyltransferase, and glycine synthase-catalyzed catabolism of glycine to N 5 , N 10 -methylene-tetrahydrofolic acid (THF), which reacts with another molecule of glycine to afford l-serine. l-Serine is transformed to acetyl-CoA via pyruvic acid. Acetyl-CoA enters the tricarboxylic acid cycle, affording 2-oxoglutaric acid, which in turn is transformed to l-glutamic acid. The l-glutamic acid enters the C5 pathway, affording ALA in A. hyalinus. A 13 C NMR spectroscopic comparison of the labeling patterns of Copro'gen III obtained after feeding of [2- 13 C]glycine, sodium [1- 13 C]acetate, and sodium [2- 13 C]acetate showed that [2- 13 C]glycine transformation and [2- 13 C]glycine catabolism in A. hyalinus proceed in the ratio of 52 and 48 %. The reaction of [2- 13 C]glycine and N 5 , N 10 -methylene-THF, that of glycine and N 5 , N 10 -[methylene- 13 C]methylene-THF generated from the [2- 13 C]glycine catabolism, and that of [2- 13 C]glycine and N 5 , N 10 -[methylene- 13 C]methylene-THF transformed the fed [2- 13 C]glycine to [1- 13 C]acetyl-CoA, [2- 13 C]acetyl-CoA, and [1,2- 13 C 2 ]acetyl-CoA in the ratios of 42, 37, and 21 %, respectively. These labeled acetyl-CoAs were then incorporated into ALA. Our results provide a quantitative picture of the pathways of biosynthetic transformation to ALA from glycine in A. hyalinus. (author)

Direct N9-arylation of purines with aryl halides

DEFF Research Database (Denmark)

Larsen, Anders Foller; Ulven, Trond

2014-01-01

An efficient method for N-arylation of purines is reported. The N-arylation is catalysed by Cu(i) and 4,7-bis(2-hydroxyethylamino)-1,10-phenanthroline (BHPhen) in aqueous DMF or ethanol. The reaction generally proceeds with high selectivity for the N(9)-position.......An efficient method for N-arylation of purines is reported. The N-arylation is catalysed by Cu(i) and 4,7-bis(2-hydroxyethylamino)-1,10-phenanthroline (BHPhen) in aqueous DMF or ethanol. The reaction generally proceeds with high selectivity for the N(9)-position....

EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

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Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

2017-03-01

The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1- 14 C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1- 14 C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

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Bo Wu

2009-07-01

Full Text Available Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole can interrupt transmission predominantly by killing microfilariae (mf larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs.Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP, which targets ferrochelatase (FC, the last step. Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA between Wolbachia and human 5

An unusual mode of DNA duplex association: Watson-Crick interaction of all-purine deoxyribonucleic acids.

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Battersby, Thomas R; Albalos, Maria; Friesenhahn, Michel J

2007-05-01

Nucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N(3)-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids.

Purines and Carotid Body: New Roles in Pathological Conditions

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Silvia V. Conde

2017-12-01

Full Text Available It is known that adenosine and adenosine-5′-triphosphate (ATP are excitatory mediators involved in carotid body (CB hypoxic signaling. The CBs are peripheral chemoreceptors classically defined by O2, CO2, and pH sensors. When hypoxia activates the CB, it induces the release of neurotransmitters from chemoreceptor cells leading to an increase in the action potentials frequency at the carotid sinus nerve (CSN. This increase in the firing frequency of the CSN is integrated in the brainstem to induce cardiorespiratory compensatory responses. In the last decade several pathologies, as, hypertension, diabetes, obstructive sleep apnea and heart failure have been associated with CB overactivation. In the first section of the present manuscript we review in a concise manner fundamental aspects of purine metabolism. The second section is devoted to the role of purines on the hypoxic response of the CB, providing the state-of-the art for the presence of adenosine and ATP receptors in the CB; for the role of purines at presynaptic level in CB chemoreceptor cells, as well as, its metabolism and regulation; at postsynaptic level in the CSN activity; and on the ventilatory responses to hypoxia. Recently, we have showed that adenosine is involved in CB hypersensitization during chronic intermittent hypoxia (CIH, which mimics obstructive sleep apnea, since caffeine, a non-selective adenosine receptor antagonist that inhibits A2A and A2B adenosine receptors, decreased CSN chemosensory activity in animals subjected to CIH. Apart from this involvement of adenosine in CB sensitization in sleep apnea, it was recently found that P2X3 ATP receptor in the CB contributes to increased chemoreflex hypersensitivity and hypertension in spontaneously hypertension rats. Therefore the last section of this manuscript is devoted to review the recent findings on the role of purines in CB-mediated pathologies as hypertension, diabetes and sleep apnea emphasizing the potential

Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major.

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Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M

2010-10-01

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations. © 2010 Blackwell Publishing Ltd.

Heterogeneity and dynamics of the ligand recognition mode in purine-sensing riboswitches.

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Jain, Niyati; Zhao, Liang; Liu, John D; Xia, Tianbing

2010-05-04

High-resolution crystal structures and biophysical analyses of purine-sensing riboswitches have revealed that a network of hydrogen bonding interactions appear to be largey responsible for discrimination of cognate ligands against structurally related compounds. Here we report that by using femtosecond time-resolved fluorescence spectroscopy to capture the ultrafast decay dynamics of the 2-aminopurine base as the ligand, we have detected the presence of multiple conformations of the ligand within the binding pockets of one guanine-sensing and two adenine-sensing riboswitches. All three riboswitches have similar conformational distributions of the ligand-bound state. The known crystal structures represent the global minimum that accounts for 50-60% of the population, where there is no significant stacking interaction between the ligand and bases of the binding pocket, but the hydrogen-bonding cage collectively provides an electronic environment that promotes an ultrafast ( approximately 1 ps) charge transfer pathway. The ligand also samples multiple conformations in which it significantly stacks with either the adenine or the uracil bases of the A21-U75 and A52-U22 base pairs that form the ceiling and floor of the binding pocket, respectively, but favors the larger adenine bases. These alternative conformations with well-defined base stacking interactions are approximately 1-1.5 kcal/mol higher in DeltaG degrees than the global minimum and have distinct charge transfer dynamics within the picosecond to nanosecond time regime. Inside the pocket, the purine ligand undergoes dynamic motion on the low nanosecond time scale, sampling the multiple conformations based on time-resolved anisotropy decay dynamics. These results allowed a description of the energy landscape of the bound ligand with intricate details and demonstrated the elastic nature of the ligand recognition mode by the purine-sensing riboswitches, where there is a dynamic balance between hydrogen bonding

Trisubstituted purine inhibitors of PDGFRα and their antileukemic activity in the human eosinophilic cell line EOL-1.

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Malínková, Veronika; Řezníčková, Eva; Jorda, Radek; Gucký, Tomáš; Kryštof, Vladimír

2017-12-15

Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G 1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Draft genome sequence of Streptomyces coelicoflavus ZG0656 reveals the putative biosynthetic gene cluster of acarviostatin family α-amylase inhibitors.

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Guo, X; Geng, P; Bai, F; Bai, G; Sun, T; Li, X; Shi, L; Zhong, Q

2012-08-01

The aims of this study are to obtain the draft genome sequence of Streptomyces coelicoflavus ZG0656, which produces novel acarviostatin family α-amylase inhibitors, and then to reveal the putative acarviostatin-related gene cluster and the biosynthetic pathway. The draft genome sequence of S. coelicoflavus ZG0656 was generated using a shotgun approach employing a combination of 454 and Solexa sequencing technologies. Genome analysis revealed a putative gene cluster for acarviostatin biosynthesis, termed sct-cluster. The cluster contains 13 acarviostatin synthetic genes, six transporter genes, four starch degrading or transglycosylation enzyme genes and two regulator genes. On the basis of bioinformatic analysis, we proposed a putative biosynthetic pathway of acarviostatins. The intracellular steps produce a structural core, acarviostatin I00-7-P, and the extracellular assemblies lead to diverse acarviostatin end products. The draft genome sequence of S. coelicoflavus ZG0656 revealed the putative biosynthetic gene cluster of acarviostatins and a putative pathway of acarviostatin production. To our knowledge, S. coelicoflavus ZG0656 is the first strain in this species for which a genome sequence has been reported. The analysis of sct-cluster provided important insights into the biosynthesis of acarviostatins. This work will be a platform for producing novel variants and yield improvement. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

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Nozaki, T; Asai, T; Sanchez, L B; Kobayashi, S; Nakazawa, M; Takeuchi, T

1999-11-05

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

Czech Academy of Sciences Publication Activity Database

Sharma, V.K.; Jelen, František; Trnková, L.

2015-01-01

Roč. 15, č. 1 (2015), s. 1564-1600 ISSN 1424-8220 Institutional support: RVO:68081707 Keywords : purine derivatives * electrochemistry of purines * carbon electrode Subject RIV: BO - Biophysics Impact factor: 2.033, year: 2015

Genetics Home Reference: purine nucleoside phosphorylase deficiency

Science.gov (United States)

... the expand/collapse boxes. Description Purine nucleoside phosphorylase deficiency is one of several disorders that damage the immune system and cause severe combined immunodeficiency (SCID). People with SCID lack virtually all immune protection from foreign invaders such as bacteria, viruses, and ...

Regulation of the Omega-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

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Marte Avranden Kjær

Full Text Available Limited availability of the n-3 fatty acids EPA and DHA have led to an interest in better understanding of the n-3 biosynthetic pathway and its regulation. The biosynthesis of alpha-linolenic acid to EPA and DHA involves several complex reaction steps including desaturation-, elongation- and peroxisomal beta-oxidation enzymes. The aims of the present experiments were to gain more knowledge on how this biosynthesis is regulated over time by different doses and fatty acid combinations. Hepatocytes isolated from salmon were incubated with various levels and combinations of oleic acid, EPA and DHA. Oleic acid led to a higher expression of the Δ6 fatty acid desaturase (fad genes Δ6fad_a, Δ6fad_b, Δ6fad_c and the elongase genes elovl2 compared with cells cultured in medium enriched with DHA. Further, the study showed rhythmic variations in expression over time. Levels were reached where a further increase in specific fatty acids given to the cells not stimulated the conversion further. The gene expression of Δ6fad_a_and Δ6fad_b responded similar to fatty acid treatment, suggesting a co-regulation of these genes, whereas Δ5fad and Δ6fad_c showed a different regulation pattern. EPA and DHA induced different gene expression patterns, especially of Δ6fad_a. Addition of radiolabelled alpha-linolenic acid to the hepatocytes confirmed a higher degree of elongation and desaturation in cells treated with oleic acid compared to cells treated with DHA. This study suggests a complex regulation of the conversion process of n-3 fatty acids. Several factors, such as that the various gene copies are differently regulated, the gene expression show rhythmic variations and gene expression only affected to a certain level, determines when you get the maximum conversion of the beneficial n-3 fatty acids.

Effect of overall feedback inhibition in unbranched biosynthetic pathways.

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Alves, R; Savageau, M A

2000-11-01

We have determined the effects of control by overall feedback inhibition on the systemic behavior of unbranched metabolic pathways with an arbitrary pattern of other feedback inhibitions by using a recently developed numerical generalization of Mathematically Controlled Comparisons, a method for comparing the function of alternative molecular designs. This method allows the rigorous determination of the changes in systemic properties that can be exclusively attributed to overall feedback inhibition. Analytical results show that the unbranched pathway can achieve the same steady-state flux, concentrations, and logarithmic gains with respect to changes in substrate, with or without overall feedback inhibition. The analytical approach also shows that control by overall feedback inhibition amplifies the regulation of flux by the demand for end product while attenuating the sensitivity of the concentrations to the same demand. This approach does not provide a clear answer regarding the effect of overall feedback inhibition on the robustness, stability, and transient time of the pathway. However, the generalized numerical method we have used does clarify the answers to these questions. On average, an unbranched pathway with control by overall feedback inhibition is less sensitive to perturbations in the values of the parameters that define the system. The difference in robustness can range from a few percent to fifty percent or more, depending on the length of the pathway and on the metabolite one considers. On average, overall feedback inhibition decreases the stability margins by a minimal amount (typically less than 5%). Finally, and again on average, stable systems with overall feedback inhibition respond faster to fluctuations in the metabolite concentrations. Taken together, these results show that control by overall feedback inhibition confers several functional advantages upon unbranched pathways. These advantages provide a rationale for the prevalence of this

Characterization of cyanobacterial hydrocarbon composition and distribution of biosynthetic pathways.

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R Cameron Coates

Full Text Available Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene and methyl group positions (3-, 4- and 5-methylheptadecane for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR and aldehyde deformylating oxygenase (ADO. The second involves a polyketide synthase (PKS pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS. Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.

Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast

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Kozmin Stanislav G

2005-06-01

Full Text Available Abstract Background N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP and 2-amino-6-hydroxylaminopurine (AHA, are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. Results We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. Conclusion We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains

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In order to determine the genetic basis for loss of fumonisin B¬2 (FB2) biosynthesis in FB2 non-producing A. niger strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified in FB2-produci...

Nucleotide Metabolism and its Control in Lactic Acid Bacteria

DEFF Research Database (Denmark)

Kilstrup, Mogens; Hammer, Karin; Jensen, Peter Ruhdal

2005-01-01

Most metabolic reactions are connected through either their utilization of nucleotides or their utilization of nucleotides or their regulation by these metabolites. In this review the biosynthetic pathways for pyrimidine and purine metabolism in lactic acid bacteria are described including...... the interconversion pathways, the formation of deoxyribonucleotides and the salvage pathways for use of exogenous precursors. The data for the enzymatic and the genetic regulation of these pathways are reviewed, as well as the gene organizations in different lactic acid bacteria. Mutant phenotypes and methods...... for manipulation of nucleotide pools are also discussed. Our aim is to provide an overview of the physiology and genetics of nucleotide metabolism and its regulation that will facilitate the interpretation of data arising from genetics, metabolomics, proteomics, and transcriptomics in lactic acid bacteria....

Research on urinary excretion of purine derivatives in ruminants: Past, present and future

International Nuclear Information System (INIS)

Chen, X.B.; Orskov, E.R.

2004-01-01

Research on urinary excretion of purine derivatives (PD), namely allantoin, uric acid, xanthine and hypoxanthine, in ruminants have been carried out with an objective to use the excretion of these purine metabolites as a parameter to estimate the intestinal flow of microbial protein. This paper reviews the published literature, from the first paper in 1931 to the current date. The current status of understanding in some key topics is discussed. The topics include: endogenous excretion, modelling the response of PD excretion to purine absorption, calculation of microbial N supply from PD excretion, use of spot urine measurement, possible use of plasma or milk PD as an alterative index, and applications in ruminant nutrition research. This review also covers the current understanding of PD excretion in different animal species, including sheep, cattle, goats, buffaloes, llamas, camels, yak and deer. Progress in analytical methods for the determination of purine derivatives is also discussed. Finally, areas of future research are highlighted. The paper stresses the need for more studies on metabolism of PD in the tissue, the kinetics of PD in the blood and physiological processes of renal excretion, so as to understand better the mechanism that accounts for the between-species and within species variation in PD excretion. Development of simpler and more rapid methods for defining the endogenous excretion and purine input-output relationship is also an area for future work. (author)

Integration of Fermentation and Organic Synthesis: Studies of Roquefortine C and Biosynthetic Derivatives

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Gober, Claire Marie

Roquefortine C is one of the most ubiquitous indoline alkaloids of fungal origin. It has been isolated from over 30 different species of Penicillium fungi and has garnered attention in recent years for its role as a biosynthetic precursor to the triazaspirocyclic natural products glandicoline B, meleagrin, and oxaline. The triazaspirocyclic motif, which encompasses three nitrogen atoms attached to one quaternary carbon forming a spirocyclic scaffold, is a unique chemical moiety that has been shown to impart a wide array of biological activity, from anti-bacterial activity and antiproliferative activity against cancer cell lines to anti-biofouling against marine organisms. Despite the promise of these compounds in the pharmaceutical and materials industries, few syntheses of triazaspirocycles exist in the literature. The biosynthesis of roquefortine C-derived triazaspirocycles, however, provides inspiration for the synthesis of these compounds, namely through a nitrone-promoted transannular rearrangement. This type of internal rearrangement has never been carried out synthetically and would provide an efficient stereoselective synthesis of triazaspirocycles. This work encompasses efforts towards elucidating the biosynthetic pathway of roquefortine C-derived triazaspirocycles as well as synthetic efforts towards the construction of triazaspirocycles. Chapter 1 will discuss a large-scale fermentation procedure for the production of roquefortine C from Penicillium crustosum. Chapters 2 and 3 explore (through enzymatic and synthetic means, respectively) the formation of the key indoline nitrone moiety required for the proposed transannular rearrangement. Finally, chapter 4 will discuss synthetic efforts towards the synthesis of triazaspirocycles. This work has considerably enhanced our understanding of the roquefortine C biosynthetic pathway and the unique chemistry of this natural product, and our efforts towards the synthesis of triazaspirocycles will facilitate the

Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway.

Directory of Open Access Journals (Sweden)

Cynthia A Dick

2011-04-01

Full Text Available Intra- and interspecific variation in flower color is a hallmark of angiosperm diversity. The evolutionary forces underlying the variety of flower colors can be nearly as diverse as the colors themselves. In addition to pollinator preferences, non-pollinator agents of selection can have a major influence on the evolution of flower color polymorphisms, especially when the pigments in question are also expressed in vegetative tissues. In such cases, identifying the target(s of selection starts with determining the biochemical and molecular basis for the flower color variation and examining any pleiotropic effects manifested in vegetative tissues. Herein, we describe a widespread purple-white flower color polymorphism in the mustard Parrya nudicaulis spanning Alaska. The frequency of white-flowered individuals increases with increasing growing-season temperature, consistent with the role of anthocyanin pigments in stress tolerance. White petals fail to produce the stress responsive flavonoid intermediates in the anthocyanin biosynthetic pathway (ABP, suggesting an early pathway blockage. Petal cDNA sequences did not reveal blockages in any of the eight enzyme-coding genes in white-flowered individuals, nor any color differentiating SNPs. A qRT-PCR analysis of white petals identified a 24-fold reduction in chalcone synthase (CHS at the threshold of the ABP, but no change in CHS expression in leaves and sepals. This arctic species has avoided the deleterious effects associated with the loss of flavonoid intermediates in vegetative tissues by decoupling CHS expression in petals and leaves, yet the correlation of flower color and climate suggests that the loss of flavonoids in the petals alone may affect the tolerance of white-flowered individuals to colder environments.

Partition coefficients of some purine derivatives and its application to pharmacokinetics.

Science.gov (United States)

Chrzanowska, M; Sobiak, J; Kuehn, M; Dorawa, E; Hermann, T

2009-12-01

Metazathioprine (MAZA), a methylated derivative of azathioprine (AZA), demonstrated the greatest values of apparent and specific partition coefficients in n-octanol/phosphate buffer at pH 5.7 and pH 7.4 among purine derivatives such as 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and AZA. Introduction of a methyl group into the imidazole ring of AZA increases lipophilic properties of MAZA compared to AZA. Mass balance of purine derivatives in n-octanol and in phosphate buffer indicated their chemical stability in those media.

1,3,5-Triazine-based analogues of purine: from isosteres to privileged scaffolds in medicinal chemistry.

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Lim, Felicia Phei Lin; Dolzhenko, Anton V

2014-10-06

Purines can be considered as the most ubiquitous and functional N-heterocyclic compounds in nature. Structural modifications of natural purines, particularly using isosteric ring systems, have been in the focus of many drug discovery programs. Fusion of 1,3,5-triazine ring with pyrrole, pyrazole, imidazole, 1,2,3-triazole or 1,2,4-triazole results in seven bicyclic heterocyclic systems isosteric to purine. Application of the isosterism concept for the development of new compounds with therapeutic potential in areas involving purinergic regulation or purine metabolism led to significant advances in medicinal chemistry of the azolo[1,3,5]triazines. These 1,3,5-triazine-based purine-like scaffolds significantly increase level of molecular diversity and allow covering chemical space in the important areas of medicinal chemistry. Some of these azolo[1,3,5]triazine systems have become privileged scaffolds in the development of inhibitors of various kinases, phosphodiesterase, xanthine oxidase, and thymidine phosphorylase, antagonists of adenosine and corticotropin-releasing hormone receptors, anticancer and antiviral agents. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters

DEFF Research Database (Denmark)

Weber, Tilmann; Blin, Kai; Duddela, Srikanth

2015-01-01

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we...... introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration...... of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products...

Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase

DEFF Research Database (Denmark)

Jensen, Kaj Frank

1978-01-01

Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared...

Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives

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Moses, Tessa; Papadopoulou, Kalliope K.

2014-01-01

Saponins are widely distributed plant natural products with vast structural and functional diversity. They are typically composed of a hydrophobic aglycone, which is extensively decorated with functional groups prior to the addition of hydrophilic sugar moieties, to result in surface-active amphipathic compounds. The saponins are broadly classified as triterpenoids, steroids or steroidal glycoalkaloids, based on the aglycone structure from which they are derived. The saponins and their biosynthetic intermediates display a variety of biological activities of interest to the pharmaceutical, cosmetic and food sectors. Although their relevance in industrial applications has long been recognized, their role in plants is underexplored. Recent research on modulating native pathway flux in saponin biosynthesis has demonstrated the roles of saponins and their biosynthetic intermediates in plant growth and development. Here, we review the literature on the effects of these molecules on plant physiology, which collectively implicate them in plant primary processes. The industrial uses and potential of saponins are discussed with respect to structure and activity, highlighting the undoubted value of these molecules as therapeutics. PMID:25286183

The radiation chemistry of the purine bases within DNA and related model compounds

International Nuclear Information System (INIS)

Cadet, J.; Berger, M.; Shaw, A.

1986-01-01

Both the direct and indirect effects of ionizing radiations are believed to contribute to the chemical changes induced in cellular DNA. Relevant information on the possible degradation pathways has been provided by studies using DNA model compounds, the major proportion of which have focused on pyrimidine components and sugar derivatives. With the development of powerful analytical tools such as high performance liquid chromatography and soft ionization mass spectrometry techniques, progress has recently been made in the elucidation of the nature of the radiation-induced chemical modifications of purine bases in DNA and related nucleosides and nucleotides. This short review details recent aspects of the radiation-induced degradation of adenine and guanine bases in DNA and its model compounds as the result of both direct and indirect effects. 11 refs., 2 figs., 1 tab

Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer.

Science.gov (United States)

Singh, Himanshu Narayan; Rajeswari, Moganty R

2016-01-01

Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich's ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed "PuRepeatFinder.pl" algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, n ≥ 200. A total of 1158 EPRs were identified in the genome which followed Wakeby distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein-protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.

Inhibition and Structure of Trichomonas vaginalis Purine Nucleoside Phosphorylase with Picomolar Transition State Analogues

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Rinaldo-Matthis,A.; Wing, C.; Ghanem, M.; Deng, H.; Wu, P.; Gupta, A.; Tyler, P.; Evans, G.; Furneaux, R.; et al.

2007-01-01

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition stte mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a K{sub m}/K{sub d} ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a K{sub m}/K{sub d} ratio of 203,300. The tight binding of DADMe-ImmA supports a late S{sub N}1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP-ImmA{center_dot}PO{sub 4} and TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4} ternary complexes differ from previous structures with substrate anologues. The tight binding with DADMe-ImmA is in part due to a 2.7 {angstrom} ionic interaction between a PO{sub 4} oxygen and the N1 cation of the hydroxypyrrolidine and is weaker in the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure at 3.5 {angstrom}. However, the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4}. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope

Engineering the spatial organization of metabolic pathways

DEFF Research Database (Denmark)

Albertsen, Line; Maury, Jerome; Bach, Lars Stougaard

One of the goals of metabolic engineering is to optimize the production of valuable metabolites in cell factories. In this context, modulating the gene expression and activity of enzymes are tools that have been extensively used. Another approach that is gaining interest is the engineering...... of the spatial organization of biosynthetic pathways. Several natural systems for ensuring optimal spatial arrangement of biosynthetic enzymes exist. Sequentially acting enzymes can for example be positioned in close proximity by attachment to cellular structures, up-concentration in membrane enclosed organelles...... or assembly into large complexes. The vision is that by positioning sequentially acting enzymes in close proximity, the cell can accelerate reaction rates and thereby prevent loss of intermediates through diffusion, degradation or competing pathways. The production of valuable metabolites in cell factories...

Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

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Sharma, Vimal Kumar; Jelen, Frantisek; Trnkova, Libuse

2015-01-01

Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications. PMID:25594595

Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

Directory of Open Access Journals (Sweden)

Vimal Kumar Sharma

2015-01-01

Full Text Available Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications.

An Expanding Role For Purine Uptake Permease (PUP -like Transporters In Plant Secondary Metabolism.

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John G. Jelesko

2012-05-01

Full Text Available For the past decade, our understanding of the plant purine uptake permease (PUP transporter family of was primarily oriented on purine nucleobase substrates and their tissue-specific expression patterns in Arabidopsis. However, a tobacco PUP-like homolog demonstrating nicotine uptake permease (NUP activity was recently shown to affect both nicotine metabolism and root cell growth. These new findings expand the physiological role for PUP-like transporters to include plant secondary metabolism. Molecular evolution analyses of PUP-like transporters indicate they are distinct group within an ancient super family of drug and metabolite transporters (DMTs. The PUP-like family originated during terrestrial plant evolution sometime between the bryophytes and the lycophytes. A phylogenetic analysis indicates that the PUP-like transporters were likely were derived from a pre-existing nucleotide sugar transporter family within the DMT super family. Within the lycophyte Selaginella, there are three paralogous groups of PUP-like transporters. One of the three PUP-like paralogous groups showed an extensive pattern of gene duplication and diversification within the angiosperm lineage, whereas the other two more ancestral PUP-like paralogous groups did not. Biochemical characterization of four closely-related PUP-like paralogs together with model-based phylogenetic analyses indicate both subfunctionalization and neofunctionalization during the molecular evolution of angiosperm PUP-like transporters. These findings suggest that members of the PUP-like family of DMT transporters are likely involved in diverse primary and secondary plant metabolic pathways.

The Purine-Utilizing Bacterium Clostridium acidurici 9a: A Genome-Guided Metabolic Reconsideration

OpenAIRE

Hartwich, Katrin; Poehlein, Anja; Daniel, Rolf

2012-01-01

Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists ...

Plasma Membrane-Located Purine Nucleotide Transport Proteins Are Key Components for Host Exploitation by Microsporidian Intracellular Parasites

Science.gov (United States)

Heinz, Eva; Hacker, Christian; Dean, Paul; Mifsud, John; Goldberg, Alina V.; Williams, Tom A.; Nakjang, Sirintra; Gregory, Alison; Hirt, Robert P.; Lucocq, John M.; Kunji, Edmund R. S.; Embley, T. Martin

2014-01-01

Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis. PMID:25474405

Plasma membrane-located purine nucleotide transport proteins are key components for host exploitation by microsporidian intracellular parasites.

Directory of Open Access Journals (Sweden)

Eva Heinz

2014-12-01

Full Text Available Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes, consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.

Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

International Nuclear Information System (INIS)

Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang; Lu, Weiqiang; Huang, Jin

2016-01-01

Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC_5_0 values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

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Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China); Lu, Weiqiang, E-mail: wqlu@bio.ecnu.edu.cn [Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Huang, Jin, E-mail: huangjin@ecust.edu.cn [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China)

2016-09-02

Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

Contents and retentions of free and total purine bases in lamb meat cooked by several household methods

Directory of Open Access Journals (Sweden)

P. Anfossi

2011-03-01

Full Text Available Concerns about the content of total and free purine bases in muscle foods and their retentions upon cooking have been since long established (Brulé et al., 1988. Recently, though, an important rôle has been acknowledged to dietary sources of preformed purines for the growth of tissues with a rapid turnover and for optimal function of the cellular immune response, up to the point that the positive features of these nutrients seem to outweigh by far the negative ones (ILSI, 1998. Scanty information exists about the total purine content of raw ovine meat, the only available sources of data being a survey by Herbel and Montag (1987 on purine and pyrimidine contents of protein-rich foods and the comprehensive collection of food composition tables compiled by Scherz and Senser (1994...

Flg22-Triggered Immunity Negatively Regulates Key BR Biosynthetic Genes.

Science.gov (United States)

Jiménez-Góngora, Tamara; Kim, Seong-Ki; Lozano-Durán, Rosa; Zipfel, Cyril

2015-01-01

In plants, activation of growth and activation of immunity are opposing processes that define a trade-off. In the past few years, the growth-promoting hormones brassinosteroids (BR) have emerged as negative regulators of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), promoting growth at the expense of defense. The crosstalk between BR and PTI signaling was described as negative and unidirectional, since activation of PTI does not affect several analyzed steps in the BR signaling pathway. In this work, we describe that activation of PTI by the bacterial PAMP flg22 results in the reduced expression of BR biosynthetic genes. This effect does not require BR perception or signaling, and occurs within 15 min of flg22 treatment. Since the described PTI-induced repression of gene expression may result in a reduction in BR biosynthesis, the crosstalk between PTI and BR could actually be negative and bidirectional, a possibility that should be taken into account when considering the interaction between these two pathways.

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA.

Science.gov (United States)

Cadet, Jean; Wagner, J Richard; Shafirovich, Vladimir; Geacintov, Nicholas E

2014-06-01

The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.

Measurement and application of purine derivatives: Creatinine ratio in spot urine samples of ruminants

International Nuclear Information System (INIS)

Chen, X.B.; Jayasuriya, M.C.N.; Makkar, H.P.S.

2004-01-01

The daily excretion of purine derivatives in urine has been used to estimate the supply of microbial protein to ruminant animals. The method provides a simple and non-invasive tool to indicate the nutritional status of farm animals. However due to the need for complete collection of urine the potential application at farm level is restricted. Research conducted under the FAO/IAEA Co-ordinated Research Project has indicated that it is possible to use the purine derivatives:creatinine ratio measured in several spot urine samples collected within a day, as an index of microbial protein supply in a banding system for farm application. Some theoretical and experimental aspects in the measurement of purine derivatives:creatinine ratio in spot urine samples and the possible application of the banding system at the farm level are discussed. (author)

Characterization of a depurinated-DNA purine-base-insertion activity from Drosophila.

Science.gov (United States)

Deutsch, W A; Spiering, A L

1985-01-01

An activity that binds preferentially to depurinated DNA and inserts purines into those sites was partially purified from Drosophila melanogaster embryos. The protein has a sedimentation coefficient of 4.9 S and is devoid of AP (apurinic/apyrimidinic) endonuclease activity. Upon incorporation of purines into apurinic DNA, the number of alkali-labile sites decreases, thus establishing the conversion of depurinated sites into normal nucleotides. The activity requires K+, and is totally inhibited by caffeine or EDTA. Guanine is specifically incorporated into partially depurinated poly(dG-dC) and adenine is specifically incorporated into poly(dA-dT), thus demonstrating the apparent template specificity of the enzyme. PMID:2417589

Biosynthetic origin of acetic acid using SNIF-NMR

International Nuclear Information System (INIS)

Boffo, Elisangela Fabiana; Ferreira, Antonio Gilberto

2006-01-01

The main purpose of this work is to describe the use of the technique Site-Specific Natural Isotopic Fractionation of hydrogen (SNIF-NMR), using 2 H and 1 H NMR spectroscopy, to investigate the biosynthetic origin of acetic acid in commercial samples of Brazilian vinegar. This method is based on the deuterium to hydrogen ratio at a specific position (methyl group) of acetic acid obtained by fermentation, through different biosynthetic mechanisms, which result in different isotopic ratios. We measured the isotopic ratio of vinegars obtained through C 3 , C 4 , and CAM biosynthetic mechanisms, blends of C 3 and C 4 (agrins) and synthetic acetic acid. (author)

Modes of Action of 3-Amino-1, 2, 4-Triazole: Current Status

Energy Technology Data Exchange (ETDEWEB)

Hilton, J. L. [United States Department of Agriculture, Belts Ville, MD (United States)

1966-05-15

3-amino-1, 2, 4-triazole (amitrole) is a selective herbicide toxic to many species of green plants and micro-organisms. The primary biochemical site of action does not appear to be the same in all organisms. Growth inhibitions are reversed by purines or, more effectively, by histidine in micro-organisms or by riboflavin in green plants. Both metabolites are products derived from purine metabolism. Therefore, a study of amitrole effects on purine metabolism in various organisms is in progress. Saccharomyces cerevisiae Meyen ex Hansen is highly sensitive to amitrole but may be protected by histidine. Amitrole inhibits incorporation of {sup 14}C from adenine-2- {sup 14}C into proteins and nucleic acids, and causes imidazoleglycerol phosphate-4C (IGP- {sup 14}C) to accumulate as a result of competitive inhibition of IGP dehydratase, one of the enzymes of the histidine biosynthetic pathway. Amitrole affinity for the enzyme is approximately 10 times that of IGP. The inhibition releases the histidine pathway from feedback control thereby permitting IGP to accumulate at the expense of the purine pool. The concept of an induced purine deficiency provides interpretation for the observation that: (a) purines provide limited protection against amitrole; (b) an inhibitor of adenine biosynthesis offers protection comparable to that of purines; and (c) that inhibition of riboflavin biosynthesis in Eremothecium ashbyii Guill. is reversed by histidine. Riboflavin protection of green plants against amitrole results primarily from chemical inactivation of amitrole by photoactivated flavins; however, some support for a physiological mechanism of protection still exists. Bacteria are approximately 1/100-th as sensitive to amitrole as yeasts. Biosynthesis of IGP dehydratase is derepressed in amitrole-treated Salmonella typhimurium (Loeffler) Cast, and Chalm. As a result of the increased enzyme level, higher concentrations of amitrole are required to affect growth. At these high

Effects of caffeine on purine metabolism and ultraviolet light-induced lethality in cultured mammalian cells

International Nuclear Information System (INIS)

Waldren, C.A.; Patterson, D.

1979-01-01

Caffeine, at doses which enhance the killing action of ultraviolet light, inhibits both de novo synthesis and the utilization of exogenous purines in cultured CHO-K1, a Chinese hamster ovary cell line. The effect is dose dependent, with a caffeine concentration of 7.5 mM producing a 90% reduction in 15 min. Interference with utilization of exogenous purines was seen as a substantial decrease in the conversion of [14C]hypoxanthine, [14C]adenine, or [14C]guanine into their respective di- and triphosphates in the presence of caffeine. Thus, one of the ways by which antimetabolites and caffeine act to enhance ultraviolet light killing may be by interference with the supply of purine nucleotides needed for repair

The oxylipin pathway in Arabidopsis.

Science.gov (United States)

Creelman, Robert A; Mulpuri, Rao

2002-01-01

Oxylipins are acyclic or cyclic oxidation products derived from the catabolism of fatty acids which regulate many defense and developmental pathways in plants. The dramatic increase in the volume of publications and reviews on these compounds since 1997 documents the increasing interest in this compound and its role in plants. Research on this topic has solidified our understanding of the chemistry and biosynthetic pathways for oxylipin production. However, more information is still needed on how free fatty acids are produced and the role of beta-oxidation in the biosynthetic pathway for oxylipins. It is also becoming apparent that oxylipin content and composition changes during growth and development and during pathogen or insect attack. Oxylipins such as jasmonic acid (JA) or 12-oxo-phytodienoic acid modulate the expression of numerous genes and influence specific aspects of plant growth, development and responses to abiotic and biotic stresses. Although oxylipins are believed to act alone, several examples were presented to illustrate that JA-induced responses are modulated by the type and the nature of crosstalk with other signaling molecules such as ethylene and salicylic acid. How oxylipins cause changes in gene expression and instigate a physiological response is becoming understood with the isolation of mutations in both positive and negative regulators in the jasmonate signaling pathway and the use of cDNA microarrays.

Prolonged fasting increases purine recycling in post-weaned northern elephant seals.

Science.gov (United States)

Soñanez-Organis, José Guadalupe; Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Aguilar, Andres; Crocker, Daniel E; Ortiz, Rudy M

2012-05-01

Northern elephant seals are naturally adapted to prolonged periods (1-2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species.

Synthesis of (purin-6-yl)acetates and 6-(2-hydroxyethyl)purines via cross-couplings of 6-chloropurines with the Reformatsky reagent

Czech Academy of Sciences Publication Activity Database

Hasník, Zbyněk; Šilhár, Peter; Hocek, Michal

2007-01-01

Roč. 48, č. 32 (2007), s. 5589-5592 ISSN 0040-4039 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : purines * nucleosides * cross-coupling reactions Subject RIV: CC - Organic Chemistry Impact factor: 2.615, year: 2007

Effects of pH, temperature, and chemical structure on the stability of S-(purin-6-yl)-L-cysteine: evidence for a novel molecular rearrangement mechanism to yield N-(purin-6-yl)-L-cysteine.

Science.gov (United States)

Elfarra, A A; Hwang, I Y

1996-01-01

The stability of S-(purin-6-yl)-L-cysteine (SPC), a kidney-selective prodrug of 6-mercaptopurine and a putative metabolite of 6-chloropurine, was investigated under various pH and temperature conditions. At room temperature, the half-life (t 1/2) of SPC at either highly acidic (pH 3.6) or basic conditions (pH 9.6) was longer than at neutral or slightly acidic or basic conditions (pH 5.7-8.75). The primary degradation product, N-(purin-6-yl)-L-cysteine (NPC), was isolated using Sephadex LH-20 chromatography and characterized by 1H NMR and FAB/MS after derivatization with 2-iodoacetic acid. These results reveal novel stability requirements and implicate the cysteinyl amino group and the purinyl N-1 nitrogen in the mechanism of SPC rearrangement to NPC. Further evidence for this hypothesis was provided by the findings that the stability of SPC in phosphate buffer (pH 7.4) at 37 degrees C was similar to that of S-(guanin-6-yl)-L-cysteine, whereas S-(purin-6-yl)-N-acetyl-L-cysteine and S-(purin-6-yl)glutathione which have their cysteine amino groups blocked were much more stable than SPC. S-(Purin-6-yl)-L-homocysteine (SPHC) was also more stable than SPC, possibly because the formation of a 6-membered ring transition state as would be expected with SPHC is kinetically less favored than the formation of a 5-membered ring transition state as would be expected with SPC. These results may explain previous in vivo metabolism results of SPC and its analogs and may contribute to a better understanding of stability of structurally related cysteine S-conjugates.

Evolution and Diversity of Biosynthetic Gene Clusters in Fusarium

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Koen Hoogendoorn

2018-06-01

Full Text Available Plant pathogenic fungi in the Fusarium genus cause severe damage to crops, resulting in great financial losses and health hazards. Specialized metabolites synthesized by these fungi are known to play key roles in the infection process, and to provide survival advantages inside and outside the host. However, systematic studies of the evolution of specialized metabolite-coding potential across Fusarium have been scarce. Here, we apply a combination of bioinformatic approaches to identify biosynthetic gene clusters (BGCs across publicly available genomes from Fusarium, to group them into annotated families and to study gain/loss events of BGC families throughout the history of the genus. Comparison with MIBiG reference BGCs allowed assignment of 29 gene cluster families (GCFs to pathways responsible for the production of known compounds, while for 57 GCFs, the molecular products remain unknown. Comparative analysis of BGC repertoires using ancestral state reconstruction raised several new hypotheses on how BGCs contribute to Fusarium pathogenicity or host specificity, sometimes surprisingly so: for example, a gene cluster for the biosynthesis of hexadehydro-astechrome was identified in the genome of the biocontrol strain Fusarium oxysporum Fo47, while being absent in that of the tomato pathogen F. oxysporum f.sp. lycopersici. Several BGCs were also identified on supernumerary chromosomes; heterologous expression of genes for three terpene synthases encoded on the Fusarium poae supernumerary chromosome and subsequent GC/MS analysis showed that these genes are functional and encode enzymes that each are able to synthesize koraiol; this observed functional redundancy supports the hypothesis that localization of copies of BGCs on supernumerary chromosomes provides freedom for evolutionary innovations to occur, while the original function remains conserved. Altogether, this systematic overview of biosynthetic diversity in Fusarium paves the way for

antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters.

Science.gov (United States)

Weber, Tilmann; Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A; Müller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko; Medema, Marnix H

2015-07-01

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants

Energy Technology Data Exchange (ETDEWEB)

Schenck, Craig A.; Holland, Cynthia K.; Schneider, Matthew R.; Men, Yusen; Lee, Soon Goo; Jez, Joseph M.; Maeda , Hiroshi A. (UW); (WU)

2017-06-26

L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

The Arabidopsis histone chaperone FACT is required for stress-induced expression of anthocyanin biosynthetic genes.

Science.gov (United States)

Pfab, Alexander; Breindl, Matthias; Grasser, Klaus D

2018-03-01

The histone chaperone FACT is involved in the expression of genes encoding anthocyanin biosynthetic enzymes also upon induction by moderate high-light and therefore contributes to the stress-induced plant pigmentation. The histone chaperone FACT consists of the SSRP1 and SPT16 proteins and associates with transcribing RNAPII (RNAPII) along the transcribed region of genes. FACT can promote transcriptional elongation by destabilising nucleosomes in the path of RNA polymerase II, thereby facilitating efficient transcription of chromatin templates. Transcript profiling of Arabidopsis plants depleted in SSRP1 or SPT16 demonstrates that only a small subset of genes is differentially expressed relative to wild type. The majority of these genes is either up- or down-regulated in both the ssrp1 and spt16 plants. Among the down-regulated genes, those encoding enzymes of the biosynthetic pathway of the plant secondary metabolites termed anthocyanins (but not regulators of the pathway) are overrepresented. Upon exposure to moderate high-light stress several of these genes are up-regulated to a lesser extent in ssrp1/spt16 compared to wild type plants, and accordingly the mutant plants accumulate lower amounts of anthocyanin pigments. Moreover, the expression of SSRP1 and SPT16 is induced under these conditions. Therefore, our findings indicate that FACT is a novel factor required for the accumulation of anthocyanins in response to light-induction.

Synthesis of (Purin-6-yl)methylphosphonate Bases and Nucleosides

Czech Academy of Sciences Publication Activity Database

Hasník, Zbyněk; Pohl, Radek; Hocek, Michal

2010-01-01

Roč. 51, č. 18 (2010), s. 2464-2466 ISSN 0040-4039 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : purines * nucleosides * phosphonates * cross-coupling Subject RIV: CC - Organic Chemistry Impact factor: 2.618, year: 2010

Efecto Antagónico in vitro de Actinomicetos Aislados de Purines de Chipaca (Bidens pilosa L. Frente a Phytophthora infestans (Mont de Bary In vitro Antagonistic Effect of Actinomycetes Isolated from Chipaca (Bidens pilosa L. Purins Against Phytophthora infestans (Mont de Bary

Directory of Open Access Journals (Sweden)

Yudy Astrid Fonseca Ardila

2011-12-01

Full Text Available Se estudió el efecto inhibidor de los actinomicetos presentes en purines o extractos fermentados de plantas de chipaca (Bidens pilosa L., sobre el crecimiento de Phytophthora infestans (Mont de Bary, causante del tizón tardío de la papa. Se elaboraron cuatro purines de flores, raíces, hojas-tallos y su mezcla. De estos purines se obtuvieron 25 aislamientos de actinomicetos, cada uno de los cuales se enfrentó con P. infestans en placas de medio de cultivo, utilizando la técnica de anillos de Gauze y estableciendo las concentraciones iniciales de esporas mediante conteos microscópicos en cámara de Neubauer. Los actinomicetos no crecieron en el purin de flores debido, posiblemente, a que en él no se utiliza suelo rizosférico o porque su pH (9 es mayor que el rango normal de crecimiento de estos microorganismos ( pH 6 -; 8. Se evidenció inhibición del crecimiento del oomycete por parte de 8 aislamientos de actinomicetos con porcentajes de inhibición entre 33,3 - 77,8%, provenientes de los purines de raíces, tallos-hojas y mezcla de partes de la planta. La mayor inhibición se obtuvo en los aislamientos AC001, AC010, AC011 y AC025 con conteos de 0,4, 6,0, 3,0, y 3,6 x10(5 esporas mL-1.Purins or liquid fermented extracts of chipaca (Bidens pilosa L. were prepared to establish the inhibitory effect of the actinomycetes found in such biopharmaceutical preparations on the growth of Phytophthora infestans (Mont de Bary, the causative of potato late blight disease. Four purins made from flowers, roots, leaf-steams and a mixture of them were prepared; 25 actinomycete isolates were obtained from these purins and their ability to resist challenge by P. infestans was ascertained in medium plates using the ring Gauze technique and establishing initial concentrations of spores by microscopic counting in Neubauer chamber. Actinomycetes did not grow in flower purin as rhizosphere soil was not used in its preparation or because this particular pH (9

Definition of the Bacillus subtilis PurR operator using genetic and bioinformatic tools and expansion of the PurR regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO

DEFF Research Database (Denmark)

Saxild, Hans Henrik; Brunstedt, K.; Nielsen, K.I.

2001-01-01

The expression of the pur operon, which encodes enzymes of the purine biosynthetic pathway in Bacillus subtilis, is subject to control by the purR gene product (PurR) and phosphoribosylpyrophosphate. This control is also exerted on the purA and purR genes. A consensus sequence for the binding...... of PurR, named the PurBox, has been suggested (M. Kilstrup, S.G. Jessing, S.B. Wichmand-Jorgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900-3906, 1998). To determine whether the expression of other genes might be regulated by PurR, we performed a search for PurBox sequences in the B. subtilis...

Synthesis and Cytotoxic Activity of Some New 2,6-Substituted Purines

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Nageswara Rao Kode

2011-07-01

Full Text Available A seriesof twenty four acyclic unsaturated 2,6-substututed purines 5a-20b were synthesized. These compounds were evaluated for cytotoxic activity against NCI-60 DTP human tumor cell line screen at 10µMconcentration. N9-[(Z-4'-chloro-2'-butenyl-1'-yl]-2,6-dichloropurine(5a, N9-[4'-chloro-2'-butynyl-1'-yl]-2,6-dichloropurine(10a, N9-[(E-2',3'-dibromo-4'-chloro-2'-butenyl-1'-yl]-6-methoxypurine(14and N9-[4'-chloro-2'-butynyl-1'-yl]-6-(4-methoxyphenyl-purine(19exhibited highly potent cytotoxic activity with GI50 values in the 1–5 µM range for most human tumor cell lines. Other compounds exhibited moderate activity.

Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

Energy Technology Data Exchange (ETDEWEB)

Timofeev, V. I., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Abramchik, Yu. A., E-mail: ugama@yandex.ru [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Zhukhlistova, N. E., E-mail: inna@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

2016-03-15

Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB-ID: 4RJ2).

Excretion of purine base derivatives after intake of bacterial protein meal in pigs

DEFF Research Database (Denmark)

Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, A.

2007-01-01

Bacterial protein meal has a high content ofprotein but also of RNA and DNA. Sixteen barrows were allocated to four diets containing increasing levels of bacterial protein meal (BPM), from weaning to 80 kg live weight, to evaluate whether the RNA and DNA contents of BPM influenced the retention...... of nitrogen. It was hypothesised that an increased intake of RNA and DNA would lead to an increased urinary excretion of purine base derivatives and increased plasma concentrations. Retention of nitrogen was unaffected by dietary content of BPM (P=0.08) and the urinary excretion of purine base derivatives...

The hormonal regulation of purine biosynthesis: control of the inosinic acid branch point

International Nuclear Information System (INIS)

Pizzichini, M.; Di Stefano, A.; Marinello, E.; Pompucci, G.

1986-01-01

This paper studies the behavior of purine biosynthesis de novo in the levator animal muscle (LAM) of adult rats, before, after castration, and after testosterone administration. The incorporation of C 14-formate into the acid-soluble bases was performed as an index of the overall rate of purine nucleotide synthesis. It is shown that castration reduces the content, the specific activity of total bases and of the single bases in the LAM, indicating an inferior turnover. The increased turnover of guanylic acid, which is always present although not as much as adenylic acid, will favor the sunthesis of RNA in the sexual organs

Transcriptome and metabolome analysis of Ferula gummosa Boiss. to reveal major biosynthetic pathways of galbanum compounds.

Science.gov (United States)

Sobhani Najafabadi, Ahmad; Naghavi, Mohammad Reza; Farahmand, Hamid; Abbasi, Alireza

2017-11-01

Ferula gummosa Boiss. is an industrial and pharmaceutical plant that has been highly recognized for its valuable oleo-gum-resin, namely galbanum. Despite the fabulous value of galbanum, very little information on the genetic and biochemical mechanisms of its production existed. In the present study, the oleo-gum-resin and four organs (root, flower, stem, and leaf) of F. gummosa were assessed in terms of metabolic compositions and the expression of genes involved in their biosynthetic pathways. Results showed that the most accumulation of resin and essential oils were occurred in the roots (13.99 mg/g) and flowers (6.01 mg/g), respectively. While the most dominant compound of the resin was β-amyrin from triterpenes, the most abundant compounds of the essential oils were α-pinene and β-pinene from monoterpenes and α-eudesmol and germacrene-D from sesquiterpenes. Transcriptome analysis was performed by RNA sequencing (RNA-seq) for the plant roots and flowers. Differential gene expression analysis showed that 1172 unigenes were differential between two organs that 934 (79.6%) of them were up-regulated in the flowers and 238 (20.4%) unigenes were up-regulated in the roots (FDR ≤0.001). The most important up-regulated unigenes in the roots were involved in the biosynthesis of the major components of galbanum, including myrcene, germacrene-D, α-terpineol, and β-amyrin. The results obtained by RNA-Seq were confirmed by qPCR. These analyses showed that different organs of F. gummosa are involved in the production of oleo-gum-resin, but the roots are more active than other organs in terms of the biosynthesis of triterpenes and some mono- and sesquiterpenes. This study provides rich molecular and biochemical resources for further studies on molecular genetics and functional genomics of oleo-gum-resin production in F. gummosa.

Stacking of purines in water: the role of dipolar interactions in caffeine.

Science.gov (United States)

Tavagnacco, L; Di Fonzo, S; D'Amico, F; Masciovecchio, C; Brady, J W; Cesàro, A

2016-05-11

During the last few decades it has been ascertained that base stacking is one of the major contributions stabilizing nucleic acid conformations. However, the understanding of the nature of the interactions involved in the stacking process remains under debate and it is a subject of theoretical and experimental studies. Structural similarity between purine bases (guanine and adenine) in DNA and the caffeine molecule makes caffeine an excellent model for the purine bases. The present study clearly shows that dipolar interactions play a fundamental role in determining stacking of purine molecules in solution. In order to reach this achievement, polarized ultraviolet Raman resonant scattering experiments have been carried out on caffeine aqueous solutions as a function of concentration and temperature. The investigation pointed out at the aggregation and solvation properties, particularly at elevated temperatures. Kubo-Anderson theory was used as a framework to investigate the non-coincidence effect (NCE) occurring in the totally symmetric breathing modes of the purine rings, and in the bending modes of the methyl groups of caffeine. The NCE concentration dependence shows that caffeine aggregation at 80 °C occurs by planar stacking of the hydrophobic faces. The data clearly indicate that dipolar interactions determine the reorientational motion of the molecules in solution and are the driving force for the stacking of caffeine. In parallel, the observed dephasing times imply a change in caffeine interactions as a function of temperature and concentration. A decrease, at low water content, of the dephasing time for the ring breathing vibration mode indicates that self-association alters the solvation structure that is detectable at low concentration. These results are in agreement with simulation predictions and serve as an important validation of the models used in those calculations.

A novel approach to select differential pathways associated with hypertrophic cardiomyopathy based on gene co‑expression analysis.

Science.gov (United States)

Chen, Xiao-Min; Feng, Ming-Jun; Shen, Cai-Jie; He, Bin; Du, Xian-Feng; Yu, Yi-Bo; Liu, Jing; Chu, Hui-Min

2017-07-01

The present study was designed to develop a novel method for identifying significant pathways associated with human hypertrophic cardiomyopathy (HCM), based on gene co‑expression analysis. The microarray dataset associated with HCM (E‑GEOD‑36961) was obtained from the European Molecular Biology Laboratory‑European Bioinformatics Institute database. Informative pathways were selected based on the Reactome pathway database and screening treatments. An empirical Bayes method was utilized to construct co‑expression networks for informative pathways, and a weight value was assigned to each pathway. Differential pathways were extracted based on weight threshold, which was calculated using a random model. In order to assess whether the co‑expression method was feasible, it was compared with traditional pathway enrichment analysis of differentially expressed genes, which were identified using the significance analysis of microarrays package. A total of 1,074 informative pathways were screened out for subsequent investigations and their weight values were also obtained. According to the threshold of weight value of 0.01057, 447 differential pathways, including folding of actin by chaperonin containing T‑complex protein 1 (CCT)/T‑complex protein 1 ring complex (TRiC), purine ribonucleoside monophosphate biosynthesis and ubiquinol biosynthesis, were obtained. Compared with traditional pathway enrichment analysis, the number of pathways obtained from the co‑expression approach was increased. The results of the present study demonstrated that this method may be useful to predict marker pathways for HCM. The pathways of folding of actin by CCT/TRiC and purine ribonucleoside monophosphate biosynthesis may provide evidence of the underlying molecular mechanisms of HCM, and offer novel therapeutic directions for HCM.

Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

Science.gov (United States)

Menendez-Bravo, Simón; Comba, Santiago; Sabatini, Martín; Arabolaza, Ana; Gramajo, Hugo

2014-07-01

Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Estimation of microbial protein supply in ruminants using urinary purine derivatives

International Nuclear Information System (INIS)

Makkar, H.P.S.; Chen, X.B.

2004-01-01

This publication presents various models, describing the quantitative excretion of purine derivatives in urine, developed for various breeds of cattle and for sheep, goat, camel and buffalo and their use for estimation of microbial protein supply in ruminant livestock. It also describes progress made over the last decade in analytical methods for determining purine derivatives, and a unique approach for estimating microbial protein supply using spot urine samples developed under the FAO/IAEA CRP. This approach of using spot urine samples dispenses with quantitative recovery of urine, enabling its use by field and extension workers for evaluation of the nutritional status of farm animals. Future areas of research are also highlighted in the book. This book is a good source of reference for research workers, students and extension workers alike

Purine derivative excretion and microbial protein synthesis in sheep ...

African Journals Online (AJOL)

In a 3 x 3 Latin square design experiment, urinary excretions of purine derivatives (allantoin N, Uric acid N, Xanthine + Hypoxanthine N) were measured and used to estimate microbial N yield in 9 sheep fed roughage- based diet supplemented with 0, 150 and 300g DM grass silage respectively. Daily urinary excretions of ...

Novel metabolic pathways in Archaea.

Science.gov (United States)

Sato, Takaaki; Atomi, Haruyuki

2011-06-01

The Archaea harbor many metabolic pathways that differ to previously recognized classical pathways. Glycolysis is carried out by modified versions of the Embden-Meyerhof and Entner-Doudoroff pathways. Thermophilic archaea have recently been found to harbor a bi-functional fructose-1,6-bisphosphate aldolase/phosphatase for gluconeogenesis. A number of novel pentose-degrading pathways have also been recently identified. In terms of anabolic metabolism, a pathway for acetate assimilation, the methylaspartate cycle, and two CO2-fixing pathways, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, have been elucidated. As for biosynthetic pathways, recent studies have clarified the enzymes responsible for several steps involved in the biosynthesis of inositol phospholipids, polyamine, coenzyme A, flavin adeninedinucleotide and heme. By examining the presence/absence of homologs of these enzymes on genome sequences, we have found that the majority of these enzymes and pathways are specific to the Archaea. Copyright © 2011 Elsevier Ltd. All rights reserved.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae.

Science.gov (United States)

Lin-Wang, Kui; Bolitho, Karen; Grafton, Karryn; Kortstee, Anne; Karunairetnam, Sakuntala; McGhie, Tony K; Espley, Richard V; Hellens, Roger P; Allan, Andrew C

2010-03-21

The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

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McGhie Tony K

2010-03-01

Full Text Available Abstract Background The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. Results We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry. Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. Conclusions This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

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Belshoff Alex C

2011-05-01

Full Text Available Abstract Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (on linr)

NARCIS (Netherlands)

Wang, Kui-Lin; Bolitho, Karen; Grafton, Karryn; Kortstee, A.J.; Karunairetnam, Sakuntala; McGhie, T.K.; Espley, R.V.; Hellens, R.P.; Allan, A.C.

2010-01-01

Background - The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the

Purine analogs as phosphatidylinositol 4-kinase III beta inhibitors

Czech Academy of Sciences Publication Activity Database

Šála, Michal; Kögler, Martin; Plačková, Pavla; Mejdrová, Ivana; Hřebabecký, Hubert; Procházková, Eliška; Strunin, Dmytro; Lee, G.; Birkuš, G.; Weber, Jan; Mertlíková-Kaiserová, Helena; Nencka, Radim

2016-01-01

Roč. 26, č. 11 (2016), s. 2706-2712 ISSN 0960-894X R&D Projects: GA ČR GA15-09310S; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : phosphatidylinositol 4-kinase * purine * PI4K III beta * antiviral agent * hepatitis C virus Subject RIV: CC - Organic Chemistry Impact factor: 2.454, year: 2016

Growth in rice cells requires de novo purine biosynthesis by the blast fungus Magnaporthe oryzae

Science.gov (United States)

Fernandez, Jessie; Yang, Kuan Ting; Cornwell, Kathryn M.; Wright, Janet D.; Wilson, Richard A.

2013-01-01

Increasing incidences of human disease, crop destruction and ecosystem perturbations are attributable to fungi and threaten socioeconomic progress and food security on a global scale. The blast fungus Magnaporthe oryzae is the most devastating pathogen of cultivated rice, but its metabolic requirements in the host are unclear. Here we report that a purine-requiring mutant of M. oryzae could develop functional appressoria, penetrate host cells and undergo the morphogenetic transition to elaborate bulbous invasive hyphae from primary hyphae, but further in planta growth was aborted. Invasive hyphal growth following rice cell ingress is thus dependent on de novo purine biosynthesis by the pathogen and, moreover, plant sources of purines are neither available to the mutant nor required by the wild type during the early biotrophic phase of infection. This work provides new knowledge about the metabolic interface between fungus and host that might be applicable to other important intracellular fungal pathogens. PMID:23928947

The Formation of Nucleobases from the Ultraviolet Photoirradiation of Purine in Simple Astrophysical Ice Analogues.

Science.gov (United States)

Materese, Christopher K; Nuevo, Michel; Sandford, Scott A

2017-08-01

Nucleobases are the informational subunits of RNA and DNA and are essential to all known forms of life. The nucleobases can be divided into two groups of molecules: the pyrimidine-based compounds that include uracil, cytosine, and thymine, and the purine-based compounds that include adenine and guanine. Previous work in our laboratory has demonstrated that uracil, cytosine, thymine, and other nonbiological, less common nucleobases can form abiotically from the UV photoirradiation of pyrimidine in simple astrophysical ice analogues containing combinations of H 2 O, NH 3 , and CH 4 . In this work, we focused on the UV photoirradiation of purine mixed with combinations of H 2 O and NH 3 ices to determine whether or not the full complement of biological nucleobases can be formed abiotically under astrophysical conditions. Room-temperature analyses of the resulting photoproducts resulted in the detection of adenine, guanine, and numerous other functionalized purine derivatives. Key Words: Pyrimidine-Nucleobases-Interstellar; Ices-Cometary; Ices-Molecular processes-Prebiotic chemistry. Astrobiology 17, 761-770.

Effect of tempe waste on excreation of purine derivatives and microbial–N supply in lactating Etawah crossbred goats

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D.A Astuti

2002-10-01

Full Text Available The aim of this study was to evaluate excretion of purine derivatives and microbial–N supply in lactating Etawah crossbred goats fed with fermented soybean waste. Sixteen first lactating goats were randomly allotted into four dietary treatment groups that received 50% king grass plus R1: 50% concentrate, R2: 25% concentrate and 25% fresh tempe waste, R3: 25% concentrate and 25% fermented tempe waste, and R4: 25% concentrate and 25% gelatinizing of liquid tempe waste. Fermented tempe waste was made by fermentation of tempe waste (seed content of soybean using Aspergillus niger, while for the gellatinizing of liquid tempe waste was made by gelatinized with maize flour. Protein balance studies were conducted during two week trial and at the end of the research. Urinary protein and purine derivatives were collected for analysis. Microbial–N supply was calculated from purine derivatives excretion. Results showed that nitrogen consumptions were significantly different between R4 and three other treatments and apparent digestible nitrogen in R3 were higher than that of R4 (P<0.05. The nitrogen retention in R1 and R3 were higher than that of R2 and R4. Urinary purine derivatives in this study showed that allantoin, xanthine and hypoxanthine in R3 were higher than that of R4, while R1 and R2 were the same and the highest uric acid excretion and total purine derivatives were observed in R3. Microbial–N supply were significantly different between all treatments where R3 was the highest. This research concluded that fermented soybean waste had the highest total purine derivatives excretion and microbial–N supply to the lactating Etawah crossbred goats.

Novel key metabolites reveal further branching of the roquefortine/meleagrin biosynthetic pathway.

Science.gov (United States)

Ries, Marco I; Ali, Hazrat; Lankhorst, Peter P; Hankemeier, Thomas; Bovenberg, Roel A L; Driessen, Arnold J M; Vreeken, Rob J

2013-12-27

Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.

Urinary excretion of purine derivatives as an index of microbial protein synthesis in the camel (Camelus dromedarius).

Science.gov (United States)

Guerouali, Abdelhai; El Gass, Youssef; Balcells, Joaquim; Belenguer, Alvaro; Nolan, John

2004-08-01

Five experiments were carried out to extend knowledge of purine metabolism in the camel (Camelus dromedarius) and to establish a model to enable microbial protein outflow from the forestomachs to be estimated from the urinary excretion of purine derivatives (PD; i.e. xanthine, hypoxanthine, uric acid, allantoin). In experiment 1, four camels were fasted for five consecutive days to enable endogenous PD excretion in urine to be determined. Total PD excretion decreased during the fasting period to 267 (SE 41.5) micromol/kg body weight (W)0.75 per d. Allantoin and xanthine + hypoxanthine were consistently 86 and 6.1 % of total urinary PD during this period but uric acid increased from 3.6 % to 7.4 %. Xanthine oxidase activity in tissues (experiment 2) was (micromol/min per g fresh tissue) 0.038 in liver and 0.005 in gut mucosa but was not detected in plasma. In experiment 3, the duodenal supply of yeast containing exogenous purines produced a linear increase in urinary PD excretion rate with the slope indicating that 0.63 was excreted in urine. After taking account of endogenous PD excretion, the relationship can be used to predict purine outflow from the rumen. From the latter prediction, and also the purine:protein ratio in bacteria determined in experiment 5, we predicted the net microbial outflow from the rumen. In experiment 4, with increasing food intake, the rate of PD excretion in the urine increased linearly by about 11.1 mmol PD/kg digestible organic matter intake (DOMI), equivalent to 95 g microbial protein/kg DOMI.

Effect of physiological status on endogenous excretion of purine derivatives in cattle

International Nuclear Information System (INIS)

Balcells, J.; Vicente, F.; Orellana-Boero, P.; Martin-Orue, S.; Gonzalez-Ronquillo, M.

2004-01-01

The present study examined the endogenous urinary excretion of purine derivatives (PD: allantoin and uric acid) in normally fed animals in different physiological states. Animals fitted with a simple rumen cannula and a T-shaped duodenal cannula were used in three separate experiments. In Experiment I, three heifers (about 8 months old; 225 ± 4.4 kg) fed a cereal based diet were used. In Experiment 11, two Friesian dry cows (about 24 months old; 696 ± 21 kg) fed at maintenance level on chopped barley straw and barley grain (50:50). In Experiment 3, three multiparous crossbreed Holstein-Friesian cows (560 ± 10 kg, average milk yield 25 ± 3.2 kg/d) in their third lactation were used. The cows were fed a mixed diet (48:52; roughage:concentrate). 15 N ammonium phosphate was infused continuously into the rumen to label microbial purine bases (PB). Duodenal flow of digesta and PB was determined using a dual marker system. After 72-80 h purine enrichment had reached plateau values in body pools and in urine. Daily endogenous PD excretion (μmol/W 0.75 ) obtained in dry cows (310 ± 31.0) were not significantly different from that obtained in growing steers (236 ± 6.0) but were consistently higher (512.4 ± 36.4) in lactating cows. (author)

Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

Science.gov (United States)

Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

2017-11-30

The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

International Nuclear Information System (INIS)

Nara, Takeshi; Hashimoto, Muneaki; Hirawake, Hiroko; Liao, Chien-Wei; Fukai, Yoshihisa; Suzuki, Shigeo; Tsubouchi, Akiko; Morales, Jorge; Takamiya, Shinzaburo; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Fan, Chia-Kwung; Inaoka, Daniel Ken; Inoue, Masayuki; Tanaka, Akiko; Harada, Shigeharu; Kita, Kiyoshi

2012-01-01

Highlights: ► An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. ► Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. ► CPSII bound with both ATC and DHO. ► ATC bound with both CPSII and DHO. ► A functional tri-enzyme complex might precede the establishment of the fused enzyme. -- Abstract: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded—and led to—gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.

Caracterización de la microbiota bacteriana presente en biorreactores de microalgas alimentados con purines

OpenAIRE

Prado Carrascal, Ana Isabel

2016-01-01

La producción intensiva ganadera, con una alta concentración de animales estabulados, conlleva la generación de grandes cantidades de residuos semilíquidos o purines, cuyo vertido no controlado puede generar contaminación tanto en los suelos como en las aguas superficiales y subterráneas, lo que provoca importantes problemas ambientales que han obligado a legislar su gestión. El tratamiento de los purines tiene por objetivo básico reducir la materia orgánica y el contenido de nutrientes, lo q...

Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

Science.gov (United States)

Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

2015-12-01

In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

The preliminary research for biosynthetic engineering by radiation fusion technology

Energy Technology Data Exchange (ETDEWEB)

Roh, Chang Hyun; Jung, U Hee; Park, Hae Ran [KAERI, Daejeon (Korea, Republic of)

2012-01-15

The purpose of this project is to elucidate the solution to the production of bioactive substance using biotransformation process from core technology of biosynthetic engineering by radiation fusion technology. And, this strategy will provide core technology for development of drugs as new concept and category. Research scopes and contents of project include 1) The development of mutant for biosynthetic engineering by radiation fusion technology 2) The development of host for biosynthetic engineering by radiation fusion technology 3) The preliminary study for biosynthetic engineering of isoflavone by radiation fusion technology. The results are as follows. Isoflavone compounds(daidzein, hydroxylated isoflavone) were analyzed by GC-MS. The study of radiation doses and p-NCA high-throughput screening for mutant development were elucidated. And, it was carried out the study of radiation doses for host development. Furthermore, the study of redox partner and construction of recombinant strain for region-specific hydroxylation(P450, redox partner). In addition, the biological effect of 6,7,4'-trihydroxyisoflavone as an anti-obesity agent was elucidated in this study.

The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice

International Nuclear Information System (INIS)

Hosek, B.; Bohaecek, J.; Sikulova, J.

1991-01-01

Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide and after whole-body gamma irradiation, applied alone or three days after CY application, In the first days after the treatment the enzyme activities were significantly depressed with the exception of ADA in the spleen, where a high elevation in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation. In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs

Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

Directory of Open Access Journals (Sweden)

Valerio Mori

Full Text Available NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT, which in mammals comprises three distinct isozymes, and NAD synthetase (NADS. First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide. In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species

DEFF Research Database (Denmark)

Nielsen, Jens Christian; Grijseels, Sietske; Prigent, Sylvain

2017-01-01

Filamentous fungi produce a wide range of bioactive compounds with important pharmaceutical applications, such as antibiotic penicillins and cholesterol-lowering statins. However, less attention has been paid to fungal secondary metabolites compared to those from bacteria. In this study, we...... sequenced the genomes of 9 Penicillium species and, together with 15 published genomes, we investigated the secondary metabolism of Penicillium and identified an immense, unexploited potential for producing secondary metabolites by this genus. A total of 1,317 putative biosynthetic gene clusters (BGCs) were......-referenced the predicted pathways with published data on the production of secondary metabolites and experimentally validated the production of antibiotic yanuthones in Penicillia and identified a previously undescribed compound from the yanuthone pathway. This study is the first genus-wide analysis of the genomic...

Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes

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Yang Haihua

2009-11-01

Full Text Available Abstract Background Nikkomycins are a group of peptidyl nucleoside antibiotics and act as potent inhibitors of chitin synthases in fungi and insects. Nikkomycin X and Z are the main components produced by Streptomyces ansochromogenes. Of them, nikkomycin Z is a promising antifungal agent with clinical significance. Since highly structural similarities between nikkomycin Z and X, separation of nikkomycin Z from the culture medium of S. ansochromogenes is difficult. Thus, generating a nikkomycin Z selectively producing strain is vital to scale up the nikkomycin Z yields for clinical trials. Results A nikkomycin Z producing strain (sanPDM was constructed by blocking the imidazolone biosynthetic pathway of nikkomycin X via genetic manipulation and yielded 300 mg/L nikkomycin Z and abolished the nikkomycin X production. To further increase the yield of nikkomycin Z, the effects of different precursors on its production were investigated. Precursors of nucleoside moiety (uracil or uridine had a stimulatory effect on nikkomycin Z production while precursors of peptidyl moiety (L-lysine and L-glutamate had no effect. sanPDM produced the maximum yields of nikkomycin Z (800 mg/L in the presence of uracil at the concentration of 2 g/L and it was approximately 2.6-fold higher than that of the parent strain. Conclusion A high nikkomycin Z selectively producing was obtained by genetic manipulation combined with precursors feeding. The strategy presented here might be applicable in other bacteria to selectively produce targeted antibiotics.

SnapShot: O-Glycosylation Pathways across Kingdoms

DEFF Research Database (Denmark)

Joshi, Hiren J.; Narimatsu, Yoshiki; Schjoldager, Katrine T.

2018-01-01

O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of......-glycans found in different organisms and their principle biosynthetic pathways...

Biosynthetic origin of the isoprene units in chromenes of Piper aduncum (Piperaceae)

International Nuclear Information System (INIS)

Leite, Ana C.; Lopes, Adriana A.; Bolzani, Vanderlan da S.; Furlan, Maysa; Kato, Massuo J.

2007-01-01

Metabolic studies involving the incorporation of [1- 13 C]-D-glucose into intact leaves of Piper aduncum (Piperaceae) have indicated that both the mevalonate (MVA) and the pyruvate-triose (MEP) non-mevalonate pathways are implicated in the biosynthesis of isoprene moieties present in methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl- 2'-butenyl)-2H-1-chromene-6-carboxylate (2). The pattern of incorporation of label from [1- 13 C]-D-glucose into these chromenes was determined by quantitative 13 C NMR spectroscopy. The results confirmed that biosynthetic compartment of 1 and 2 could either be the plastid and/ or the cytosol or, possibly, an additional compartment such as the plastid inter-membrane space. (author)

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

Science.gov (United States)

Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, María Angeles; Sabater-Jara, Ana Belén

2018-04-21

Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight -1 ) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota

Decoding the similarities and differences among mycobacterial species.

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Sony Malhotra

2017-08-01

Full Text Available Mycobacteriaceae comprises pathogenic species such as Mycobacterium tuberculosis, M. leprae and M. abscessus, as well as non-pathogenic species, for example, M. smegmatis and M. thermoresistibile. Genome comparison and annotation studies provide insights into genome evolutionary relatedness, identify unique and pathogenicity-related genes in each species, and explore new targets that could be used for developing new diagnostics and therapeutics. Here, we present a comparative analysis of ten-mycobacterial genomes with the objective of identifying similarities and differences between pathogenic and non-pathogenic species. We identified 1080 core orthologous clusters that were enriched in proteins involved in amino acid and purine/pyrimidine biosynthetic pathways, DNA-related processes (replication, transcription, recombination and repair, RNA-methylation and modification, and cell-wall polysaccharide biosynthetic pathways. For their pathogenicity and survival in the host cell, pathogenic species have gained specific sets of genes involved in repair and protection of their genomic DNA. M. leprae is of special interest owing to its smallest genome (1600 genes and ~1300 psuedogenes, yet poor genome annotation. More than 75% of the pseudogenes were found to have a functional ortholog in the other mycobacterial genomes and belong to protein families such as transferases, oxidoreductases and hydrolases.

Development of Equation Based on Urinary Purine Derivatives to Estimate Rumen Microbial Protein Production in Goats

International Nuclear Information System (INIS)

Jetana, Thongsuk; Abdullah, Norhani; Liang, Boo Juan; Syed Salim, Syed Jalaludin; Ho, Wan Yin

2003-06-01

Three experiments were conducted at the farm of the Universiti Putra Malaysia, Serdang, Selangor, Malaysia, to establish a model as an index for estimating rumen microbial protein production. In Experiment 1, six Ferral male goats (wt. 40.2±4.6 kg) were used to determine the endogenous purine derivatives (PD) excreted in the urine by fasting. In Experiment 2, four Ferral male goats (wt. 39.6±1.8 kg) were used to measure the proportion of plasma PD excreted in the urine by using [ 14 C]-uric acid as a marker at two levels of feed intake (40% and 80% voluntary intake), using an incomplete 2x4 Latin square experimental design. The feed consisted of 40% oil palm frond and 60% concentrate (OPFC). In Experiment 3, four Ferral male goats fed (OPFC)) were slaughtered and rumen contents were taken for measurements of purine and total nitrogen contents of mixed rumen microbes. The results showed that endogenous PD (allantoin, uric acid, xanthine and hypoxanthine) excreted in the urine obtained by the fasting trial was 202±17 μmol/kg BW 0 . 75 d - 1. The average percentage recovery of plasma PD excretion in the urine by using [ 14 C)-uric acid as a marker was 83±2.0% (cv=6.88, ranged 76.3-91.4%, n=8). Percentage recovery was not affected by levels of feed intake. The ratio of purine N: total N in the mixed rumen liquid associated bacteria (LAB) was 0.085. In this study, a preliminary model for goats was established by using the information from the recovery of labeled PD [ 14 C]-uric acid and the fasting PD excretion. The model obtained was Y 0.83X + 0.202 x BW 0 . 75 , where Y = PD excretion in the urine (mmol/d) X PD absorption at small intestine (mmol/d) BW 0 . 75 = Metabolic body weight (kg) Thus the microbial nitrogen based on total PD (MNpd) can be calculated as follows: MNpd = 70 x X = 0.992 x X (g/d) 0.085 x 0.83 x 1000 where 0.085 is the ratio of purine-N: total N in mixed rumen microbes, 0.83 is the average of digestibility of microbial purine from published

Integrating computational methods to retrofit enzymes to synthetic pathways.

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Brunk, Elizabeth; Neri, Marilisa; Tavernelli, Ivano; Hatzimanikatis, Vassily; Rothlisberger, Ursula

2012-02-01

Microbial production of desired compounds provides an efficient framework for the development of renewable energy resources. To be competitive to traditional chemistry, one requirement is to utilize the full capacity of the microorganism to produce target compounds with high yields and turnover rates. We use integrated computational methods to generate and quantify the performance of novel biosynthetic routes that contain highly optimized catalysts. Engineering a novel reaction pathway entails addressing feasibility on multiple levels, which involves handling the complexity of large-scale biochemical networks while respecting the critical chemical phenomena at the atomistic scale. To pursue this multi-layer challenge, our strategy merges knowledge-based metabolic engineering methods with computational chemistry methods. By bridging multiple disciplines, we provide an integral computational framework that could accelerate the discovery and implementation of novel biosynthetic production routes. Using this approach, we have identified and optimized a novel biosynthetic route for the production of 3HP from pyruvate. Copyright © 2011 Wiley Periodicals, Inc.

Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

Science.gov (United States)

Nielsen, Agnieszka Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos; Wlodarczyk, Artur Jacek; Gnanasekaran, Thiyagarajan; Perestrello Ramos H de Jesus, Maria; King, Brian Christopher; Bakowski, Kamil; Jensen, Poul Erik

2016-07-01

Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Synthesis and NMR of {sup 15}N-labeled DNA fragments

Energy Technology Data Exchange (ETDEWEB)

Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

1994-12-01

DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

Evolutionary systems biology of amino acid biosynthetic cost in yeast.

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Michael D Barton

2010-08-01

Full Text Available Every protein has a biosynthetic cost to the cell based on the synthesis of its constituent amino acids. In order to optimise growth and reproduction, natural selection is expected, where possible, to favour the use of proteins whose constituents are cheaper to produce, as reduced biosynthetic cost may confer a fitness advantage to the organism. Quantifying the cost of amino acid biosynthesis presents challenges, since energetic requirements may change across different cellular and environmental conditions. We developed a systems biology approach to estimate the cost of amino acid synthesis based on genome-scale metabolic models and investigated the effects of the cost of amino acid synthesis on Saccharomyces cerevisiae gene expression and protein evolution. First, we used our two new and six previously reported measures of amino acid cost in conjunction with codon usage bias, tRNA gene number and atomic composition to identify which of these factors best predict transcript and protein levels. Second, we compared amino acid cost with rates of amino acid substitution across four species in the genus Saccharomyces. Regardless of which cost measure is used, amino acid biosynthetic cost is weakly associated with transcript and protein levels. In contrast, we find that biosynthetic cost and amino acid substitution rates show a negative correlation, but for only a subset of cost measures. In the economy of the yeast cell, we find that the cost of amino acid synthesis plays a limited role in shaping transcript and protein expression levels compared to that of translational optimisation. Biosynthetic cost does, however, appear to affect rates of amino acid evolution in Saccharomyces, suggesting that expensive amino acids may only be used when they have specific structural or functional roles in protein sequences. However, as there appears to be no single currency to compute the cost of amino acid synthesis across all cellular and environmental

TD-DFT investigation of the magnetic circular dichroism spectra of some purine and pyrimidine bases of nucleic acids.

Science.gov (United States)

Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick; Santoro, Fabrizio; Improta, Roberto; Coriani, Sonia

2015-05-28

We present a computational study of the magnetic circular dichroism (MCD) spectra in the 200-300 nm wavelength region of purine and its derivative hypoxanthine, as well as of the pyrimidine bases of nucleic acids uracil, thymine, and cytosine, using the B3LYP and CAM-B3LYP functionals. Solvent effects are investigated within the polarizable continuum model and by inclusion of explicit water molecules. In general, the computed spectra are found to be in good agreement with the experimental ones, apart from some overall blue shifts. Both the pseudo-A term shape of the MCD spectra of the purines and the B term shape of the spectra of pyrimidine bases are reproduced. Our calculations also correctly reproduce the reversed phase of the MCD bands in purine compared to that of its derivatives present in nucleic acids. Solvent effects are sizable and system specific, but they do not in general alter the qualitative shape of the spectra. The bands are dominated by the bright π → π* transitions, and our calculations in solution nicely reproduce their energy differences, improving the estimates obtained in the gas phase. Shoulders are predicted for purine and uracil due to n → π* excitations, but they are too weak to be observed in the experiment.

Effect of training load structure on purine metabolism in middle-distance runners.

Science.gov (United States)

Zieliński, Jacek; Kusy, Krzysztof; Rychlewski, Tadeusz

2011-09-01

There are no studies analyzing the effect of training loads on purine metabolism during long training periods. The study's purpose was to evaluate the effect of training load changes and subsequent detraining on purine metabolism in middle-distance runners during a 1-yr cycle. In four characteristic points of the training cycle, loads assigned to five intensity zones, pre- and postexercise plasma hypoxanthine (Hx) and uric acid, and erythrocyte Hx-guanine phosphoribosyltransferase (HGPRT) activity were determined in 11 male middle-distance runners at the national level, practicing competitive sport for 8.1 ± 0.3 yr and with a mean age of 22.3 ± 0.7 yr, body mass of 73.0 ± 3.4 kg, and body height of 180 ± 2.2 cm. In the competition phase (CP), training loads in aerobic compensation and threshold zones decreased by 65.4% and by 20.5%, respectively. At the same time, anaerobic training loads increased by 132.5% in the VO(2max) zone and by 74.6% in the lactic acid tolerance zone. Postexercise Hx decreased significantly in CP by 6.2 μmol·L(-1). and increased in the transition phase (TP) by 17.4 μmol·L(-1). Both pre- and postexercise HGPRT activity increased significantly in CP by 9.3 nmol·mg(-1)·h(-1). and by 4.9 nmol·mg(-1)·h(-1). , respectively, and decreased significantly in TP by 10.6 nmol·mg(-1)·h(-1). and by 12.0 nmol·mg(-1)·h(-1). , respectively. A significant uric acid increase of 54 μmol·L(-1). was revealed merely in TP. The effect of anaerobic training on purine metabolism is significant despite of a very short total duration of anaerobic loads. Elevated preexercise HGPRT activity in CP suggests adaptation changes consisting in a "permanent readiness" for purine salvage. The detraining in TP leads to reverse adaptation changes. Probably, plasma Hx concentration and erythrocyte HGPRT activity may be considered as a useful measure of training status.

Linking fungal secondary metabolites and pathways to their genes in Aspergillus

DEFF Research Database (Denmark)

Petersen, Lene Maj

. oryzae metabolites, however, revealed the chemical link between the two species. In two parallel projects, involving A. niger and A. aculeatus respectively, the polyketide 6-methyl salicylic acid (6-MSA), and corresponding biosynthetic pathways, were investigated. In A. niger, 6-MSA was converted...

Optimization of the operation of separation of the solids in suspension of the purines (Part I); Optimizacion de la operacion de separacion de los solidos en suspension de los purines (Parte I)

Energy Technology Data Exchange (ETDEWEB)

Bosque Hernandez, J. L. del; Martin Sanchez, J. L.

2004-07-01

The swinish cattle raising you can calculate in our country m 18 million heads. Locating the Spanish cattle cabin in the second position of the European Union. This sector is of great importance in the economy, having big possibilities of growth, due to the great existent demand. However, the generated residuals, denominated purines, they suppose a great obstacle for the expansion of this sector. The purines mammoth production and other cattle residuals outline a serious problem of environmental contamination; for what is necessary to establish the approaches for an appropriate treatment of the same ones, with the purpose of achieving the elimination of the problems of environmental contamination that you/they generate and, in special way, those contamination of the waters. (Author) 5 refs.

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl., a Non-Model Plant with Potent Laxative Properties.

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Nagaraja Reddy Rama Reddy

Full Text Available Senna (Cassia angustifolia Vahl. is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG, Cluster of Orthologous Gene (COG and Gene Ontology (GO. Out of the total transcripts, 40138 (95.0% and 36349 (97.7% from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf and 32077 (mature leaf transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7% CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties.

Science.gov (United States)

Rama Reddy, Nagaraja Reddy; Mehta, Rucha Harishbhai; Soni, Palak Harendrabhai; Makasana, Jayanti; Gajbhiye, Narendra Athamaram; Ponnuchamy, Manivel; Kumar, Jitendra

2015-01-01

Senna (Cassia angustifolia Vahl.) is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090)', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various

Catalytic carbene transfer allows the direct customization of cyclic purine dinucleotides.

Science.gov (United States)

Fei, Na; Häussinger, Daniel; Blümli, Seraina; Laventie, Benoît-Joseph; Bizzini, Lorenzo D; Zimmermann, Kaspar; Jenal, Urs; Gillingham, Dennis

2014-08-11

We describe a simple method for the direct modification of nucleobases in cyclic purine dinucleotides, important signalling molecules in both prokaryotes and eukaryotes. The method tolerates all members of the cyclic dinucleotide family and could be used to modulate their function or introduce useful side-chains such as fluorophores and photo-crosslinking groups.

Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440

Science.gov (United States)

Molina‐Henares, M. Antonia; García‐Salamanca, Adela; Molina‐Henares, A. Jesús; De La Torre, Jesús; Herrera, M. Carmen; Ramos, Juan L.; Duque, Estrella

2009-01-01

Summary Pseudomonas putida KT2440 is a non‐pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini‐Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene‐encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB‐like genes are present in the host chromosome. PMID:21261884

Chapter 7. Cloning and analysis of natural product pathways.

Science.gov (United States)

Gust, Bertolt

2009-01-01

The identification of gene clusters of natural products has lead to an enormous wealth of information about their biosynthesis and its regulation, and about self-resistance mechanisms. Well-established routine techniques are now available for the cloning and sequencing of gene clusters. The subsequent functional analysis of the complex biosynthetic machinery requires efficient genetic tools for manipulation. Until recently, techniques for the introduction of defined changes into Streptomyces chromosomes were very time-consuming. In particular, manipulation of large DNA fragments has been challenging due to the absence of suitable restriction sites for restriction- and ligation-based techniques. The homologous recombination approach called recombineering (referred to as Red/ET-mediated recombination in this chapter) has greatly facilitated targeted genetic modifications of complex biosynthetic pathways from actinomycetes by eliminating many of the time-consuming and labor-intensive steps. This chapter describes techniques for the cloning and identification of biosynthetic gene clusters, for the generation of gene replacements within such clusters, for the construction of integrative library clones and their expression in heterologous hosts, and for the assembly of entire biosynthetic gene clusters from the inserts of individual library clones. A systematic approach toward insertional mutation of a complete Streptomyces genome is shown by the use of an in vitro transposon mutagenesis procedure.

Direct determination of tautomerism in purine derivatives by low-temperature NMR spectroscopy

Czech Academy of Sciences Publication Activity Database

Sečkářová, P.; Marek, R.; Maliňáková, K.; Kolehmainen, E.; Hocková, Dana; Hocek, Michal; Sklenář, V.

2004-01-01

Roč. 45, č. 33 (2004), s. 6259-6263 ISSN 0040-4039 R&D Projects: GA MŠk LN00A016 Institutional research plan: CEZ:AV0Z4055905 Keywords : purine * NMR * tautomerism Subject RIV: CC - Organic Chemistry Impact factor: 2.484, year: 2004

Absorption and Intermediary Metabolism of Purines and Pyrimidines in Lactating Dairy Cows

DEFF Research Database (Denmark)

Nielsen, Charlotte Stentoft; Røjen, Betina Amdisen; Jensen, Søren Krogh

2015-01-01

About 20 % of ruminal microbial N in dairy cows derives from purines and pyrimidines; however, their intermediary metabolism and contribution to the overall N metabolism has sparsely been described. In the present study, the postprandial patterns of net portal-drained viscera (PDV) and hepatic...

Biosynthetic origin of the isoprene units in chromenes of Piper aduncum (Piperaceae)

Energy Technology Data Exchange (ETDEWEB)

Leite, Ana C.; Lopes, Adriana A.; Bolzani, Vanderlan da S.; Furlan, Maysa [UNESP, Araraquara, SP (Brazil). Inst. de Quimica]. E-mail: maysaf@iq.unesp.br; Kato, Massuo J. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica

2007-07-01

Metabolic studies involving the incorporation of [1-{sup 13}C]-D-glucose into intact leaves of Piper aduncum (Piperaceae) have indicated that both the mevalonate (MVA) and the pyruvate-triose (MEP) non-mevalonate pathways are implicated in the biosynthesis of isoprene moieties present in methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl- 2'-butenyl)-2H-1-chromene-6-carboxylate (2). The pattern of incorporation of label from [1- {sup 13}C]-D-glucose into these chromenes was determined by quantitative {sup 13}C NMR spectroscopy. The results confirmed that biosynthetic compartment of 1 and 2 could either be the plastid and/ or the cytosol or, possibly, an additional compartment such as the plastid inter-membrane space. (author)

Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.

Science.gov (United States)

Rodriguez, Alberto; Martínez, Juan A; Millard, Pierre; Gosset, Guillermo; Portais, Jean-Charles; Létisse, Fabien; Bolivar, Francisco

2017-06-01

Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Efecto antagónico in vitro de actinomicetos aislados de purines de chipaca (bidens pilosa l.) frente a phytophthora infestans (mont) de bary

OpenAIRE

Fonseca Ardila, Yudy Astrid; Castellanos Suárez, Diana Edith; León Sicard, Tomás Enrique

2011-01-01

Se estudió el efecto inhibidor de los actinomicetos presentes en purines o extractos fermentados de plantas de chipaca (Bidens pilosa L.), sobre el crecimiento de Phytophthora infestans (Mont) de Bary, causante del tizón tardío de la papa. Se elaboraron cuatro purines de flores, raíces, hojas-tallos y su mezcla. De estos purines se obtuvieron 25 aislamientos de actinomicetos, cada uno de los cuales se enfrentó con P. infestans en placas de medio de cultivo, utilizando la técnica de anillos de...

Recent advances in biosynthetic modeling of nitric oxide reductases and insights gained from nuclear resonance vibrational and other spectroscopic studies

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, Saumen; Reed, Julian; Sage, Timothy; Branagan, Nicole C.; Petrik, Igor D.; Miner, Kyle D.; Hu, Michael Y.; Zhao, Jiyong; Alp, E. Ercan; Lu, Yi

2015-10-05

This Forum Article focuses on recent advances in structural and spectroscopic studies of biosynthetic models of nitric oxide reductases (NORs). NORs are complex metalloenzymes found in the denitrification pathway of Earth's nitrogen cycle where they catalyze the proton-dependent twoelectron reduction of nitric oxide (NO) to nitrous oxide (N₂O). While much progress has been made in biochemical and biophysical studies of native NORs and their variants, a. clear mechanistic understanding of this important metalloenzyme related to its function is still elusive. We report herein UV vis and nuclear resonance vibrational spectroscopy (NRVS) studies of mononitrosylated intermediates of the NOR reaction of a biosynthetic model. The ability to selectively substitute metals at either heme or nonheme metal sites allows the introduction of independent ⁵⁷Fe probe atoms at either site, as well as allowing the preparation of analogues of stable reaction intermediates by replacing either metal with a redox inactive metal. Together with previous structural and spectroscopic results, we summarize insights gained from studying these biosynthetic models toward understanding structural features responsible for the NOR activity and its mechanism. As a result, the outlook on NOR modeling is also discussed, with an emphasis on the design of models capable of catalytic turnovers designed based on close mimics of the secondary coordination sphere of native NORs.

Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism.

Science.gov (United States)

Xie, Zhengzhi; Ma, Xiaoqiang; Gang, David R

2009-01-01

Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules-groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants.

Gene transcript profiles of the TIA biosynthetic pathway in response to ethylene and copper reveal their interactive role in modulating TIA biosynthesis in Catharanthus roseus.

Science.gov (United States)

Pan, Ya-Jie; Liu, Jia; Guo, Xiao-Rui; Zu, Yuan-Gang; Tang, Zhong-Hua

2015-05-01

Research on transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis of the medicinal plant, Catharanthus roseus, has largely been focused on gene function and not clustering analysis of multiple genes at the transcript level. Here, more than ten key genes encoding key enzyme of alkaloid synthesis in TIA biosynthetic pathways were chosen to investigate the integrative responses to exogenous elicitor ethylene and copper (Cu) at both transcriptional and metabolic levels. The ethylene-induced gene transcripts in leaves and roots, respectively, were subjected to principal component analysis (PCA) and the results showed the overall expression of TIA pathway genes indicated as the Q value followed a standard normal distribution after ethylene treatments. Peak gene expression was at 15-30 μM of ethephon, and the pre-mature leaf had a higher Q value than the immature or mature leaf and root. Treatment with elicitor Cu found that Cu up-regulated overall TIA gene expression more in roots than in leaves. The combined effects of Cu and ethephon on TIA gene expression were stronger than their separate effects. It has been documented that TIA gene expression is tightly regulated by the transcriptional factor (TF) ethylene responsive factor (ERF) and mitogen-activated protein kinase (MAPK) cascade. The loading plot combination with correlation analysis for the genes of C. roseus showed that expression of the MPK gene correlated with strictosidine synthase (STR) and strictosidine b-D-glucosidase(SGD). In addition, ERF expression correlated with expression of secologanin synthase (SLS) and tryptophan decarboxylase (TDC), specifically in roots, whereas MPK and myelocytomatosis oncogene (MYC) correlated with STR and SGD genes. In conclusion, the ERF regulates the upstream pathway genes in response to heavy metal Cu mainly in C. roseus roots, while the MPK mainly participates in regulating the STR gene in response to ethylene in pre-mature leaf. Interestingly, the

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways

DEFF Research Database (Denmark)

Vavitsas, Konstantinos; Rue, Emil Østergaard; Stefánsdóttir, Lára Kristín

2017-01-01

BACKGROUND: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity...... different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids...

Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

Energy Technology Data Exchange (ETDEWEB)

Geiger, Jim

2013-11-30

structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.

Determining Antifungal Target Sites in the Sterol Pathway of the Yeast Candida and Saccharomyces

National Research Council Canada - National Science Library

Bard, Martin

1998-01-01

... as in topical infections which lead to significant losses in work-place productivity. The work reported here seeks to identify new target sites in the sterol biosynthetic pathway against which new antifungal compounds might be developed...

Loss of nitric oxide-mediated inhibition of purine neurotransmitter release in the colon in the absence of interstitial cells of Cajal.

Science.gov (United States)

Durnin, Leonie; Lees, Andrea; Manzoor, Sheerien; Sasse, Kent C; Sanders, Kenton M; Mutafova-Yambolieva, Violeta N

2017-11-01

Regulation of colonic motility depends on the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor-α-positive (PDGFRα + ) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have suggested a decreased nitrergic and increased purinergic neurotransmission in Kit W /Kit W-v ( W/W v ) mice that display lesions in ICC-IM along the gastrointestinal tract. However, contributions of NO to these phenotypes have not been evaluated. We used small-chamber superfusion assays and HPLC to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD + )/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP), and metabolites from the tunica muscularis of human, monkey, and murine colons and circular muscle of monkey colon, and we tested drugs that modulate NO levels or blocked NO receptors. NO inhibited EFS-evoked release of purines in the colon via presynaptic neuromodulation. Colons from W/W v , Nos1 -/- , and Prkg1 -/- mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/W v mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/W v mouse colons demonstrated reduced Nos1 expression and reduced NO release. Our results suggest that enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provide novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility. NEW & NOTEWORTHY This is the first study investigating the role of nitric

Cross-coupling reactions of unprotected halopurine bases, nucleosides, nucleotides and nucleoside triphosphates with 4-boronophenylalanine in water. Synthesis of (purin-8-yl)- and (purin-6-yl)phenylalanines

Czech Academy of Sciences Publication Activity Database

Čapek, Petr; Pohl, Radek; Hocek, Michal

2006-01-01

Roč. 4, č. 11 (2006), s. 2278-2284 ISSN 1477-0520 R&D Projects: GA AV ČR(CZ) 1QS400550501; GA MŠk(CZ) 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : amino acids * purines * nucleosides * cross-coupling reactions Subject RIV: CC - Organic Chemistry Impact factor: 2.874, year: 2006

Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution

Energy Technology Data Exchange (ETDEWEB)

Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo 184-8588 (Japan); Fujii, Kentaro; Yokoya, Akinari [Advanced Science Research Center, Japan Atomic Energy Agency, Tokai-mura, Naka-gun, Ibaraki 319-1195 (Japan); Fukuda, Yoshihiro; Saitoh, Yuji [Synchrotron Radiation Research Center, Japan Atomic Energy Agency, Sayo-gun, Hyougo 679-5148 (Japan)

2014-08-07

The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5{sup ′}-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5{sup ′}-monophosphate, and adenosine 5{sup ′}-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

Adaptive evolution to a high purine and fat diet of carnivorans revealed by gut microbiomes and host genomes.

Science.gov (United States)

Zhu, Lifeng; Wu, Qi; Deng, Cao; Zhang, Mengjie; Zhang, Chenglin; Chen, Hua; Lu, Guoqing; Wei, Fuwen

2018-05-01

Carnivorous members of the Carnivora reside at the apex of food chains and consume meat-only diets, rich in purine, fats and protein. Here, we aimed to identify potential adaptive evolutionary signatures compatible with high purine and fat metabolism based on analysis of host genomes and symbiotic gut microbial metagenomes. We found that the gut microbiomes of carnivorous Carnivora (e.g., Felidae, Canidae) clustered in the same clade, and other clades comprised omnivorous and herbivorous Carnivora (e.g., badgers, bears and pandas). The relative proportions of genes encoding enzymes involved in uric acid degradation were higher in the gut microbiomes of meat-eating carnivorans than plant-eating species. Adaptive amino acid substitutions in two enzymes, carnitine O-palmitoyltransferase 1 (CPT1A) and lipase F (LIPF), which play a role in fat digestion, were identified in Felidae-Candidae species. Carnivorous carnivorans appear to endure diets high in purines and fats via gut microbiomic and genomic adaptations. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Protein design for pathway engineering.

Science.gov (United States)

Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

2014-02-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

9-Benzyl-6-benzylsulfanyl-9H-purin-2-amine

Directory of Open Access Journals (Sweden)

Maywan Hariono

2014-03-01

Full Text Available In the title compound, C19H17N5S, the dihedral angles between the purine ring system (r.m.s. deviation = 0.009 Å and the S-bound and methylene-bound phenyl rings are 74.67 (8 and 71.28 (7°, respectively. In the crystal, inversion dimers linked by pairs of N—H...N hydrogen bonds generate R22(8 loops. C—H...N interactions link the dimers into (100 sheets.

Increase in urinary purines and pyrimidines in patients with methylmalonic aciduria combined with homocystinuria.

Science.gov (United States)

Porcu, Simona; Corda, Marcella; Lilliu, Franco; Contini, Liliana; Era, Benedetta; Traldi, Pietro; Fais, Antonella

2010-06-03

Methylmalonic aciduria combined with homocystinuria (MMA-HC) is the biochemical trait of a metabolic disorder resulting from impaired conversion of dietary cobalamin (cbl, or vitamin B12) to its two metabolically active forms. Effects on urinary purine and pyrimidine levels have not been described for this condition. Urine samples were collected from three patients with methylmalonic aciduria combined with homocystinuria and from 70 healthy subjects. Urinary purine and pyrimidine levels were quantitated by the use of LC/UV-Vis and LC/ESI/MS. Higher urine levels of pyrimidines were detected with both methods in patients compared to controls. Methylmalonic aciduria with homocystinuria is due to deficiency of the enzyme, cobalamin reductase. The enzyme defect leads to altered hepatic metabolism, which appears to modify circulating pyrimidine levels. Copyright 2010 Elsevier B.V. All rights reserved.

Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria.

Science.gov (United States)

Dittmann, Elke; Gugger, Muriel; Sivonen, Kaarina; Fewer, David P

2015-10-01

Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with intricate chemical structures and potent biological activities. The bulk of these natural products are known from just a handful of genera. Recent efforts have elucidated the mechanisms underpinning the biosynthesis of a diverse array of natural products from cyanobacteria. Many of the biosynthetic mechanisms are unique to cyanobacteria or rarely described from other organisms. Advances in genome sequence technology have precipitated a deluge of genome sequences for cyanobacteria. This makes it possible to link known natural products to biosynthetic gene clusters but also accelerates the discovery of new natural products through genome mining. These studies demonstrate that cyanobacteria encode a huge variety of cryptic gene clusters for the production of natural products, and the known chemical diversity is likely to be just a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms. Copyright © 2015. Published by Elsevier Ltd.

Key roles of Arf small G proteins and biosynthetic trafficking for animal development.

Science.gov (United States)

Rodrigues, Francisco F; Harris, Tony J C

2017-04-14

Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.

Substituent and noncovalent interaction effects in the reactivity of purine derivatives with tetracarboxylato-dirhodium(II) units. Rationalization of a rare binding mode via N3.

Science.gov (United States)

Amo-Ochoa, Pilar; Castillo, Oscar; Harrington, Ross W; Zamora, Félix; Houlton, Andrew

2013-02-18

Reactions between [Rh(2)(CH(3)COO)(4)] with 2,6-diaminopurine (HDap) or 6-chloro-2-aminopurine (HClap) and [Rh(2)((CH(3))(3)CCOO)(4)] with HClap produce, three new dirhodium(II) carboxylate complexes of the general form, [Rh(2)(RCOO)(4)(Purine)(2)] (R = CH(3), (CH(3))(3)C). Single crystal X-ray diffraction studies confirm that in all cases the purine coordinates to the axial position of the dirhodium(II)tetracarboxylate unit. However, while the complex obtained with HDap features the typical purine binding mode via N(7), complexes containing HClap show unusual N3 coordination. This is an extremely rare instance of an unrestricted purine binding via N3. Some rationalization of these data is offered based on a series of DFT calculations.

The purine efflux pump PbuE in Bacillus subtilis modulates expression of the PurR and G-box (XptR) regulons by adjusting the purine base pool size

DEFF Research Database (Denmark)

Nygaard, P.; Saxild, Hans Henrik

2005-01-01

a functional PbuE pump. In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE. A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration...

Antinociceptive effect of purine nucleotides.

Science.gov (United States)

Mello, C F; Begnini, J; De-La-Vega, D D; Lopes, F P; Schwartz, C C; Jimenez-Bernal, R E; Bellot, R G; Frussa-Filho, R

1996-10-01

The antinociceptive effect of purine nucleotides administered systematically (sc) was determined using the formalin and writhing tests in adult male albino mice. The mechanisms underlying nucleotide-induced antinociception were investigated by preinjecting the animals (sc) with specific antagonists for opioid (naloxone, 1 mg/kg), purinergic P1 (caffeine, 5, 10, of 30 mg/kg); theophylline, 10 mg/kg) or purinergic P2 receptors (suramin, 100 mg/kg; Coomassie blue, 30-300 mg/kg; quinidine, 10 mg/kg). Adenosine, adenosine monophosphate (AMP), diphosphate (ADP) and triphosphate (ATP) caused a reduction in the number of writhes and in the time of licking the formalin-injected paw. Naloxone had no effect on adenosine- or adenine nucleotide-induced antinociception. Caffeine (30 mg/kg) and theophylline (10 mg/kg) reversed the antinociceptive action of adenosine and adenine nucleotide derivatives in both tests. P2 antagonists did not reverse adenine nucleotide-induced antinociception. These results suggest that antinociceptive effect of adenine nucleotides is mediated by adenosine.

MUTATIONAL SYNERGISM BETWEEN RADIATIONS AND METHYLATED PURINES IN ESCHERICHIA COLI

Science.gov (United States)

Doneson, Ira N.; Shankel, Delbert M.

1964-01-01

Doneson, Ira N. (University of Kansas, Lawrence), and Delbert M. Shankel. Mutational synergism between radiations and methylalted purines in Escherichia coli. J. Bacteriol. 87:61–67. 1964.—A synergistic mutational effect was demonstrated between low doses of ultraviolet light and the methylated purines caffeine, theophylline, and theobromine. Caffeine produced the greatest effect and theobromine the least effect. The magnitude of the synergism was inversely related to the ultraviolet dosage. A large percentage of the synergistic effect could be “photoprevented” by exposure of the ultraviolet-treated cells to white light prior to exposure to the analogues. The consequence of the combined treatment occurred only when the chemical treatment followed the ultraviolet treatment. Furthermore, it was necessary to administer the chemical treatment soon after the ultraviolet treatment or the mutants were “lost.” When cells were treated with low dosages of ultraviolet light and of X irradiation (X ray), the result was merely additive, and combinations of X ray and chemical treatment yielded no synergism. Synchronous growth studies indicated that a particular growth stage of the organisms was most susceptible to the synergistic effect. The mutation studied was that of Escherichia coli B/r to high-level streptomycin resistance. PMID:14102875

Astrocyte dysfunction following molybdenum-associated purine loading could initiate Parkinson's disease with dementia.

Science.gov (United States)

Bourke, Christopher A

2018-01-01

Sporadic or idiopathic Parkinson's disease is a movement disorder with a worldwide distribution, a long pre-clinical latent period and a frequent association with dementia. The combination of molybdenum deficiency and purine ingestion could explain the movement disorder, the distribution, the latent period and the dementia association. Recent studies in sheep have shown that molybdenum deficiency enables some dietary purines to accumulate in the central nervous system. This causes astrocyte dysfunction, altered neuromodulation and eventually irreversible central nervous system disease. Humans and sheep share the ability to salvage purines and this ability places humans at risk when they ingest xanthosine, inosine, adenosine and guanosine. Adenosine ingestion in molybdenum-deficient humans will lead to adenosine loading and potentially a disturbance to the A2a adenosine receptors in the nigro-striatum. This could result in Parkinson's disease. Guanosine ingestion in molybdenum-deficient humans will lead to guanosine loading and potentially a disturbance to the guanosine receptors in the hippocampus, amygdala and ventral striatum. This could result in dementia. The molybdenum content of the average daily diet in the United States is 0.07 ppm and in the United Kingdom 0.04 ppm. Central nervous system disease occurs in sheep at <0.04 ppm. Consistent with the role proposed for molybdenum deficiency in Parkinson's disease is the observation that affected individuals have elevated sulfur amino acid levels, depressed sulfate levels, and depressed uric acid levels. Likewise the geographical distribution of Parkinson's dementia complex on Guam corresponds with the distribution of molybdenum-deficient soils hence molybdenum-deficient food gardens on that island.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (on linr)

OpenAIRE

Wang, Kui-Lin; Bolitho, Karen; Grafton, Karryn; Kortstee, A.J.; Karunairetnam, Sakuntala; McGhie, T.K.; Espley, R.V.; Hellens, R.P.; Allan, A.C.

2010-01-01

Background - The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all th...

Aspergillus nidulans as a platform for discovery and characterization of complex biosynthetic pathways

DEFF Research Database (Denmark)

Anyaogu, Diana Chinyere

in industrial applications for the productionof these bioactive compounds and other chemicals as well as for enzyme production. Especially Aspergillusniger and Aspergillus oryzae are used as industrial workhorses for the production of various enzymes. Manyof the secreted proteins are glycosylated, indicating...... aspharmaceuticals. Access to this unexploited reservoir is hampered as many of the clusters are silent orbarely expressed under laboratory conditions. Methods for activating these pathways are thereforeessential for pathway discovery and elucidation.  Filamentous fungi and Aspergillus species in particular are used...... that glycosylation plays an important role in thesecretory pathway. Thus, understanding the role and process of glycosylation will enable directedglycoengineering in Aspergilli to improve protein production and expand the repertoire of proteins, whichcan be produced by these fungi. Aspergillus nidulans has been used...

An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue.

Science.gov (United States)

Yu, Dayu; Xu, Fuchao; Valiente, Jonathan; Wang, Siyuan; Zhan, Jixun

2013-01-01

A putative indigoidine biosynthetic gene cluster was located in the genome of Streptomyces chromofuscus ATCC 49982. The silent 9.4-kb gene cluster consists of five open reading frames, named orf1, Sc-indC, Sc-indA, Sc-indB, and orf2, respectively. Sc-IndC was functionally characterized as an indigoidine synthase through heterologous expression of the enzyme in both Streptomyces coelicolor CH999 and Escherichia coli BAP1. The yield of indigoidine in E. coli BAP1 reached 2.78 g/l under the optimized conditions. The predicted protein product of Sc-indB is unusual and much larger than any other reported IndB-like protein. The N-terminal portion of this enzyme resembles IdgB and the C-terminal portion is a hypothetical protein. Sc-IndA and/or Sc-IndB were co-expressed with Sc-IndC in E. coli BAP1, which demonstrated the involvement of Sc-IndB, but not Sc-IndA, in the biosynthetic pathway of indigoidine. The yield of indigoidine was dramatically increased by 41.4 % (3.93 g/l) when Sc-IndB was co-expressed with Sc-IndC in E. coli BAP1. Indigoidine is more stable at low temperatures.

Anion-exchange analysis of isotopically labelled nucleotides, nucleosides, and bases in metabolic disorders

International Nuclear Information System (INIS)

Nissinen, E.A.O.

1987-01-01

This paper on the importance of cellular purines and pyrimidines is evidenced by the multitude of diseases, such as hyperuricemia, orotic aciduria, gout, Lesch-Nyhan syndrome, immunodeficiencies with B- and T-cell dysfunctions, etc. which result from aberrant metabolism. In addition, the use of purine and pyrimidine analogs in chemotherapy is of growing interest. Purine metabolism consists of a complex network of biochemical pathway. These pathways are under complicated feedback regulation and there also exists a close relationship between purine and pyrimidine metabolism. In addition, these pathways interact with those of the carbohydrate, amino acid, and energy metabolism. Since metabolic pathways are closely interrelated, a change in the concentration of a particular metabolite may lead to many changes in the overall metabolic profiles. For instance, in the area of nucleotide metabolism, the inhibition of IMP dehydrogenase by mycophenolic acid leads to various changes in both purine and pyrimidine nucleotide pools. Inhibition of de nova purine biosynthesis by methotrexate leads to many changes in purine and pyrimidine ribonucleotides and deoxyribonucleotides. Thus, the simultaneous measurement of all cellular purine and pyrimidine metabolites from individuals whose metabolism is altered, either by a metabolic disease or by the action of drugs, may further our understanding of cellular metabolism

Giant virus Megavirus chilensis encodes the biosynthetic pathway for uncommon acetamido sugars.

Science.gov (United States)

Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G

2014-08-29

Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The evolutionary fate of the genes encoding the purine catabolic enzymes in hominoids, birds, and reptiles.

Science.gov (United States)

Keebaugh, Alaine C; Thomas, James W

2010-06-01

Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes.

Synthesis of some glycoside analogs and related compounds from 9-amino-6-(methylthio)-9H-purine.

Science.gov (United States)

Temple, C; Kussner, C L; Montgomery, J A

1975-12-01

Additional information on the anticancer activity of 9-amino-9H-purine-6(1H)-thione and its derivatives was sought by the synthesis of some 9-(substituted amino)-6-(methylthio)-9H-purines in which the 9-substituent contained functional groups capable of either reversible or irreversible binding with an enzymatic site. Condensation of 9-amino-6-(methylthio)-9H-purine (1) with some carbonyl compounds followed by hydride reduction of the azomethine linkage in the intermediates leads to the 2-pyrrolylmethyl (8), 2,3,4-trihydroxybutyl (10), and the 1,5-dihydroxy-2- and 3-pentyl (11 and 12) compounds. A 4-hydroxybutyl derivative (13) was obtained by alkylation of 18, the 9-acetyl derivative of 1, with 4-chlorobutyl acetate followed by saponification. The cyclization of 13 and 11 with a sulfonyl chloride gave the 9-pyrrolidin-1-yl (27) and the 9-[2-(tosyloxymethyl)pyrrolidin-1-yl] (28), respectively. Acylation of 1 with ethyl L-2-pyrrolidine-5-carboxylate and ethyl 1-methyl-5-pyrrolidone-3-carboxylate, respectively, in Me2SO containing NaH gave the corresponding amides 15 and 17. Alkylation of 18 with 1-bromo-2-chloroethane and epichlorohydrin gave the N-(2-chloroethyl) and N-(1,2-epoxy-3-propyl) derivatives 19 and 20. The chloro group of the chlorobutyl derivative of 18 was displaced with KSCN and NaN3, respectively, to give the thiocyanate and azido derivatives 23 and 24. Hydrogenation of the latter gave the amine (25), which was acylated with ethyl chloroformate to give the (ethoxycarbonyl)amino compound 26. None of these compounds showed activity against L1210 leukemia cells implanted ip in mice on a single-dose schedule, suggesting that the activity observed in the simpler 9-aminopurines resulted from cleavage of the hydrazino linkage to give pH-purine-6(1H)-thione.

Reprogramming One-Carbon Metabolic Pathways To Decouple l-Serine Catabolism from Cell Growth in Corynebacterium glutamicum.

Science.gov (United States)

Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Yu; Hu, Qitiao; Chai, Xin; Wang, Bo; Liu, Shuwen; Wen, Tingyi

2018-02-16

l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13 CH 2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

Science.gov (United States)

Suzuki, T; Takahashi, E

1975-01-01

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage. PMID:1147906

Promzea: a pipeline for discovery of co-regulatory motifs in maize and other plant species and its application to the anthocyanin and phlobaphene biosynthetic pathways and the Maize Development Atlas.

Science.gov (United States)

Liseron-Monfils, Christophe; Lewis, Tim; Ashlock, Daniel; McNicholas, Paul D; Fauteux, François; Strömvik, Martina; Raizada, Manish N

2013-03-15

The discovery of genetic networks and cis-acting DNA motifs underlying their regulation is a major objective of transcriptome studies. The recent release of the maize genome (Zea mays L.) has facilitated in silico searches for regulatory motifs. Several algorithms exist to predict cis-acting elements, but none have been adapted for maize. A benchmark data set was used to evaluate the accuracy of three motif discovery programs: BioProspector, Weeder and MEME. Analysis showed that each motif discovery tool had limited accuracy and appeared to retrieve a distinct set of motifs. Therefore, using the benchmark, statistical filters were optimized to reduce the false discovery ratio, and then remaining motifs from all programs were combined to improve motif prediction. These principles were integrated into a user-friendly pipeline for motif discovery in maize called Promzea, available at http://www.promzea.org and on the Discovery Environment of the iPlant Collaborative website. Promzea was subsequently expanded to include rice and Arabidopsis. Within Promzea, a user enters cDNA sequences or gene IDs; corresponding upstream sequences are retrieved from the maize genome. Predicted motifs are filtered, combined and ranked. Promzea searches the chosen plant genome for genes containing each candidate motif, providing the user with the gene list and corresponding gene annotations. Promzea was validated in silico using a benchmark data set: the Promzea pipeline showed a 22% increase in nucleotide sensitivity compared to the best standalone program tool, Weeder, with equivalent nucleotide specificity. Promzea was also validated by its ability to retrieve the experimentally defined binding sites of transcription factors that regulate the maize anthocyanin and phlobaphene biosynthetic pathways. Promzea predicted additional promoter motifs, and genome-wide motif searches by Promzea identified 127 non-anthocyanin/phlobaphene genes that each contained all five predicted promoter

Aplicaci??n de humedales artificiales para la depuraci??n de purines de granjas porcinas

OpenAIRE

Blanco Rubio, Iv??n

2014-01-01

204 p. El principal objetivo de este trabajo de investigaci??n es proporcionar informaci??n relevante sobre el funcionamiento y dimensionado de los sistemas de humedales artificiales de tipo horizontal para el tratamiento de la fracci??n l??quida de purines de cerdo

Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system.

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Tomáš Pluskal

Full Text Available Ergothioneine is a small, sulfur-containing metabolite (229 Da synthesized by various species of bacteria and fungi, which can accumulate to millimolar levels in tissues or cells (e.g. erythrocytes of higher eukaryotes. It is commonly marketed as a dietary supplement due to its proposed protective and antioxidative functions. In this study we report the genes forming the two-step ergothioneine biosynthetic pathway in the fission yeast, Schizosaccharomyces pombe. We identified the first gene, egt1+ (SPBC1604.01, by sequence homology to previously published genes from Neurospora crassa and Mycobacterium smegmatis. We showed, using metabolomic analysis, that the Δegt1 deletion mutant completely lacked ergothioneine and its precursors (trimethyl histidine/hercynine and hercynylcysteine sulfoxide. Since the second step of ergothioneine biosynthesis has not been characterized in eukaryotes, we examined four putative homologs (Nfs1/SPBC21D10.11c, SPAC11D3.10, SPCC777.03c, and SPBC660.12c of the corresponding mycobacterial enzyme EgtE. Among deletion mutants of these genes, only one (ΔSPBC660.12c, designated Δegt2 showed a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibited no phenotypic defects during vegetative growth or quiescence. To effectively study the role of ergothioneine, we constructed an egt1+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. We employed three versions of the nmt1 promoter with increasing strength of expression and confirmed corresponding accumulations of ergothioneine. We quantified the intracellular concentration of ergothioneine in S. pombe (0.3, 157.4, 41.6, and up to 1606.3 µM in vegetative, nitrogen-starved, glucose-starved, and egt1+-overexpressing cells, respectively and described its gradual accumulation under long

Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

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Sarah J Fletcher

Full Text Available Tight junctions (TJs link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes. By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways.

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Audray Dugrand-Judek

Full Text Available Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the "grapefruit juice effect". Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus

Inhibition of DNA glycosylases via small molecule purine analogs.

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Aaron C Jacobs

Full Text Available Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.

Can Crystal Symmetry and Packing Influence the Active Site Conformation of Homohexameric Purine Nucleoside Phosphorylases?

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Marija Luić

2016-06-01

Full Text Available It is generaly believed that enzymes retain most of their functionality in the crystal form due to the large solvent content of protein crystals. This is facilitated by the fact that their natural environment in solution is not too far from the one found in the crystal form. Nevertheless, if the nature of the enzyme is such to require conformational changes, overcoming of the crystal packing constraints may prove to be too difficult. Such conformational change is present in one class of enzymes (purine nucleoside phosphorylases, that is the subject of our scientific interest for many years. The influence of crystal symmetry and crystal packing on the conformation of the active sites in the case of homohexameric purine nucleoside phosphorylases is presented and analysed. This work is licensed under a Creative Commons Attribution 4.0 International License.

Flow of nucleic acids from the rumen and recovery of purine derivatives in the urine of cattle and buffaloes

International Nuclear Information System (INIS)

Soejono, M.; Yusiati, L.M.; Bachrudin, Z.; Budhi, S.P.S.; Widyobroto, B.P.; Utomo, R.

2004-01-01

An experiment was conducted to determine the flow of nucleic acids from the rumen to duodenum. Two duodenal-cannulated each of male Ongole cattle and buffaloes aged three to four years were used and fed a mixture of king grass and rice bran (70:30 DM basis). At 95% and 60% of the voluntary intake in three weeks each before morning feeding period. Cr-mordanted alfalfa was used as a marker. The excretion of allantoin, uric acid, purine derivatives, and creatinine and the PDC index were higher in cattle than buffaloes at both levels of intake (P < 0.05). There were no difference between cattle and buffaloes with regard to the flow of RNA when expressed on digestible organic matter intake (DOMI), flow rate of RNA/kg DOMI, or flow rate of crude protein/kg DOMI. It can be concluded that the differences in urine excretion of purine derivatives between cattle and buffalo is not due to the differences in the amount of rumen microbial protein synthesis, but due to differences in purine metabolism between cattle and buffaloes. (author)

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

Science.gov (United States)

Horsman, Geoff P.; Chen, Yihua; Thorson, Jon S.; Shen, Ben

2010-01-01

Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence. PMID:20534556

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

Science.gov (United States)

Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

2016-06-12

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Crosstalk of Autophagy and the Secretory Pathway and Its Role in Diseases.

Science.gov (United States)

Zahoor, Muhammad; Farhan, Hesso

2018-01-01

The secretory and autophagic pathways are two fundamental, evolutionary highly conserved endomembrane processes. Typically, secretion is associated with biosynthesis and delivery of proteins. In contrast, autophagy is usually considered as a degradative pathway. Thus, an analogy to metabolic pathways is evident. Anabolic (biosynthetic) and catabolic (degradative) pathways are usually intimately linked and intertwined, and likewise, the secretory and autophagy pathways are intertwined. Investigation of this link is an emerging area of research, and we will provide an overview of some of the major advances that have been made to contribute to understanding of how secretion regulates autophagy and vice versa. Finally, we will highlight evidence that supports a potential involvement of the autophagy-secretion crosstalk in human diseases. © 2018 Elsevier Inc. All rights reserved.

Emergent biosynthetic capacity in simple microbial communities.

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Hsuan-Chao Chiu

2014-07-01

Full Text Available Microbes have an astonishing capacity to transform their environments. Yet, the metabolic capacity of a single species is limited and the vast majority of microorganisms form complex communities and join forces to exhibit capabilities far exceeding those achieved by any single species. Such enhanced metabolic capacities represent a promising route to many medical, environmental, and industrial applications and call for the development of a predictive, systems-level understanding of synergistic microbial capacity. Here we present a comprehensive computational framework, integrating high-quality metabolic models of multiple species, temporal dynamics, and flux variability analysis, to study the metabolic capacity and dynamics of simple two-species microbial ecosystems. We specifically focus on detecting emergent biosynthetic capacity--instances in which a community growing on some medium produces and secretes metabolites that are not secreted by any member species when growing in isolation on that same medium. Using this framework to model a large collection of two-species communities on multiple media, we demonstrate that emergent biosynthetic capacity is highly prevalent. We identify commonly observed emergent metabolites and metabolic reprogramming patterns, characterizing typical mechanisms of emergent capacity. We further find that emergent secretion tends to occur in two waves, the first as soon as the two organisms are introduced, and the second when the medium is depleted and nutrients become limited. Finally, aiming to identify global community determinants of emergent capacity, we find a marked association between the level of emergent biosynthetic capacity and the functional/phylogenetic distance between community members. Specifically, we demonstrate a "Goldilocks" principle, where high levels of emergent capacity are observed when the species comprising the community are functionally neither too close, nor too distant. Taken together

SYNTHESIS OF NEW TWO DERIVATIVES OF 6-MERCAPTOPURINE (6MP) 6-[5-PYRIDINE-4-YL- 1, 2, 3, 4-OXADIAZOLE-2-YL)DITHIOL]-9H-PURINE (38) AND 9H-PURINE-6-YL-BENYLDITHIOCARBAMATE (45) WITH CYTOTOXICITY RESULTS FROM THE NATIONAL CANCER INSTITUTE'S ANTICANCER DRUG SCREEN

OpenAIRE

Mohammed Hassan Mohammed et al

2012-01-01

6-[(5-pyridine-yl-1, 2, 3, 4-oxadiazole-2-yl)dithiol]-9H-purine (38) and 9H-purine-6yl-benzyldithiocarbamate (45) are synthesized as possible prodrugs for 6-mercaptopurine(6-MP). The generation of the compounds 38, 42, 45 and 48 were accomplished following multistep reaction procedures. The reaction and purity of the products were checked by TLC, the structure of the final compounds and their intermediates were confirmed by their melting points, infra red spectroscopy and whether differences ...

Novel modified purine bases and nucleosides: new methodologies of synthesis and biological activity

Czech Academy of Sciences Publication Activity Database

Hocek, Michal

-, č. 49 (2005), s. 29-30 ISSN 0261-3166. [International Symposium on Nucleic Acids Chemistry /4./. Fukuoka, 20.09.2005-22.09.2005] R&D Projects: GA ČR(CZ) GA203/03/0035; GA MŠk(CZ) 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : purines * nucleosides * synthesis Subject RIV: CC - Organic Chemistry

Targeting the cell cycle and the PI3K pathway: a possible universal strategy to reactivate innate tumor suppressor programmes in cancer cells.

Science.gov (United States)

David-Pfeuty, Thérèse; Legraverend, Michel; Ludwig, Odile; Grierson, David S

2010-04-01

Corruption of the Rb and p53 pathways occurs in virtually all human cancers. This could be because it lends oncogene-bearing cells a surfeit of Cdk activity and growth, enabling them to elaborate strategies to evade tumor-suppressive mechanisms and divide inappropriately. Targeting both Cdk activities and the PI3K pathway might be therefore a potentially universal means to palliate their deficiency in cancer cells. We showed that the killing efficacy of roscovitine and 16 other purines and potentiation of roscovitine-induced apoptosis by the PI3K inhibitor, LY294002, decreased with increasing corruption of the Rb and p53 pathways. Further, we showed that purines differing by a single substitution, which exerted little lethal effect on distant cell types in rich medium, could display widely-differing cytotoxicity profiles toward the same cell types in poor medium. Thus, closely-related compounds targeting similar Cdks may interact with different targets that could compete for their interaction with therapeutically-relevant Cdk targets. In the perspective of clinical development in association with the PI3K pathway inhibitors, it might thus be advisable to select tumor cell type-specific Cdk inhibitors on the basis of their toxicity in cell-culture-based assays performed at a limiting serum concentration sufficient to suppress their interaction with undesirable crossreacting targets whose range and concentration would depend on the cell genotype.

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms

Czech Academy of Sciences Publication Activity Database

Coesel, S.; Oborník, Miroslav; Varela, J.; Falciatore, A.; Bowler, C.

2008-01-01

Roč. 3, č. 8 (2008), s. 1-16 E-ISSN 1932-6203 R&D Projects: GA AV ČR IAA500220502 Institutional research plan: CEZ:AV0Z60220518 Keywords : marine diatoms * carotenoid pathway * evolution Subject RIV: EB - Genetics ; Molecular Biology

In silico tools for the analysis of antibiotic biosynthetic pathways

DEFF Research Database (Denmark)

Weber, Tilmann

2014-01-01

Natural products of bacteria and fungi are the most important source for antimicrobial drug leads. For decades, such compounds were exclusively found by chemical/bioactivity-guided screening approaches. The rapid progress in sequencing technologies only recently allowed the development of novel...... screening methods based on the genome sequences of potential producing organisms. The basic principle of such genome mining approaches is to identify genes, which are involved in the biosynthesis of such molecules, and to predict the products of the identified pathways. Thus, bioinformatics methods...... and tools are crucial for genome mining. In this review, a comprehensive overview is given on programs and databases for the identification and analysis of antibiotic biosynthesis gene clusters in genomic data....

Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures.

Science.gov (United States)

Rodas-Junco, Beatriz A; Cab-Guillén, Yahaira; Muñoz-Sánchez, J Armando; Vázquez-Flota, Felipe; Monforte-González, Miriam; Hernández-Sotomayor, S M Teresa

2013-10-01

Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

2,6,9-Trisubstituted purines as CRK3 kinase inhibitors with antileishmanial activity in vitro

Czech Academy of Sciences Publication Activity Database

Řezníčková, Eva; Popa, Alexandr; Gucký, Tomáš; Zatloukal, Marek; Havlíček, Libor; Bazgier, Václav; Berka, K.; Jorda, Radek; Popa, Igor; Nasereddin, A.; Jaffe, Ch. L.; Kryštof, Vladimír; Strnad, Miroslav

2015-01-01

Roč. 25, č. 11 (2015), s. 2298-2301 ISSN 0960-894X R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Purine * Cyclin-dependent kinase * Leishmania Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.486, year: 2015

On the biosynthetic origin of carminic acid

DEFF Research Database (Denmark)

Rasmussen, Silas A.; Kongstad, Kenneth T; Khorsand-Jamal, Paiman

2018-01-01

provides solid evidence of a polyketide, rather than a shikimate, origin of coccid pigments. Based on the newly identified compounds, we present a detailed biosynthetic scheme that accounts for the formation of carminic acid (CA) in D. coccus and all described coccid pigments which share a flavokermesic...... distribution suggests a common evolutionary origin for the trait in all coccid dye producing insect species....

Chapter 3: Omics Advances of Biosynthetic Pathways of Isoprenoid Production in Microalgae

Energy Technology Data Exchange (ETDEWEB)

Paniagua-Michel, J.; Subramanian, Venkataramanan

2017-01-01

In this chapter, the current status of microalgal isoprenoids and the role of omics technologies, or otherwise specified, in bioproducts optimization and applications are reviewed. Emphasis is focused in the metabolic pathways of microalgae involved in the production of commercially important products, namely, hydrocarbons and biofuels, nutraceuticals, and pharmaceuticals.

The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C.

Science.gov (United States)

Wilkinson, G F; Purkiss, J R; Boarder, M R

1993-03-01

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3. The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADP beta S while adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) gave the largest maximal response. 4. The concentration-effect curve to UTP was additive in the presence of either 2MeSATP or ADP beta S. However, the concentration-effect curves to ATP gamma S reached the same maximum in the presence or absence of UTP. 5. Suramin, at concentrations between 10 microM and 100 microM was a competitive antagonist for the response to ADP beta S and 2MeSATP but not the response to UTP. 6. The results show that there are two separate, co-existing, receptor populations: P2y-purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C-linked receptors.

Formal modeling and analysis of the hexosamine biosynthetic pathway: role of O-linked N-acetylglucosamine transferase in oncogenesis and cancer progression

Directory of Open Access Journals (Sweden)

Muhammad Tariq Saeed

2016-09-01

Full Text Available The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT, an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer.

Purine nucleoside phosphorylase deficiency in two unrelated Saudi patients

International Nuclear Information System (INIS)

Alangari, Abdullah; AlHarbi, Abdullah; AlGhonaium Abdulaziz; Santisteban, Ines; Hershfield, Michael

2009-01-01

Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder that results in combined immunodeficiency, neurologic dysfunction and autoimmunity. PNP deficiency has never been reported from Saudi Arabia or in patients with an Arabic ethnic background. We report on two Saudi girls with PNP deficiency. Both showed severe lymphopenia and neurological involvement. Sequencing of the PNP gene of one girl revealed a novel missense mutation Pro146>Leu in exon 4 due to a change in the codon from CCT>CTT. Expression of PNP (146L) cDNA in E coli indicated that the mutation greatly reduced, but did not completely eliminate PNP activity. (author)

Interactions of trimeric purine nucleoside phosphorylases with ground state analogues - calorimetric and fluorimetric studies

Czech Academy of Sciences Publication Activity Database

Wielgus-Kutrowska, B.; Frank, J.; Holý, Antonín; Koellner, G.; Bzowska, A.

2003-01-01

Roč. 22, 5/8 (2003), s. 1695-1698 ISSN 1525-7770 Grant - others:PCSR(PL) 6 P04A04416; PCSR(PL) 3 P04A03524 Institutional research plan: CEZ:AV0Z4055905 Keywords : purine nucleoside phosphorylase * fluorescence Subject RIV: CC - Organic Chemistry Impact factor: 0.813, year: 2003

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

Science.gov (United States)

Schultz, Eric P.; Vasquez, Ernesto E.; Scott, William G.

2014-01-01

The hammerhead ribozyme catalyzes RNA cleavage via acid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK a of the substituted purine; in both cases inosine, which is similar to G in pK a and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

Science.gov (United States)

Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

2014-09-01

The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

Science.gov (United States)

Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

2016-08-03

Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purine and its analogues and radiation damage in Bacillus megaterium spores

Energy Technology Data Exchange (ETDEWEB)

Powers, E.L.

1986-12-01

As an extension of results obtained from radiation studies on caffeine both in other laboratories and more recently in this laboratory using the bacterial spore as the test system, six compounds with chemical structures closely resembling that of caffeine were tested as radiation modifiers. Of these compounds, purine, adenine and hypoxanthine resembled caffeine in sensitizing spores to radiation, while theobromine, xanthine and theophylline did not. These responses are discussed in relation to the electron sequestration hypothesis of cellular sensitization to high-energy radiation.

Fabrication of biosynthetic vascular prostheses by 193-nm excimer laser radiation

Science.gov (United States)

Husinsky, Wolfgang; Csek, Ch.; Bartel, A.; Grabenwoeger, M.; Fitzal, F.; Wolner, Ernst

1998-05-01

This study was undertaken to investigate the feasibility of transmural capillary ingrowth into the inner surface of biosynthetic vascular prostheses (OmniflowTM) through perforations created by an excimer-laser, thus inducing an endothelial cell coverage. The biosynthetic vascular prostheses (10 cm length, 6 mm (phi) ) were perforated with an excimer laser ((phi) of the holes 50 - 100 micrometer, distance 4 mm) and implanted into the carotid arteries of 8 sheep. The laser tissue interaction process of 193 nm radiation ensures minimal thermal damage to the prostheses. They were compared to untreated OmniflowTM prostheses implanted at the contralateral side. Three months after implantation the prostheses were explanted and evaluated by gross morphology, histological examination and scanning electron microscopy. Scanning electron microscopy showed endothelial cells in the midgraft portion of all perforated prostheses, whereas collagen fibers, fibrin meshwork and activated platelets formed the inner layer in 6 out of 8 untreated OmniflowTM prostheses. It can be concluded, that spontaneous endothelialization of biosynthetic vascular prostheses can be achieved by transmural capillary ingrowth through perforations in the wall of the prostheses in an experimental sheep model.

What is the evidence for the use of biologic or biosynthetic meshes in abdominal wall reconstruction?

Science.gov (United States)

Köckerling, F; Alam, N N; Antoniou, S A; Daniels, I R; Famiglietti, F; Fortelny, R H; Heiss, M M; Kallinowski, F; Kyle-Leinhase, I; Mayer, F; Miserez, M; Montgomery, A; Morales-Conde, S; Muysoms, F; Narang, S K; Petter-Puchner, A; Reinpold, W; Scheuerlein, H; Smietanski, M; Stechemesser, B; Strey, C; Woeste, G; Smart, N J

2018-04-01

Although many surgeons have adopted the use of biologic and biosynthetic meshes in complex abdominal wall hernia repair, others have questioned the use of these products. Criticism is addressed in several review articles on the poor standard of studies reporting on the use of biologic meshes for different abdominal wall repairs. The aim of this consensus review is to conduct an evidence-based analysis of the efficacy of biologic and biosynthetic meshes in predefined clinical situations. A European working group, "BioMesh Study Group", composed of invited surgeons with a special interest in surgical meshes, formulated key questions, and forwarded them for processing in subgroups. In January 2016, a workshop was held in Berlin where the findings were presented, discussed, and voted on for consensus. Findings were set out in writing by the subgroups followed by consensus being reached. For the review, 114 studies and background analyses were used. The cumulative data regarding biologic mesh under contaminated conditions do not support the claim that it is better than synthetic mesh. Biologic mesh use should be avoided when bridging is needed. In inguinal hernia repair biologic and biosynthetic meshes do not have a clear advantage over the synthetic meshes. For prevention of incisional or parastomal hernias, there is no evidence to support the use of biologic/biosynthetic meshes. In complex abdominal wall hernia repairs (incarcerated hernia, parastomal hernia, infected mesh, open abdomen, enterocutaneous fistula, and component separation technique), biologic and biosynthetic meshes do not provide a superior alternative to synthetic meshes. The routine use of biologic and biosynthetic meshes cannot be recommended.

Integrating nitric oxide into salicylic acid and jasmonic acid/ ethylene plant defense pathways.

Science.gov (United States)

Mur, Luis A J; Prats, Elena; Pierre, Sandra; Hall, Michael A; Hebelstrup, Kim H

2013-01-01

Plant defense against pests and pathogens is known to be conferred by either salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) pathways, depending on infection or herbivore-grazing strategy. It is well attested that SA and JA/ET pathways are mutually antagonistic allowing defense responses to be tailored to particular biotic stresses. Nitric oxide (NO) has emerged as a major signal influencing resistance mediated by both signaling pathways but no attempt has been made to integrate NO into established SA/JA/ET interactions. NO has been shown to act as an inducer or suppressor of signaling along each pathway. NO will initiate SA biosynthesis and nitrosylate key cysteines on TGA-class transcription factors to aid in the initiation of SA-dependent gene expression. Against this, S-nitrosylation of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) will promote the NPR1 oligomerization within the cytoplasm to reduce TGA activation. In JA biosynthesis, NO will initiate the expression of JA biosynthetic enzymes, presumably to over-come any antagonistic effects of SA on JA-mediated transcription. NO will also initiate the expression of ET biosynthetic genes but a suppressive role is also observed in the S-nitrosylation and inhibition of S-adenosylmethionine transferases which provides methyl groups for ET production. Based on these data a model for NO action is proposed but we have also highlighted the need to understand when and how inductive and suppressive steps are used.

Poly(alizarin red)/Graphene modified glassy carbon electrode for simultaneous determination of purine and pyrimidine

International Nuclear Information System (INIS)

Ba Xi; Luo Liqiang; Ding Yaping; Zhang Zhen; Chu Yuliang; Wang Bijun; Ouyang Xiaoqian

2012-01-01

Graphical abstract: DPVs of PAR/Graphene/GCE (a) and the bare GCE (c) in 0.1 M PBS containing 50.0 μM G, 50.0 μM A, 100.0 μM T and 100.0 μM C, (b) PAR/Graphene/GCE in 0.1 M PBS. Highlights: ► The sensor exhibited well-separated peaks and low detection limit. ► The sensor possesses high sensitivity and wide linear range. ► The sensor was used for simultaneous detection of G, A, T and C successfully. ► The sensor was applied in a fish sperm DNA sample with satisfactory results. ► The proposed sensor has good stability and reproducibility. - Abstract: In this work, a poly(alizarin red)/Graphene composite film modified glassy carbon electrode (PAR/Graphene/GCE) was prepared for simultaneous determination of four DNA bases (guanine, adenine, thymine and cytosine) without any pretreatment. The morphology and interface property of PAR/Graphene films were examined by scanning electron microscopy and electrochemical impedance spectroscopy. The PAR/Graphene/GCE exhibited excellent electrocatalytic activity toward purine (guanine and adenine) and pyrimidine (thymine and cytosine) in 0.1 M phosphate buffer solution (pH 7.4). Under optimum conditions, differential pulse voltammetry was used to detect the oxidation of purine and pyrimidine. The results showed that PAR/Graphene/GCE exhibited well-separated peaks, low detection limit, high sensitivity and wide linear range for simultaneous detection of purine and pyrimidine. The proposed sensor also has good stability and reproducibility. Furthermore, the modified electrode was applied for the detection of DNA bases in a fish sperm DNA sample with satisfactory results.

Purine and its analogues and radiation damage in Bacillus megaterium spores

International Nuclear Information System (INIS)

Powers, E.L.

1986-01-01

As an extension of results obtained from radiation studies on caffeine both in other laboratories and more recently in this laboratory using the bacterial spore as the test system, six compounds with chemical structures closely resembling that of caffeine were tested as radiation modifiers. Of these compounds, purine, adenine and hypoxanthine resembled caffeine in sensitizing spores to radiation, while theobromine, xanthine and theophylline did not. These responses are discussed in relation to the electron sequestration hypothesis of cellular sensitization to high-energy radiation. (author)

Rhodosporidium toruloides BANNO: Dose-response relationship, mutagenic efficiency and spectrum of mutants of auxotrophy-producing mutations induced by ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Boettcher, F.; Samsonova, I.A.

1978-01-01

The kinetics, efficiency, and specificity of induction of forward mutations to auxotrophy by ultraviolet light (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in stationary phase cells of Rhodosporidium (Rhodotorula) wild strain Rg1. In comparison to the spontaneous level the frequency of auxotrophic mutants was increased more than 1000 times by both mutagens, however, the mutagenic efficiency of MNNG was higher than that of UV. We found that the forward mutation rate is a linear function of the applicated UV and MNNG doses in the range to 600 J m -2 or 25 mM x min, respectively. The 35 studied biosynthetic pathways to amino acids, purines, pyrimidines, and vitamins are genetically blocked at different frequencies, but there is not any significant difference between UV and MNNG induced frequencies of mutants with a specific requirement. However, in difference to the approximately equal distribution of the MNNG-induced nic mutants among the genetic blocks of the tryptophan-nicotinamide pathway, UV-induced nic mutants occurred with a higher frequency in the genes of the tryptophan pyrrolase and the 3-hydroxykynureninase than in the genes of the other enzymes of the pathway. (author)

Theoretical and experimental studies on the corrosion inhibition potentials of some purines for aluminum in 0.1 M HCl.

Science.gov (United States)

Eddy, Nnabuk O; Momoh-Yahaya, H; Oguzie, Emeka E

2015-03-01

Experimental aspect of the corrosion inhibition potential of adenine (AD), guanine (GU) and, hypoxanthine (HYP) was carried out using weight loss, potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) methods while the theoretical aspect of the work was carried out by calculations of semi-empirical parameters (for AM1, MNDO, CNDO, PM3 and RM1 Hamiltonians), Fukui functions and inhibitor-metal interaction energies. Results obtained from the experimental studies were in good agreement and indicated that adenine (AD), guanine (GU) and hypoxanthine (HYP) are good adsorption inhibitors for the corrosion of aluminum in solutions of HCl. Data obtained from electrochemical experiment revealed that the studied purines functioned by adsorption on the aluminum/HCl interface and inhibited the cathodic half reaction to a greater extent and anodic half reaction to a lesser extent. The adsorption of the purines on the metal surface was found to be exothermic and spontaneous. Deviation of the adsorption characteristics of the studied purines from the Langmuir adsorption model was compensated by the fitness of Flory Huggins and El Awardy et al. adsorption models. Quantum chemical studies revealed that the experimental inhibition efficiencies of the studied purines are functions of some quantum chemical parameters including total energy of the molecules (TE), energy gap (E L-H), electronic energy of the molecule (EE), dipole moment and core-core repulsion energy (CCR). Fukui functions analysis through DFT and MP2 theories indicated slight complications and unphysical results. However, results obtained from calculated Huckel charges, molecular orbital and interaction energies, the adsorption of the inhibitors proceeded through the imine nitrogen (N5) in GU, emanine nitrogen (N7) in AD and the pyridine nitrogen (N5) in HPY.

De Novo Biosynthesis of Glutarate via α-Keto Acid Carbon Chain Extension and Decarboxylation Pathway in Escherichia coli.

Science.gov (United States)

Wang, Jian; Wu, Yifei; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

2017-10-20

Microbial based bioplastics are promising alternatives to petroleum based synthetic plastics due to their renewability and economic feasibility. Glutarate is one of the most potential building blocks for bioplastics. The recent biosynthetic routes for glutarate were mostly based on the l-lysine degradation pathway from Pseudomonas putida that required lysine either by feeding or lysine overproduction via genetic manipulations. Herein, we established a novel glutarate biosynthetic pathway by incorporation of a "+1" carbon chain extension pathway from α-ketoglutarate (α-KG) in combination with α-keto acid decarboxylation pathway in Escherichia coli. Introduction of homocitrate synthase (HCS), homoaconitase (HA) and homoisocitrate dehydrogenase (HICDH) from Saccharomyces cerevisiae into E. coli enabled "+1" carbon extension from α-KG to α-ketoadipate (α-KA), which was subsequently converted into glutarate by a promiscuous α-keto acid decarboxylase (KivD) and a succinate semialdehyde dehydrogenase (GabD). The recombinant E. coli coexpressing all five genes produced 0.3 g/L glutarate from glucose. To further improve the titers, α-KG was rechanneled into carbon chain extension pathway via the clustered regularly interspersed palindromic repeats system mediated interference (CRISPRi) of essential genes sucA and sucB in tricarboxylic acid (TCA) cycle. The final strain could produce 0.42 g/L glutarate, which was increased by 40% compared with the parental strain.

Insulin Biosynthetic Interaction Network Component, TMEM24, Facilitates Insulin Reserve Pool Release

Directory of Open Access Journals (Sweden)

Anita Pottekat

2013-09-01

Full Text Available Insulin homeostasis in pancreatic β cells is now recognized as a critical element in the progression of obesity and type II diabetes (T2D. Proteins that interact with insulin to direct its sequential synthesis, folding, trafficking, and packaging into reserve granules in order to manage release in response to elevated glucose remain largely unknown. Using a conformation-based approach combined with mass spectrometry, we have generated the insulin biosynthetic interaction network (insulin BIN, a proteomic roadmap in the β cell that describes the sequential interacting partners of insulin along the secretory axis. The insulin BIN revealed an abundant C2 domain-containing transmembrane protein 24 (TMEM24 that manages glucose-stimulated insulin secretion from a reserve pool of granules, a critical event impaired in patients with T2D. The identification of TMEM24 in the context of a comprehensive set of sequential insulin-binding partners provides a molecular description of the insulin secretory pathway in β cells.

Bacterial natural product biosynthetic domain composition in soil correlates with changes in latitude on a continent-wide scale.

Science.gov (United States)

Lemetre, Christophe; Maniko, Jeffrey; Charlop-Powers, Zachary; Sparrow, Ben; Lowe, Andrew J; Brady, Sean F

2017-10-31

Although bacterial bioactive metabolites have been one of the most prolific sources of lead structures for the development of small-molecule therapeutics, very little is known about the environmental factors associated with changes in secondary metabolism across natural environments. Large-scale sequencing of environmental microbiomes has the potential to shed light on the richness of bacterial biosynthetic diversity hidden in the environment, how it varies from one environment to the next, and what environmental factors correlate with changes in biosynthetic diversity. In this study, the sequencing of PCR amplicons generated using primers targeting either ketosynthase domains from polyketide biosynthesis or adenylation domains from nonribosomal peptide biosynthesis was used to assess biosynthetic domain composition and richness in soils collected across the Australian continent. Using environmental variables collected at each soil site, we looked for environmental factors that correlated with either high overall domain richness or changes in the domain composition. Among the environmental variables we measured, changes in biosynthetic domain composition correlate most closely with changes in latitude and to a lesser extent changes in pH. Although it is unclear at this time the exact mix of factors that may drive the relationship between biosynthetic domain composition and latitude, from a practical perspective the identification of a latitudinal basis for differences in soil metagenome biosynthetic domain compositions should help guide future natural product discovery efforts. Published under the PNAS license.

Overlapping riboflavin supply pathways in bacteria.

Science.gov (United States)

García-Angulo, Víctor Antonio

2017-03-01

Riboflavin derivatives are essential cofactors for a myriad of flavoproteins. In bacteria, flavins importance extends beyond their role as intracellular protein cofactors, as secreted flavins are a key metabolite in a variety of physiological processes. Bacteria obtain riboflavin through the endogenous riboflavin biosynthetic pathway (RBP) or by the use of importer proteins. Bacteria frequently encode multiple paralogs of the RBP enzymes and as for other micronutrient supply pathways, biosynthesis and uptake functions largely coexist. It is proposed that bacteria shut down biosynthesis and would rather uptake riboflavin when the vitamin is environmentally available. Recently, the overlap of riboflavin provisioning elements has gained attention and the functions of duplicated paralogs of RBP enzymes started to be addressed. Results point towards the existence of a modular structure in the bacterial riboflavin supply pathways. Such structure uses subsets of RBP genes to supply riboflavin for specific functions. Given the importance of riboflavin in intra and extracellular bacterial physiology, this complex array of riboflavin provision pathways may have developed to contend with the various riboflavin requirements. In riboflavin-prototrophic bacteria, riboflavin transporters could represent a module for riboflavin provision for particular, yet unidentified processes, rather than substituting for the RBP as usually assumed.

The effect of plane of nutrition on the urinary purine derivative excretion in sheep and goats

International Nuclear Information System (INIS)

Poshiwa, X.; Tigere, A.; Ngongoni, N.T.; Manyuchi, B.; Chakoma, C.

2004-01-01

An experiment was conducted to determine the effect of plane of nutrition on purine derivative excretion and to develop a set of model equations that relate intake to the purine derivative (PD) excretion in sheep and goats. Four male Sabi sheep and four male Small-East African goats (four months old) were used. The trial was a 4 x 4 Latin square cross-over design to examine the response of PD excretion to feed intake. The four diets consisted of star grass (Cynodon nlemfuensis) hay fed ad libitum, and at 85, 70 or 55% of ad libitum. Total PD excretion increased with the increase in feed intake for both sheep and goats. However, the increase did not reach statistical significance (P > 0.05). The model equations relating digestible organic matter intake (X) to PD excretion (Y) were Y = 2.97 X + 0.15 (R 2 =0.72) and Y = 5.86 X - 0.33 (R 2 =0.99) for sheep and goats respectively. (author)

Significant differences in gene expression and key genetic components associated with high growth vigor in populus section tacamahaca as revealed by comparative transcriptome analysis

International Nuclear Information System (INIS)

Cheng, S.; Chen, M.; Li, Y.; Wang, J.; Sun, X.; Wang, J.

2017-01-01

To identify genetic components involved in high growth vigor in F1 Populus section Tacamahaca hybrid plants, high and low vigor plants showing significant differences in apical dominance during a rapid growth period were selected. Apical bud transcriptomes of high and low-growth-vigor hybrids and their parents were analyzed using high-throughput RNA sequencing on an Illumina HiSeq 2000 platform. A total of 5,542 genes were differently expressed between high growth vigor hybrid and its parents, the genes were significantly enriched in pathways related to processes such as photosynthesis, pyrimidine ribonucleotide biosynthetic processes and nucleoside metabolic processes. There were 1410 differentially expressed genes between high and low growth vigor hybrid, the genes were mainly involved in photosynthesis, chlorophyll biosynthetic process, carbon fixation in photosynthetic organisms, porphyrin and chlorophyll metabolism and nitrogen metabolism. Moreover, a k-core of a gene co-expression network analysis was performed to identify the potential functions of genes related to high growth vigor. The functions of 8 selected candidate genes were associated mainly with circadian rhythm, water transport, cellulose catabolic processes, sucrose biosynthesis, pyrimidine ribonucleotide biosynthesis, purine nucleotide biosynthesis, meristem maintenance, and carbohydrate metabolism. Our results may contribute to a better understanding of the molecular basis of high growth vigor in hybrids and its regulation. (author)

Poly(alizarin red)/Graphene modified glassy carbon electrode for simultaneous determination of purine and pyrimidine

Energy Technology Data Exchange (ETDEWEB)

Ba Xi; Luo Liqiang [Department of Chemistry, Shanghai University, Shanghai 200444 (China); Ding Yaping, E-mail: wdingyp@sina.com [Department of Chemistry, Shanghai University, Shanghai 200444 (China); Zhang Zhen [Department of Chemistry, Shanghai University, Shanghai 200444 (China); Chu Yuliang [Instrumental Analysis and Research Center, Shanghai University, Shanghai 200444 (China); Wang Bijun; Ouyang Xiaoqian [Department of Chemistry, Shanghai University, Shanghai 200444 (China)

2012-11-08

Graphical abstract: DPVs of PAR/Graphene/GCE (a) and the bare GCE (c) in 0.1 M PBS containing 50.0 {mu}M G, 50.0 {mu}M A, 100.0 {mu}M T and 100.0 {mu}M C, (b) PAR/Graphene/GCE in 0.1 M PBS. Highlights: Black-Right-Pointing-Pointer The sensor exhibited well-separated peaks and low detection limit. Black-Right-Pointing-Pointer The sensor possesses high sensitivity and wide linear range. Black-Right-Pointing-Pointer The sensor was used for simultaneous detection of G, A, T and C successfully. Black-Right-Pointing-Pointer The sensor was applied in a fish sperm DNA sample with satisfactory results. Black-Right-Pointing-Pointer The proposed sensor has good stability and reproducibility. - Abstract: In this work, a poly(alizarin red)/Graphene composite film modified glassy carbon electrode (PAR/Graphene/GCE) was prepared for simultaneous determination of four DNA bases (guanine, adenine, thymine and cytosine) without any pretreatment. The morphology and interface property of PAR/Graphene films were examined by scanning electron microscopy and electrochemical impedance spectroscopy. The PAR/Graphene/GCE exhibited excellent electrocatalytic activity toward purine (guanine and adenine) and pyrimidine (thymine and cytosine) in 0.1 M phosphate buffer solution (pH 7.4). Under optimum conditions, differential pulse voltammetry was used to detect the oxidation of purine and pyrimidine. The results showed that PAR/Graphene/GCE exhibited well-separated peaks, low detection limit, high sensitivity and wide linear range for simultaneous detection of purine and pyrimidine. The proposed sensor also has good stability and reproducibility. Furthermore, the modified electrode was applied for the detection of DNA bases in a fish sperm DNA sample with satisfactory results.

Enzyme organization in the proline biosynthetic pathway of Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Gamper, H; Moses, V

1974-01-01

The conversion of glutamic acid to proline by an Escherichia coli extract was studied. The activity was dependent upon the presence of ATP and NADPH and was largely unaffected by the presence of NH/sub 3/ or imidazole. The first two pathway enzymes appear to exist as a complex which stabilizes a labile intermediate postulated as ..gamma..-glutamyl phosphate. Attempted synthesis of this compound was unsuccessful due to its spontaneous cyclization to 2-pyrrolidone 5-carboxylate. Dissociation of the enzyme complex upon dilution of the extract is presumed responsible for an experimentally observed dilution effect. E. coli pro/sub A//sup -/ and pro/sub B//sup -/ auxotroph extracts failed to complement one another in the biosynthesis of proline. This is attributed to the lack of a dynamic equilibrium between the complex and its constituent enzymes. In vivo studies with E. coli showed no evidence for metabolic channeling in the final reaction of proline synthesis, the reduction of ..delta../sup 1/-pyrroline 5-carboxylate.

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

Science.gov (United States)

Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

2014-04-09

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Urinary purine derivatives as a tool to estimate dry matter intake in cattle: a meta-analysis

Science.gov (United States)

The objectives of this study were: 1) to investigate the relationship between dry matter intake (DMI) and urinary purine derivatives (PD) excretion in order to develop equations to predict DMI, and 2) to determine the endogenous excretion of PD for beef and dairy cattle using a meta-analytic approac...

Evidence for a Saponin Biosynthesis Pathway in the Body Wall of the Commercially Significant Sea Cucumber Holothuria scabra.

Science.gov (United States)

Mitu, Shahida Akter; Bose, Utpal; Suwansa-Ard, Saowaros; Turner, Luke H; Zhao, Min; Elizur, Abigail; Ogbourne, Steven M; Shaw, Paul Nicholas; Cummins, Scott F

2017-11-07

The sea cucumber (phylum Echinodermata) body wall is the first line of defense and is well known for its production of secondary metabolites; including vitamins and triterpenoid glycoside saponins that have important ecological functions and potential benefits to human health. The genes involved in the various biosynthetic pathways are unknown. To gain insight into these pathways in an echinoderm, we performed a comparative transcriptome analysis and functional annotation of the body wall and the radial nerve of the sea cucumber Holothuria scabra ; to define genes associated with body wall metabolic functioning and secondary metabolite biosynthesis. We show that genes related to signal transduction mechanisms were more highly represented in the H. scabra body wall, including genes encoding enzymes involved in energy production. Eight of the core triterpenoid biosynthesis enzymes were found, however, the identity of the saponin specific biosynthetic pathway enzymes remains unknown. We confirm the body wall release of at least three different triterpenoid saponins using solid phase extraction followed by ultra-high-pressure liquid chromatography-quadrupole time of flight-mass spectrometry. The resource we have established will help to guide future research to explore secondary metabolite biosynthesis in the sea cucumber.

Evidence for a Saponin Biosynthesis Pathway in the Body Wall of the Commercially Significant Sea Cucumber Holothuria scabra

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Shahida Akter Mitu

2017-11-01

Full Text Available The sea cucumber (phylum Echinodermata body wall is the first line of defense and is well known for its production of secondary metabolites; including vitamins and triterpenoid glycoside saponins that have important ecological functions and potential benefits to human health. The genes involved in the various biosynthetic pathways are unknown. To gain insight into these pathways in an echinoderm, we performed a comparative transcriptome analysis and functional annotation of the body wall and the radial nerve of the sea cucumber Holothuria scabra; to define genes associated with body wall metabolic functioning and secondary metabolite biosynthesis. We show that genes related to signal transduction mechanisms were more highly represented in the H. scabra body wall, including genes encoding enzymes involved in energy production. Eight of the core triterpenoid biosynthesis enzymes were found, however, the identity of the saponin specific biosynthetic pathway enzymes remains unknown. We confirm the body wall release of at least three different triterpenoid saponins using solid phase extraction followed by ultra-high-pressure liquid chromatography-quadrupole time of flight-mass spectrometry. The resource we have established will help to guide future research to explore secondary metabolite biosynthesis in the sea cucumber.

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae flowers to deduce monoterpene biosynthesis pathway

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Wu Tian-Shung

2006-07-01

Full Text Available Abstract Background Floral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs, and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis. Results The main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST. Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway.

Science.gov (United States)

Hsiao, Yu-Yun; Tsai, Wen-Chieh; Kuoh, Chang-Sheng; Huang, Tian-Hsiang; Wang, Hei-Chia; Wu, Tian-Shung; Leu, Yann-Lii; Chen, Wen-Huei; Chen, Hong-Hwa

2006-07-13

Floral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis. The main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives. This systems biology program combined

Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction

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Schulz Burkhard

2010-12-01

Full Text Available Abstract Background Plant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs, are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive. Results Here, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2 and ENHANCER OF SHOOT REGENERATION2 (ESR2, were also lower in dwf7-1 as compared with wild type. Conclusions Taken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

Bioenergetic coupling between membrane transport systems and biosynthetic pathways essential for cell cycle progression

International Nuclear Information System (INIS)

Leister, K.J.; Cutry, A.F.; Wenner, C.E.

1986-01-01

Recently, it has been shown that there exists a point in the cell cycle (approximately 2 h prior to S phase entry) when (Na + /K + )ATPase pump activity is no longer needed for progression through the cycle. These data suggests that pump activity is critical in the biosynthetic processes which enables the cell to proceed through the G 1 phase. A scheme is proposed which is currently being tested that (Na + /K + )ATPase pump activity serves as the driving force in the regulation of other membrane transport processes critical for cell proliferation. For example, in post-confluent quiescent C3H-10T1/2 fibroblasts, when [K + ]/sub o/ is lowered just below the K/sub m/ of the pump for K + there is a 10-fold increase in 3 H-uridine uptake into both acid soluble and insoluble cell fractions. By modulation of the pump in this manner, glucose utilization is enhanced whereas inhibition of the pump by ouabain suppresses glucose utilization. In both methods of affecting the pump, 3 H-leucine incorporation is inhibited. Electron acceptors that influence the redox state of the cell have been shown to both stimulate or inhibit cell cycle progression. Under conditions where [K + ]/sub o/ is lowered, the nucleoside uptake responses observed were modified by electron acceptors depending on the ability to oxidize NAD(P)H directly or to interact with a cytochrome-like component, (e.g. phenazine methosulfate) reversed the enhanced uridine uptake and p-phenylene diamine further enhanced the uridine uptake response. These findings suggest that a plasma membrane redox system (presumably cyt-c like) is linked to nucleoside transport which is subject to (Na + /K + )ATPase activity

Estimating rumen microbial protein supply for indigenous ruminants using nuclear and purine excretion techniques in Indonesia

International Nuclear Information System (INIS)

Soejono, M.; Yusiati, L.M.; Budhi, S.P.S.; Widyobroto, B.P.; Bachrudin, Z.

1999-01-01

The microbial protein supply to ruminants can be estimated based on the amount of purine derivatives (PD) excreted in the urine. Four experiments were conducted to evaluate the PD excretion method for Bali and Ongole cattle. In the first experiment, six male, two year old Bali cattle (Bos sondaicus) and six Ongole cattle (Bos indicus) of similar sex and age, were used to quantify the endogenous contribution to total PD excretion in the urine. In the second experiment, four cattle from each breed were used to examine the response of PD excretion to feed intake. 14 C-uric acid was injected in one single dose to define the partitioning ratio of renal:non-renal losses of plasma PD. The third experiment was conducted to examine the ratio of purine N:total N in mixed rumen microbial population. The fourth experiment measured the enzyme activities of blood, liver and intestinal tissues concerned with PD metabolism. The results of the first experiment showed that endogenous PD excretion was 145 ± 42.0 and 132 ± 20.0 μmol/kg W 0.75 /d, for Bali and Ongole cattle, respectively. The second experiment indicated that the proportion of plasma PD excreted in the urine of Bali and Ongole cattle was 0.78 and 0.77 respectively. Hence, the prediction of purine absorbed based on PD excretion can be stated as Y = 0.78 X + 0.145 W 0.75 and Y = 0.77 X + 0.132 W 0.75 for Bali and Ongole cattle, respectively. The third experiment showed that there were no differences in the ratio of purine N:total N in mixed rumen microbes of Bali and Ongole cattle (17% vs 18%). The last experiment, showed that intestinal xanthine oxidase activity of Bali cattle was lower than that of Ongole cattle (0.001 vs 0.015 μmol uric acid produced/min/g tissue) but xanthine oxidase activity in the blood and liver of Bali cattle was higher than that of Ongole cattle (3.48 vs 1.34 μmol/min/L plasma and 0.191 vs 0.131 μmol/min/g liver tissue). Thus, there was no difference in PD excretion between these two breeds

Regio- and Enantioselective N-Allylations of Imidazole, Benzimidazole, and Purine Heterocycles Catalyzed by Single-Component Metallacyclic Iridium Complexes

Science.gov (United States)

Stanley, Levi M.

2010-01-01

Highly regio- and enantioselective iridium-catalyzed N-allylations of benzimidazoles, imidazoles, and purines have been developed. N-Allylated benzimidazoles and imidazoles were isolated in high yields (up to 97%) with high branched-to-linear selectivity (up to 99:1) and enantioselectivity (up to 98% ee) from the reactions of benzimidazole and imidazole nucleophiles with unsymmetrical allylic carbonates in the presence of single component, ethylene-bound, metallacyclic iridium catalysts. N-Allylated purines were also obtained in high yields (up to 91%) with high N9:N7 selectivity (up to 96:4), high branched-to-linear selectivity (98:2), and high enantioselectivity (up to 98% ee) under similar conditions. The reactions encompass a range of benzimidazole, imidazole, and purine nucleophiles, as well as a variety of unsymmetrical aryl, heteroaryl, and aliphatic allylic carbonates. Competition experiments between common amine nucleophiles and the heterocyclic nitrogen nucleophiles studied in this work illustrate the effect of nucleophile pKa on the rate of iridium-catalyzed N-allylation reactions. Kinetic studies on the allylation of benzimidazole catalyzed by metallacyclic iridium-phosphoramidite complexes, in combination with studies on the deactivation of these catalysts in the presence of heterocyclic nucleophiles, provide insight into the effects of the structure of the phosphoramidite ligands on the stability of the metallacyclic catalysts. The data obtained from these studies has led to the development of N-allylations of benzimidazoles and imidazoles in the absence of an exogenous base. PMID:19480431

Subcellular location of the enzymes of purine breakdown in the yeast Candida famata grown on uric acid

NARCIS (Netherlands)

Large, Peter J.; Waterham, Hans R.; Veenhuis, Marten

1990-01-01

The subcellular location of the enzymes of purine breakdown in the yeast Candida famata, which grows on uric acid as sole carbon and nitrogen source, has been examined by subcellular fractionation methods. Uricase was confirmed as being peroxisomal, but the other three enzymes, allantoinase,

78 kDa receptor for Man6P-independent lysosomal enzyme targeting: Biosynthetic transport from endoplasmic reticulum to 'high-density vesicles'

International Nuclear Information System (INIS)

Gonzalez-Noriega, Alfonso; Ortega Cuellar, Daniel D.; Michalak, Colette

2006-01-01

Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH 4 Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor

Differential effects of 17 alpha-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat

NARCIS (Netherlands)

Koopen, NR; Post, SM; Wolters, H; Havinga, R; Stellaard, F; Boverhof, R; Kuipers, F; Princen, HMG

Effects of 17 alpha-ethinylestradiol (EE) on the neutral and acidic biosynthetic pathways of bile salt (BS) synthesis were evaluated in rats with an intact enterohepatic circulation and in rats with long-term bile diversion to induce BS synthesis, For this purpose, bile salt pool composition,

The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C.

OpenAIRE

Wilkinson, G. F.; Purkiss, J. R.; Boarder, M. R.

1993-01-01

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3....

Purine 3':5'-cyclic nucleotides with the nucleobase in a syn orientation: cAMP, cGMP and cIMP.

Science.gov (United States)

Řlepokura, Katarzyna Anna

2016-06-01

Purine 3':5'-cyclic nucleotides are very well known for their role as the secondary messengers in hormone action and cellular signal transduction. Nonetheless, their solid-state conformational details still require investigation. Five crystals containing purine 3':5'-cyclic nucleotides have been obtained and structurally characterized, namely adenosine 3':5'-cyclic phosphate dihydrate, C10H12N5O6P·2H2O or cAMP·2H2O, (I), adenosine 3':5'-cyclic phosphate 0.3-hydrate, C10H12N5O6P·0.3H2O or cAMP·0.3H2O, (II), guanosine 3':5'-cyclic phosphate pentahydrate, C10H12N5O7P·5H2O or cGMP·5H2O, (III), sodium guanosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H11N5O7P(-)·4H2O or Na(cGMP)·4H2O, (IV), and sodium inosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H10N4O7P(-)·4H2O or Na(cIMP)·4H2O, (V). Most of the cyclic nucleotide zwitterions/anions [two from four cAMP present in total in (I) and (II), cGMP in (III), cGMP(-) in (IV) and cIMP(-) in (V)] are syn conformers about the N-glycosidic bond, and this nucleobase arrangement is accompanied by Crib-H...Npur hydrogen bonds (rib = ribose and pur = purine). The base orientation is tuned by the ribose pucker. An analysis of data obtained from the Cambridge Structural Database made in the context of syn-anti conformational preferences has revealed that among the syn conformers of various purine nucleotides, cyclic nucleotides and dinucleotides predominate significantly. The interactions stabilizing the syn conformation have been indicated. The inter-nucleotide contacts in (I)-(V) have been systematized in terms of the chemical groups involved. All five structures display three-dimensional hydrogen-bonded networks.

Genes encoding enzymes of the lignin biosynthesis pathway in Eucalyptus

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Ricardo Harakava

2005-01-01

Full Text Available Eucalyptus ESTs libraries were screened for genes involved in lignin biosynthesis. This search was performed under the perspective of recent revisions on the monolignols biosynthetic pathway. Eucalyptus orthologues of all genes of the phenylpropanoid pathway leading to lignin biosynthesis reported in other plant species were identified. A library made with mRNAs extracted from wood was enriched for genes involved in lignin biosynthesis and allowed to infer the isoforms of each gene family that play a major role in wood lignin formation. Analysis of the wood library suggests that, besides the enzymes of the phenylpropanoids pathway, chitinases, laccases, and dirigent proteins are also important for lignification. Colocalization of several enzymes on the endoplasmic reticulum membrane, as predicted by amino acid sequence analysis, supports the existence of metabolic channeling in the phenylpropanoid pathway. This study establishes a framework for future investigations on gene expression level, protein expression and enzymatic assays, sequence polymorphisms, and genetic engineering.

Synthesis of 9,9′-[1,2-Ethanediylbis(oxymethylene]bis-2-amino-1,9-dihydro-6H-purin-6-one, an Impurity of Acyclovir

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Juan José Vaquero

2012-07-01

Full Text Available The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene]bis-2-amino-1,9-dihydro-6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial N-(9-acetyl-6-oxo-1H-purin-2-ylacetamide, the process uses an acid catalysed phase transfer catalysis (PTC process to produce the selective alkylation at the 9 position of the guanine ring.

Structure of the Bacillus anthracis dTDP- L -rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)

Energy Technology Data Exchange (ETDEWEB)

Baumgartner, Jackson; Lee, Jesi; Halavaty, Andrei S.; Minasov, George; Anderson, Wayne F.; Kuhn, Misty L. (NWU); (SFSU)

2017-10-30

L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), fromBacillus anthraciswas determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.

Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters.

Science.gov (United States)

Kinashi, Haruyasu

2011-01-01

Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells.

Science.gov (United States)

Zuccarini, Mariachiara; Giuliani, Patricia; Buccella, Silvana; Di Liberto, Valentina; Mudò, Giuseppa; Belluardo, Natale; Carluccio, Marzia; Rossini, Margherita; Condorelli, Daniele Filippo; Rathbone, Michel Piers; Caciagli, Francesco; Ciccarelli, Renata; Di Iorio, Patrizia

2017-12-01

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.

Essential pathway identification: from in silico analysis to potential antifungal targets in Aspergillus fumigatus

DEFF Research Database (Denmark)

Thykær, Jette; Andersen, Mikael Rørdam; Baker, S. E.

2009-01-01

with the reactions, we identified orthologous candidate essential genes in Aspergillus fumigatus. Our predictions are validated in part by the modes of action for some antifungal drugs and by molecular genetic studies of essential genes in A. fumigatus and other fungi. The use of metabolic models to predict...... of 1190 biochemically unique reactions that are associated with 871 open reading frames. Through a systematic in silico deletion of single metabolic reactions using this model, several essential metabolic pathways were identified for A. niger. A total of 138 reactions were identified as being essential...... biochemical reactions during growth on a minimal glucose medium. The majority of the reactions grouped into essential biochemical pathways covering cell wall biosynthesis, amino acid biosynthesis, energy metabolism and purine and pyrimidine metabolism. Based on the A. niger open reading frames associated...

De novo transcriptome assembly and the putative biosynthetic pathway of steroidal sapogenins of Dioscorea composita.

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Xia Wang

Full Text Available The plant Dioscorea composita has important applications in the medical and energy industries, and can be used for the extraction of steroidal sapogenins (important raw materials for the synthesis of steroidal drugs and bioethanol production. However, little is known at the genetic level about how sapogenins are biosynthesized in this plant. Using Illumina deep sequencing, 62,341 unigenes were obtained by assembling its transcriptome, and 27,720 unigenes were annotated. Of these, 8,022 unigenes were mapped to 243 specific pathways, and 531 unigenes were identified to be involved in 24 secondary metabolic pathways. 35 enzymes, which were encoded by 79 unigenes, were related to the biosynthesis of steroidal sapogenins in this transcriptome database, covering almost all the nodes in the steroidal pathway. The results of real-time PCR experiments on ten related transcripts (HMGR, MK, SQLE, FPPS, DXS, CAS, HMED, CYP51, DHCR7, and DHCR24 indicated that sapogenins were mainly biosynthesized by the mevalonate pathway. The expression of these ten transcripts in the tuber and leaves was found to be much higher than in the stem. Also, expression in the shoots was low. The nucleotide and protein sequences and conserved domains of four related genes (HMGR, CAS, SQS, and SMT1 were highly conserved between D. composita and D. zingiberensis; but expression of these four genes is greater in D. composita. However, there is no expression of these key enzymes in potato and no steroidal sapogenins are synthesized.

An Efficient and Facile Methodology for Bromination of Pyrimidine and Purine Nucleosides with Sodium Monobromoisocyanurate (SMBI

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Roger Stromberg

2013-10-01

Full Text Available An efficient and facile strategy has been developed for bromination of nucleosides using sodium monobromoisocyanurate (SMBI. Our methodology demonstrates bromination at the C-5 position of pyrimidine nucleosides and the C-8 position of purine nucleosides. Unprotected and also several protected nucleosides were brominated in moderate to high yields following this procedure.

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

Science.gov (United States)

Unal, Meral; Palavan-Unsal, Narcin; Tufekci, M A

2008-03-01

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

Distribution coefficients of purine alkaloids in water-ammonium sulfate-alkyl acetate-dialkyl phthalate systems

Science.gov (United States)

Korenman, Ya. I.; Krivosheeva, O. A.; Mokshina, N. Ya.

2012-12-01

The distribution of purine alkaloids (caffeine, theobromine, theophylline) was studied in the systems: alkyl acetates-dialkyl phtalate-salting-out agent (ammonium sulfate). The quantitative characteristics of the extraction-distribution coefficients ( D) and the degree of extraction ( R, %) are calculated. The relationships between the distribution coefficients of alkaloids and the length of the hydrocarbon radical in the molecule of alkyl acetate (dialkyl phtalate) are determined. The possibility of predicting the distribution coefficients is demonstrated.

Enteric Glia Mediate Neuron Death in Colitis Through Purinergic Pathways That Require Connexin-43 and Nitric OxideSummary

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Isola A.M. Brown

2016-01-01

Full Text Available Background & Aims: The concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R; triggering adenosine triphosphate (ATP release via neuronal pannexin-1 channels that subsequently recruits intracellular calcium ([Ca2+]i in surrounding enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation. Methods: We studied neuroinflammation in vivo using the 2,4-dinitrobenzene sulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse intestine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43 [GFAP::CreERT2+/−/Cx43f/f] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO synthase (iNOS−/− were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Ca2+ and NO imaging were used to monitor glial [Ca2+]i and [NO]i. Results: Purinergic activation of enteric glia drove [Ca2+]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO. Conclusions: Our results show that the activation of glial cells in the context of neuroinflammation kills enteric neurons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute to the development of motility disorders. Keywords: Enteric Nervous System, Hemichannels

Purine Metabolism in Acute Cerebral Ischemia

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Ye. V. Oreshnikov

2008-01-01

Full Text Available Objective: to study the specific features of purine metabolism in clinically significant acute cerebral ischemia. Subjects and materials. Three hundred and fifty patients with the acutest cerebral ischemic stroke were examined. The parameters of gas and electrolyte composition, acid-base balance, the levels of malonic dialdehyde, adenine, guanine, hypox-anthine, xanthine, and uric acid, and the activity of xanthine oxidase were determined in arterial and venous bloods and spinal fluid. Results. In ischemic stroke, hyperuricemia reflects the severity of cerebral metabolic disturbances, hemodynamic instability, hypercoagulation susceptiility, and the extent of neurological deficit. In ischemic stroke, hyperuri-corachia is accompanied by the higher spinal fluid levels of adenine, guanine, hypoxanthine, and xanthine and it is an indirect indicator of respiratory disorders of central genesis, systemic acidosis, hypercoagulation susceptibility, free radical oxidation activation, the intensity of a stressor response to cerebral ischemia, cerebral metabolic disturbances, the depth of reduced consciousness, and the severity of neurological deficit. Conclusion. The high venous blood activity of xanthine oxidase in ischemic stroke is associated with the better neurological parameters in all follow-up periods, the better early functional outcome, and lower mortality rates. Key words: hyperuricemia, stroke, xanthine oxidase, uric acid, cerebral ischemia.

Distribution of secondary metabolite biosynthetic gene clusters in 343 Fusarium genomes

Science.gov (United States)

Fusarium consists of over 200 phylogenetically distinct species, many of which cause important crop diseases and/or produce mycotoxins and other secondary metabolites (SMs). Some fusaria also cause opportunistic infections in humans and other animals. To investigate the distribution of biosynthetic ...

Biosynthetic routes of hydroxylated carotenoids (xanthophylls) in Marchantia polymorpha, and production of novel and rare xanthophylls through pathway engineering in Escherichia coli.

Science.gov (United States)

Takemura, Miho; Maoka, Takashi; Misawa, Norihiko

2015-03-01

MpBHY codes for a carotene β-ring 3(,3')-hydroxylase responsible for both zeaxanthin and lutein biosynthesis in liverwort. MpCYP97C functions as an ε-ring hydroxylase (zeinoxanthin 3'-hydroxylase) to produce lutein in liverwort. Xanthophylls are oxygenated or hydroxylated carotenes that are most abundant in the light-harvesting complexes of plants. The plant-type xanthophylls consist of α-xanthophyll (lutein) and β-xanthophylls (zeaxanthin, antheraxanthin, violaxanthin and neoxanthin). The α-xanthophyll and β-xanthophylls are derived from α-carotene and β-carotene by carotene hydroxylase activities, respectively. β-Ring 3,3'-hydroxylase that mediates the route of zeaxanthin from β-carotene via β-cryptoxanthin is present in higher plants and is encoded by the BHY (BCH) gene. On the other hand, CYP97A (or BHY) and CYP97C genes are responsible for β-ring 3-hydroxylation and ε-ring 3'-hydroxylation, respectively, in routes from α-carotene to lutein. To elucidate the evolution of the biosynthetic routes of such hydroxylated carotenoids from carotenes in land plants, we identified and functionally analyzed carotenoid hydroxylase genes of liverwort Marchantia polymorpha L. Three genes homologous to higher plants, BHY, CYP97A, and CYP97C, were isolated and named MpBHY, MpCYP97A, and MpCYP97C, respectively. MpBHY was found to code for β-ring hydroxylase, which is responsible for both routes starting from β-carotene and α-carotene. MpCYP97C functioned as an ε-ring hydroxylase not for α-carotene but for zeinoxanthin, while MpCYP97A showed no hydroxylation activity for β-carotene or α-carotene. These findings suggest the original functions of the hydroxylation enzymes of carotenes in land plants, which are thought to diversify in higher plants. In addition, we generated recombinant Escherichia coli cells, which produced rare and novel carotenoids such as α-echinenone and 4-ketozeinoxanthin, through pathway engineering using bacterial carotenogenic genes

A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

Energy Technology Data Exchange (ETDEWEB)

Fan, J.; Xu, C.; Andre, C.

2011-06-23

Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

Intrinsic Apoptosis Pathway in Fallopian Tube Epithelial Cells Induced by Cladribine

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Ewelina Wawryk-Gawda

2014-01-01

Full Text Available Cladribine is a purine nucleoside analog which initiates the apoptotic mechanism within cells. Moreover, the available data confirms that cladribine, with the participation of the p53 protein, as well as the proapoptotic proteins from the Bcl-2 family, also induces the activation of the intrinsic apoptosis pathway. However, while there has been a lot of research devoted to the effect of cladribine on lymphatic system cells, little is known about the impact of cladribine on the reproductive system. The aim of our study was to evaluate apoptosis in oviduct epithelial cells sourced from 15 different female rats. In so doing, the sections were stained with caspases 3, 9, and 8. Results suggest that cladribine also induces apoptosis in the oviduct epithelial cells by way of the intrinsic pathway. Indeed, the discontinuing of the administration of cladribine leads to a reduction in the amount of apoptotic cells in the oviduct epithelium.

Signal Transduction Pathways that Regulate CAB Gene Expression

Energy Technology Data Exchange (ETDEWEB)

Chory, Joanne

2004-12-31

The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

Signal Transduction Pathways that Regulate CAB Gene Expression

Energy Technology Data Exchange (ETDEWEB)

Chory, Joanne

2006-01-16

The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

KAUST Repository

Ross, Avena C.

2013-01-23

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus. © 2012 American Chemical Society.

Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

KAUST Repository

Ross, Avena C.; Xü , Ying; Lu, Liang; Kersten, Roland D.; Shao, Zongze; Al-Suwailem, Abdulaziz M.; Dorrestein, Pieter C.; Qian, Peiyuan; Moore, Bradley S.

2013-01-01

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus. © 2012 American Chemical Society.

Study of copper and purine-copper complexes on modified carbon electrodes by cyclic and elimination voltammetry

Czech Academy of Sciences Publication Activity Database

Trnková, L.; Zerzánková, L.; Dyčka, F.; Mikelová, R.; Jelen, František

2008-01-01

Roč. 8, č. 1 (2008), s. 429-444 ISSN 1424-8220 R&D Projects: GA AV ČR(CZ) IAA100040602; GA AV ČR(CZ) IAA400040804 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : copper-purine complexes * paraffin-impregnated graphite electrode * mercury-film electrode Subject RIV: BO - Biophysics Impact factor: 1.870, year: 2008

Oligomeric interface modulation causes misregulation of purine 5'-nucleotidase in relapsed leukemia

Czech Academy of Sciences Publication Activity Database

Hnízda, Aleš; Škerlová, Jana; Fábry, Milan; Pachl, Petr; Šinalová, Martina; Vrzal, Lukáš; Man, Petr; Novák, Petr; Řezáčová, Pavlína; Veverka, Václav

2016-01-01

Roč. 14, Oct 19 (2016), č. článku 91. ISSN 1741-7007 R&D Projects: GA ČR GA15-06582S; GA MŠk(CZ) LK11205; GA MŠk(CZ) LO1304; GA MŠk LO1302; GA MŠk(CZ) LD15089; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388963 ; RVO:68378050 ; RVO:61388971 Keywords : nucleotidase * cancer mutations * relapsed ALL * purine metabolism * allosteric regulation Subject RIV: CE - Biochemistry; EE - Microbiology, Virology (MBU-M) Impact factor: 6.779, year: 2016 http://bmcbiol.biomedcentral.com/articles/10.1186/s12915-016-0313-y

Purine-related metabolites and their converting enzymes are altered in frontal, parietal and temporal cortex at early stages of Alzheimer's disease pathology.

Science.gov (United States)

Alonso-Andrés, Patricia; Albasanz, José Luis; Ferrer, Isidro; Martín, Mairena

2018-01-24

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5'-nucleotidase (5'-NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age-matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5'-NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β-amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines. © 2018 International Society of Neuropathology.

Transcription factor VdCmr1 is required for pigment production, protection from UV irradiation, and regulates expression of melanin biosynthetic genes in Verticillium dahliae.

Science.gov (United States)

Wang, Yonglin; Hu, Xiaoping; Fang, Yulin; Anchieta, Amy; Goldman, Polly H; Hernandez, Gustavo; Klosterman, Steven J

2018-04-01

Verticillium dahliae is a soilborne fungus that causes vascular wilt diseases on numerous plant species worldwide. The production of darkly melanized microsclerotia is crucial in the disease cycle of V. dahliae, as these structures allow for long-term survival in soil. Previously, transcriptomic and genomic analysis identified a cluster of genes in V. dahliae that encodes some dihydroxynaphthalene (DHN) melanin biosynthetic pathway homologues found in related fungi. In this study, we explored the roles of cluster-specific transcription factor VdCmr1, as well as two other genes within the cluster encoding a polyketide synthase (VdPKS1) and a laccase (VdLac1), enzymes at initial and endpoint steps in DHN melanin production. The results revealed that VdCmr1 and VdPKS1 are required for melanin production, but neither is required for microsclerotia production. None of the three genes were required for pathogenesis on tobacco and lettuce. Exposure of ΔVdCmr1 and wild-type strains to UV irradiation, or to high temperature (40 °C), revealed an approx. 50 % reduction of survival in the ΔVdCmr1 strain, relative to the wild-type strain, in response to either condition. Expression profiles revealed that expression of some melanin biosynthetic genes are in part dependent on VdCmr1. Combined data indicate VdCmr1 is a key regulator of melanin biosynthesis, and that via regulation of melanogenesis, VdCmr1 affects survival of V. dahliae in response to abiotic threats. We conclude with a model showing regulation of VdCmr1 by a high osmolarity glycerol response (Hog)-type MAP kinase pathway.

Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus

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Clark Shawn M

2013-01-01

Full Text Available Abstract Background Bitter acids (e.g. humulone are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP pathway. We used RNA sequencing (RNA-seq to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic and reverse (catabolic reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial and

The optimized microwave-assisted decomposition of formamides and its synthetic utility in the amination reactions of purines

Czech Academy of Sciences Publication Activity Database

Čechová, Lucie; Jansa, Petr; Šála, Michal; Dračínský, Martin; Holý, Antonín; Janeba, Zlatko

2011-01-01

Roč. 67, č. 5 (2011), s. 866-871 ISSN 0040-4020 R&D Projects: GA MŠk 1M0508; GA AV ČR KJB400550903 Institutional research plan: CEZ:AV0Z40550506 Keywords : microwave-assisted synthesis * DMF * formamides * dehalogenative amination * purines Subject RIV: CC - Organic Chemistry Impact factor: 3.025, year: 2011

Modeling compositional dynamics based on GC and purine contents of protein-coding sequences

KAUST Repository

Zhang, Zhang

2010-11-08

Background: Understanding the compositional dynamics of genomes and their coding sequences is of great significance in gaining clues into molecular evolution and a large number of publically-available genome sequences have allowed us to quantitatively predict deviations of empirical data from their theoretical counterparts. However, the quantification of theoretical compositional variations for a wide diversity of genomes remains a major challenge.Results: To model the compositional dynamics of protein-coding sequences, we propose two simple models that take into account both mutation and selection effects, which act differently at the three codon positions, and use both GC and purine contents as compositional parameters. The two models concern the theoretical composition of nucleotides, codons, and amino acids, with no prerequisite of homologous sequences or their alignments. We evaluated the two models by quantifying theoretical compositions of a large collection of protein-coding sequences (including 46 of Archaea, 686 of Bacteria, and 826 of Eukarya), yielding consistent theoretical compositions across all the collected sequences.Conclusions: We show that the compositions of nucleotides, codons, and amino acids are largely determined by both GC and purine contents and suggest that deviations of the observed from the expected compositions may reflect compositional signatures that arise from a complex interplay between mutation and selection via DNA replication and repair mechanisms.Reviewers: This article was reviewed by Zhaolei Zhang (nominated by Mark Gerstein), Guruprasad Ananda (nominated by Kateryna Makova), and Daniel Haft. 2010 Zhang and Yu; licensee BioMed Central Ltd.

Modeling compositional dynamics based on GC and purine contents of protein-coding sequences

KAUST Repository

Zhang, Zhang; Yu, Jun

2010-01-01

Background: Understanding the compositional dynamics of genomes and their coding sequences is of great significance in gaining clues into molecular evolution and a large number of publically-available genome sequences have allowed us to quantitatively predict deviations of empirical data from their theoretical counterparts. However, the quantification of theoretical compositional variations for a wide diversity of genomes remains a major challenge.Results: To model the compositional dynamics of protein-coding sequences, we propose two simple models that take into account both mutation and selection effects, which act differently at the three codon positions, and use both GC and purine contents as compositional parameters. The two models concern the theoretical composition of nucleotides, codons, and amino acids, with no prerequisite of homologous sequences or their alignments. We evaluated the two models by quantifying theoretical compositions of a large collection of protein-coding sequences (including 46 of Archaea, 686 of Bacteria, and 826 of Eukarya), yielding consistent theoretical compositions across all the collected sequences.Conclusions: We show that the compositions of nucleotides, codons, and amino acids are largely determined by both GC and purine contents and suggest that deviations of the observed from the expected compositions may reflect compositional signatures that arise from a complex interplay between mutation and selection via DNA replication and repair mechanisms.Reviewers: This article was reviewed by Zhaolei Zhang (nominated by Mark Gerstein), Guruprasad Ananda (nominated by Kateryna Makova), and Daniel Haft. 2010 Zhang and Yu; licensee BioMed Central Ltd.

Ancient horizontal gene transfer from bacteria enhances biosynthetic capabilities of fungi.

Directory of Open Access Journals (Sweden)

Imke Schmitt

Full Text Available Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive.We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication.Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase.

[Separation of purines, pyrimidines, pterins and flavonoids on magnolol-bonded silica gel stationary phase by high performance liquid chromatography].

Science.gov (United States)

Chen, Hong; Li, Laishen; Zhang, Yang; Zhou, Rendan

2012-10-01

A new magnolol-bonded silica gel stationary phase (MSP) was used to separate the basic drugs including four purines, eight pyrimidines, four pterins and five flavonoids as polar representative samples by high performance liquid chromatography (HPLC). To clarify the separation mechanism, a commercial ODS column was also tested under the same chromatographic conditions. The high selectivities and fast baseline separations of the above drugs were achieved by using simple mobile phases on MSP. Although there is no end-caped treatment, the peak shapes of basic drugs containing nitrogen such as purines, pyrimidines and pterins were rather symmetrical on MSP, which indicated the the magnolol as ligand with multi-sites could shield the side effect of residual silanol groups on the surface of silica gel. Although somewhat different in the separation resolution, it was found that the elution orders of some drugs were generally similar on both MSP and ODS. The hydrophobic interaction should play a significant role in the separations of the above basic drugs, which was attributed to their reversed-phase property in the nature. However, MSP could provide the additional sites for many polar solutes, which was a rational explanation for the high selectivity of MSP. For example, in the separation of purines, pyrimidines and pterins on MSP, hydrogen-bonding and dipole-dipole interactions played leading roles besides hydrophobic interaction. Some solute molecules (such as mercaptopurine, vitexicarpin) and MSP can form the strong pi-pi stacking in the separation process. All enhanced the retention and improved the separation selectivity of MSP, which facilitated the separation of the basic drugs.

Involvement of 2-C-methyl-D-erythritol-4-phosphate pathway in biosynthesis of aphidicolin-like tetracyclic diterpene of Scoparia dulcis.

Science.gov (United States)

Nkembo, Marguerite Kasidimoko; Lee, Jung-Bum; Nakagiri, Takeshi; Hayashi, Toshimitsu

2006-05-01

Specific inhibitors of the MVA pathway (pravastatin) and the MEP pathway (fosmidomycin) were used to interfere with the biosynthetic flux which leads to the production of aphidicolin-like diterpene in leaf organ cultures of Scoparia dulcis. Treatment of leaf organs with fosmidomycin resulted in dose dependent inhibition of chlorophylls, carotenoids, scopadulcic acid B (SDB) and phytol production, and no effect on sterol production was observed. In response to the pravastatin treatment, a significant decrease in sterol and perturbation of SDB production was observed.

Rational synthetic pathway refactoring of natural products biosynthesis in actinobacteria.

Science.gov (United States)

Tan, Gao-Yi; Liu, Tiangang

2017-01-01

Natural products (NPs) and their derivatives are widely used as frontline treatments for many diseases. Actinobacteria spp. are used to produce most of NP antibiotics and have also been intensively investigated for NP production, derivatization, and discovery. However, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in Actinobacteria, especially in the cases of genome mining and heterologous expression, it is often difficult to rationally and systematically engineer synthetic pathways to maximize biosynthetic efficiency. With the emergence of new tools and methods in metabolic engineering, the synthetic pathways of many chemicals, such as fatty acids and biofuels, in model organisms (e.g. Escherichia coli ), have been refactored to realize precise and flexible control of production. These studies also offer a promising approach for synthetic pathway refactoring in Actinobacteria. In this review, the great potential of Actinobacteria as a microbial cell factory for biosynthesis of NPs is discussed. To this end, recent progress in metabolic engineering of NP synthetic pathways in Actinobacteria are summarized and strategies and perspectives to rationally and systematically refactor synthetic pathways in Actinobacteria are highlighted. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Synthesis of carbon-11-labeled imidazopyridine- and purine-thioacetamide derivatives as new potential PET tracers for imaging of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1).

Science.gov (United States)

Gao, Mingzhang; Wang, Min; Zheng, Qi-Huang

2016-03-01

The target tracer carbon-11-labeled imidazopyridine- and purine-thioacetamide derivatives, N-(3-[(11)C]methoxy-4-methoxyphenyl)-2-((5-methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)acetamide (3-[(11)C]4a) and N-(4-[(11)C]methoxy-3-methoxyphenyl)-2-((5-methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)acetamide (4-[(11)C]4a); 2-((6-amino-9H-purin-8-yl)thio)-N-(3-[(11)C]methoxy-4-methoxyphenyl)acetamide (3-[(11)C]8a) and 2-((6-amino-9H-purin-8-yl)thio)-N-(4-[(11)C]methoxy-3-methoxyphenyl)acetamide (4-[(11)C]8a), were prepared by O-[(11)C]methylation of their corresponding precursors with [(11)C]CH3OTf under basic condition (2N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50-60% radiochemical yields based on [(11)C]CO2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185-555GBq/μmol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

Directory of Open Access Journals (Sweden)

Jungsuwadee Paiboon

2011-02-01

Full Text Available Abstract Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC and Adenosine Triphosphate Binding Cassette (ABC superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. Conclusions We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and

Diurnal and circadian expression profiles of glycerolipid biosynthetic genes in Arabidopsis.

Science.gov (United States)

Nakamura, Yuki; Andrés, Fernando; Kanehara, Kazue; Liu, Yu-chi; Coupland, George; Dörmann, Peter

2014-01-01

Glycerolipid composition in plant membranes oscillates in response to diurnal change. However, its functional significance remained unclear. A recent discovery that Arabidopsis florigen FT binds diurnally oscillating phosphatidylcholine molecules to promote flowering suggests that diurnal oscillation of glycerolipid composition is an important input in flowering time control. Taking advantage of public microarray data, we globally analyzed the expression pattern of glycerolipid biosynthetic genes in Arabidopsis under long-day, short-day, and continuous light conditions. The results revealed that 12 genes associated with glycerolipid metabolism showed significant oscillatory profiles. Interestingly, expression of most of these genes followed circadian profiles, suggesting that glycerolipid biosynthesis is partially under clock regulation. The oscillating expression profile of one representative gene, PECT1, was analyzed in detail. Expression of PECT1 showed a circadian pattern highly correlated with that of the clock-regulated gene GIGANTEA. Thus, our study suggests that a considerable number of glycerolipid biosynthetic genes are under circadian control.

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.

Science.gov (United States)

Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L; Busby, Scott A; Griffin, Patrick R; Pathak, Manish C; Ortlund, Eric A; Moore, David D

2011-05-25

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

Synthesis and spectroscopy of clay intercalated Cu(II) bio-monomer complexes: coordination of Cu(II) with purines and nucleotides

NARCIS (Netherlands)

Weckhuysen, B.M.; Leeman, H.; Schoonheydt, R.A.

1999-01-01

The spectroscopic properties of Cu(bio-monomer)nm+ complexes [BM=bio-monomer (purine, adenine, guanine, hypoxanthine, 5-ADP and 5-GMP)] in saponite clays have been investigated by diffuse reflectance spectroscopy (DRS) in the UV-Vis-NIR region and electron paramagnetic resonance (EPR) at X-band.

TD-DFT Investigation of the Magnetic Circular Dichroism Spectra of Some Purine and Pyrimidine Bases of Nucleic Acids

DEFF Research Database (Denmark)

Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick

2015-01-01

We present a computational study of the magnetic circular dichroism (MCD) spectra in the 200–300 nm wavelength region of purine and its derivative hypoxanthine, as well as of the pyrimidine bases of nucleic acids uracil, thymine, and cytosine, using the B3LYP and CAM–B3LYP functionals. Solvent...

Alteration of Mevalonate Pathway in Rat Splenic Lymphocytes: Possible Role in Cytokines Secretion Regulated by L-Theanine

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Chengjian Li

2018-01-01

Full Text Available L-Theanine is a nonprotein amino acid in tea, and its immunomodulatory function has been confirmed. This study aimed to investigate the effect of L-theanine addition on cytokines secretion in rat splenic lymphocytes and explore its potential immunomodulatory effects on the mevalonate biosynthetic pathway. Our results showed that L-theanine treatment did not influence the proliferation and division indexes of the splenic lymphocytes subsets. Interestingly, L-theanine treatment had regulated the contents of IFN-γ, IL-2, IL-4, IL-10, IL-12, and TNF-α  (P<0.001 except IL-6 and upregulated the mRNA and protein expression of Ras-related protein Rap-1A (Rap1A, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR, and farnesyl diphosphate synthase (FDPs (P<0.001. Additionally, there was a positive correlation between Rap1A and HMGCR proteins expression and IFN-γ, IL-4, and IL-6 levels. In conclusion, L-theanine regulated the secretion of cytokines probably by activating expression of Rap1A and HMGCR proteins involved in the mevalonate biosynthetic pathway in rat splenic lymphocytes. Therefore, L-theanine might be a promising potential drug candidate as immunopotentiator.

Identification and analysis of the paulomycin biosynthetic gene cluster and titer improvement of the paulomycins in Streptomyces paulus NRRL 8115.

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Jine Li

Full Text Available The paulomycins are a group of glycosylated compounds featuring a unique paulic acid moiety. To locate their biosynthetic gene clusters, the genomes of two paulomycin producers, Streptomyces paulus NRRL 8115 and Streptomyces sp. YN86, were sequenced. The paulomycin biosynthetic gene clusters were defined by comparative analyses of the two genomes together with the genome of the third paulomycin producer Streptomyces albus J1074. Subsequently, the identity of the paulomycin biosynthetic gene cluster was confirmed by inactivation of two genes involved in biosynthesis of the paulomycose branched chain (pau11 and the ring A moiety (pau18 in Streptomyces paulus NRRL 8115. After determining the gene cluster boundaries, a convergent biosynthetic model was proposed for paulomycin based on the deduced functions of the pau genes. Finally, a paulomycin high-producing strain was constructed by expressing an activator-encoding gene (pau13 in S. paulus, setting the stage for future investigations.

Expression of Xanthophyll Biosynthetic Genes during Light-Dependent Chloroplast Differentiation1

Science.gov (United States)

Woitsch, Sonja; Römer, Susanne

2003-01-01

In higher plants, etioplast to chloroplast differentiation is characterized by dramatic ultrastructural changes of the plastid and a concomitant increase in chlorophylls and carotenoids. Whereas the formation and function of carotenes and their oxygenated derivatives, the xanthophylls, have been well studied, little is known about the regulation of the genes involved in xanthophyll biosynthesis. Here, we analyze the expression of three xanthophyll biosynthetic genes (i.e. β-carotene hydroxylase [bhy], zeaxanthin epoxidase [zep], and violaxanthin de-epoxidase [vde]) during de-etiolation of seedlings of tobacco (Nicotiana tabacum L. cv Samsun) under different light conditions. White-light illumination caused an increase in the amount of all corresponding mRNAs. The expression profiles of bhy and zep not only resembled each other but were also similar to the pattern of a gene encoding a major light-harvesting protein of photosystem II. This finding indicates a coordinated synthesis during formation of the antenna complex. In contrast, the expression pattern of vde was clearly different. Furthermore, the gene expression of bhy was shown to be modulated after illumination with different white-light intensities. The expression of all xanthophyll biosynthetic genes under examination was up-regulated upon exposure to red, blue, and white light. Gene expression of bhy and vde but not of zep was more pronounced under red-light illumination, pointing at an involvement of the phytochrome system. Expression analysis in the presence of the photosynthetic electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone indicated a redox control of transcription of two of the xanthophyll biosynthetic genes (bhy and zep). PMID:12857831

Structural Analysis of DFG-in and DFG-out Dual Src-Abl Inhibitors Sharing a Common Vinyl Purine Template

Energy Technology Data Exchange (ETDEWEB)

Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Sawyer, Tomi K.; Shakespeare, William C.; Clackson, Tim; Zhu, Xiaotian; Dalgarno, David C. (ARIAD)

2010-09-30

Bcr-Abl is the oncogenic protein tyrosine kinase responsible for chronic myeloid leukemia (CML). Treatment of the disease with imatinib (Gleevec) often results in drug resistance via kinase mutations at the advanced phases of the disease, which has necessitated the development of new mutation-resistant inhibitors, notably against the T315I gatekeeper mutation. As part of our efforts to discover such mutation resistant Abl inhibitors, we have focused on optimizing purine template kinase inhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are also potent Src inhibitors. Here we present crystal structures of Abl bound by two such inhibitors, based on a common N9-arenyl purine, and that represent both DFG-in and -out binding modes. In each structure the purine template is bound deeply in the adenine pocket and the novel vinyl linker forms a non-classical hydrogen bond to the gatekeeper residue, Thr315. Specific template substitutions promote either a DFG-in or -out binding mode, with the kinase binding site adjusting to optimize molecular recognition. Bcr-Abl T315I mutant kinase is resistant to all currently marketed Abl inhibitors, and is the focus of intense drug discovery efforts. Notably, our DFG-out inhibitor, AP24163, exhibits modest activity against this mutant, illustrating that this kinase mutant can be inhibited by DFG-out class inhibitors. Furthermore our DFG-out inhibitor exhibits dual Src-Abl activity, absent from the prototypical DFG-out inhibitor, imatinib as well as its analog, nilotinib. The data presented here provides structural guidance for the further design of novel potent DFG-out class inhibitors against Src, Abl and Abl T315I mutant kinases.

A Multitarget Approach toward the Development of 8-Substituted Purines for Photoprotection and Prevention of UV-Related Damage.

Science.gov (United States)

Djuidje, Ernestine N; Dissette, Valeria; Bino, Alessia; Benetti, Simonetta; Balzarini, Jan; Liekens, Sandra; Manfredini, Stefano; Vertuani, Silvia; Baldisserotto, Anna

2017-05-22

Ultraviolet (UV) light is the most abundant and significant modifiable risk factor for skin cancer and many other skin diseases such as early photo-aging. Across the solar radiation spectrum, UV light is the main cause behind skin problems. In the search for novel photoprotective compounds, a new series of 8-substituted purines were synthesized from commercially available 6-hydroxy-4,5-diaminopyrimidine hemisulfate or 4,5-diaminopyrimidine. All title compounds were investigated for their UV filtering, antioxidant, antifungal, and antiproliferative activities. For the photoprotection assays we used a diffuse transmittance technique to determine the sun protection factor (SPF) in vitro, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion reducing antioxidant power (FRAP) tests for evaluating the antioxidant activity of the more potent compounds. Among them, 8-(2,5-dihydroxyphenyl)-7H-purin-6-ol (compound 26) proved to be a good radical scavenger and is also endowed with broad-spectrum UVA filtering capabilities, suitable for further development as a protective molecule. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ni2+-binding RNA motifs with an asymmetric purine-rich internal loop and a G-A base pair.

Science.gov (United States)

Hofmann, H P; Limmer, S; Hornung, V; Sprinzl, M

1997-01-01

RNA molecules with high affinity for immobilized Ni2+ were isolated from an RNA pool with 50 randomized positions by in vitro selection-amplification. The selected RNAs preferentially bind Ni2+ and Co2+ over other cations from first series transition metals. Conserved structure motifs, comprising about 15 nt, were identified that are likely to represent the Ni2+ binding sites. Two conserved motifs contain an asymmetric purine-rich internal loop and probably a mismatch G-A base pair. The structure of one of these motifs was studied with proton NMR spectroscopy and formation of the G-A pair at the junction of helix and internal loop was demonstrated. Using Ni2+ as a paramagnetic probe, a divalent metal ion binding site near this G-A base pair was identified. Ni2+ ions bound to this motif exert a specific stabilization effect. We propose that small asymmetric purine-rich loops that contain a G-A interaction may represent a divalent metal ion binding site in RNA. PMID:9409620

A G-C-rich palindromic structural motif and a stretch of single-stranded purines are required for optimal packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA.

Science.gov (United States)

Jaballah, Soumeya Ali; Aktar, Suriya J; Ali, Jahabar; Phillip, Pretty Susan; Al Dhaheri, Noura Salem; Jabeen, Aayesha; Rizvi, Tahir A

2010-09-03

During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5' end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason-Pfizer monkey virus (MPMV) contains packaging sequences within the 5' untranslated region (UTR) and Gag. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region "A", immediately downstream of the primer binding site, is composed of 50 nt, whereas region "B" is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G-C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process. Copyright (c) 2010 Elsevier Ltd

Mutation of a Rice Gene Encoding a Phenylalanine Biosynthetic Enzyme Results in Accumulation of Phenylalanine and Tryptophan[W

Science.gov (United States)

Yamada, Tetsuya; Matsuda, Fumio; Kasai, Koji; Fukuoka, Shuichi; Kitamura, Keisuke; Tozawa, Yuzuru; Miyagawa, Hisashi; Wakasa, Kyo

2008-01-01

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size. PMID:18487352

Adaptive responses to cefotaxime treatment in ESBL-producing Escherichia coli and the possible use of significantly regulated pathways as novel secondary targets

DEFF Research Database (Denmark)

Møller, Thea S. B.; Rau, Martin Holm; Bonde, Charlotte S

2016-01-01

The aim of the study was to determine how ESBL-producing Escherichia coli change the expression of metabolic and biosynthesis genes when adapting to inhibitory concentrations of cefotaxime. Secondly, it was investigated whether significantly regulated pathways constitute putative secondary targets......-fold). Inhibition and/or mutations in other genes that were significantly regulated, belonging to energy synthesis, purine synthesis, proline uptake or potassium uptake, also rendered the resistant bacteria more susceptible to cefotaxime. The results show that ESBL-producing E. coli adapt to treatment...

Evolutionary rate patterns of the Gibberellin pathway genes

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Zhang Fu-min

2009-08-01

Full Text Available Abstract Background Analysis of molecular evolutionary patterns of different genes within metabolic pathways allows us to determine whether these genes are subject to equivalent evolutionary forces and how natural selection shapes the evolution of proteins in an interacting system. Although previous studies found that upstream genes in the pathway evolved more slowly than downstream genes, the correlation between evolutionary rate and position of the genes in metabolic pathways as well as its implications in molecular evolution are still less understood. Results We sequenced and characterized 7 core structural genes of the gibberellin biosynthetic pathway from 8 representative species of the rice tribe (Oryzeae to address alternative hypotheses regarding evolutionary rates and patterns of metabolic pathway genes. We have detected significant rate heterogeneity among 7 GA pathway genes for both synonymous and nonsynonymous sites. Such rate variation is mostly likely attributed to differences of selection intensity rather than differential mutation pressures on the genes. Unlike previous argument that downstream genes in metabolic pathways would evolve more slowly than upstream genes, the downstream genes in the GA pathway did not exhibited the elevated substitution rate and instead, the genes that encode either the enzyme at the branch point (GA20ox or enzymes catalyzing multiple steps (KO, KAO and GA3ox in the pathway had the lowest evolutionary rates due to strong purifying selection. Our branch and codon models failed to detect signature of positive selection for any lineage and codon of the GA pathway genes. Conclusion This study suggests that significant heterogeneity of evolutionary rate of the GA pathway genes is mainly ascribed to differential constraint relaxation rather than the positive selection and supports the pathway flux theory that predicts that natural selection primarily targets enzymes that have the greatest control on fluxes.

Nicotinamide riboside kinase structures reveal new pathways to NAD+.

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Wolfram Tempel

2007-10-01

Full Text Available The eukaryotic nicotinamide riboside kinase (Nrk pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+ by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

Metabolic pathways of lung inflammation revealed by high-resolution metabolomics (HRM) of H1N1 influenza virus infection in mice.

Science.gov (United States)

Chandler, Joshua D; Hu, Xin; Ko, Eun-Ju; Park, Soojin; Lee, Young-Tae; Orr, Michael; Fernandes, Jolyn; Uppal, Karan; Kang, Sang-Moo; Jones, Dean P; Go, Young-Mi

2016-11-01

Influenza is a significant health concern worldwide. Viral infection induces local and systemic activation of the immune system causing attendant changes in metabolism. High-resolution metabolomics (HRM) uses advanced mass spectrometry and computational methods to measure thousands of metabolites inclusive of most metabolic pathways. We used HRM to identify metabolic pathways and clusters of association related to inflammatory cytokines in lungs of mice with H1N1 influenza virus infection. Infected mice showed progressive weight loss, decreased lung function, and severe lung inflammation with elevated cytokines [interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ] and increased oxidative stress via cysteine oxidation. HRM showed prominent effects of influenza virus infection on tryptophan and other amino acids, and widespread effects on pathways including purines, pyrimidines, fatty acids, and glycerophospholipids. A metabolome-wide association study (MWAS) of the aforementioned inflammatory cytokines was used to determine the relationship of metabolic responses to inflammation during infection. This cytokine-MWAS (cMWAS) showed that metabolic associations consisted of distinct and shared clusters of 396 metabolites highly correlated with inflammatory cytokines. Strong negative associations of selected glycosphingolipid, linoleate, and tryptophan metabolites with IFN-γ contrasted strong positive associations of glycosphingolipid and bile acid metabolites with IL-1β, TNF-α, and IL-10. Anti-inflammatory cytokine IL-10 had strong positive associations with vitamin D, purine, and vitamin E metabolism. The detailed metabolic interactions with cytokines indicate that targeted metabolic interventions may be useful during life-threatening crises related to severe acute infection and inflammation. Copyright © 2016 the American Physiological Society.

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

Science.gov (United States)

Dailey, Harry A; Dailey, Tamara A; Gerdes, Svetlana; Jahn, Dieter; Jahn, Martina; O'Brian, Mark R; Warren, Martin J

2017-03-01

The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized. Copyright © 2017 American Society for Microbiology.

Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold

DEFF Research Database (Denmark)

Henriques de Jesus, Maria Perestrello Ramos; Nielsen, Agnieszka Janina Zygadlo; Mellor, Silas Busck

2017-01-01

to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s....... Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase...... in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold...

Ionization of Purine Tautomers in Nucleobases, Nucleosides, and Nucleotides: From the Gas Phase to the Aqueous Environment

Czech Academy of Sciences Publication Activity Database

Pluhařová, Eva; Jungwirth, Pavel; Bradforth, S. E.; Slavíček, P.

2011-01-01

Roč. 115, č. 5 (2011), s. 1294-1305 ISSN 1520-6106 R&D Projects: GA MŠk LC512; GA ČR GA203/08/0114 Grant - others:GA ČR(CZ) GA203/09/0422 Program:GA Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA bases * purines * ab initio Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.696, year: 2011

The effect of exhaustive exercise on the concentration of purine nucleotides and their metabolites in erythrocytes

OpenAIRE

E Skotnicka; I Baranowska-Bosiacka; W Dudzińska; M Suska; R Nowak; K Krupecki; AJ Hłyńczak

2008-01-01

In this study we tried to obtain a complete overview of purine nucleotide metabolism in erythrocytes before and during an incremental, intermittent exhaustive exercise bout protocol for sportsmen (high-performance rowers) and untrained, healthy, active volunteers. Erythrocyte levels of the main nucleotides (ATP, ADP, AMP, GTP, GDP, GMP, IMP, NAD and NADP ), nucleosides (Ado, Guo, Ino) and the base Hyp were measured using the HPLC method. The parameters that can be deducted from their concent...

Prediction of rumen microbial outflow based on urinary excretion of purine derivatives

International Nuclear Information System (INIS)

Nolan, J.V.

1999-01-01

The method for predicting microbial protein outflow from the rumen based on the excretion of purine derivatives (PD) in the urine is being increasingly used by nutritionists. In contrast to methods that depend on estimates of digesta flow, the PD method does not require animals to be fitted surgically with cannulae into the gut, and studies can be performed with minimal disturbance to the experimental animals. Methods of analysis of PD have been improved and standardized. Certain assumptions, however, are required that could lead to errors when this method is used to predict microbial protein outflow from the rumen. The need for further investigation of these assumptions by means of isotopic tracers and other techniques is examined. (author)

Thermodynamics of metabolic pathways for penicillin production: Analysis of thermodynamic feasibility and free energy changes during fed-batch cultivation

DEFF Research Database (Denmark)

Pissarra, P.D.; Nielsen, Jens Bredal

1997-01-01

This paper describes the thermodynamic analysis of pathways related to penicillin production in Penicillium chrysogenum. First a thermodynamic feasibility analysis is performed of the L-lysine pathway of which one of the precursors for penicillin biosynthesis (alpha-aminoadipic acid......) is an intermediate. It is found that the L-lysine pathway in P. chrysogenum is thermodynamically feasible and that the calculated standard Gibbs free energy values of the two enzymes controlling the pathway flux indicate that they operate far from equilibrium. It is therefore proposed that the regulation of alpha......-aminoadipate reductase by lysine is important to maintain a high concentration of alpha-aminoadipate in order to direct the carbon flux to penicillin production. Secondly the changes in Gibbs free energy in the penicillin biosynthetic pathway during fed-batch cultivation were studied. The analysis showed that all...

Errores innatos del metabolismo de las purinas y otras enfermedades relacionadas Inborn purine metabolism errors and other related diseases

Directory of Open Access Journals (Sweden)

Jiovanna Contreras Roura

2012-06-01

Full Text Available Los errores innatos en el metabolismo de las purinas son trastornos hereditarios complejos de gran impacto clínico, que presentan síntomas variables de acuerdo con el tipo de enfermedad. Pueden presentarse problemas renales de origen desconocido, retardo mental con manifestaciones neurológicas, retardo del crecimiento, infecciones recurrentes, automutilación, inmunodeficiencias, anemia hemolítica inexplicable, artritis gotosa, historia familiar, consanguinidad y reacciones adversas a fármacos que son análogos de las purinas. Las investigaciones de estas enfermedades comienzan generalmente con la cuantificación del ácido úrico en suero y en orina, por ser el producto final del metabolismo de las purinas en humanos. La dieta y el consumo de medicamentos, entre otras condiciones patológicas, fisiológicas y clínicas, también pueden modificar los niveles de este compuesto. Esta revisión pretende divulgar información de los errores innatos en el metabolismo de las purinas, y facilitar la interpretación de los niveles del ácido úrico y otros marcadores bioquímicos útiles en el diagnóstico de estas enfermedades. Se incluyen tablas que relacionan estas enfermedades con los niveles de excreción de ácido úrico y otros marcadores bioquímicos, las enzimas alteradas, los síntomas clínicos, el modo de herencia y, en algunos casos, el tratamiento propuesto. Este trabajo nos permite afirmar que las variaciones en los niveles del ácido úrico y la presencia de otros marcadores bioquímicos en orina, constituyen una herramienta importante en la pesquisa de algunos errores innatos en el metabolismo de las purinas, así como de otras condiciones patológicas relacionadas.Inborn purine metabolism errors are complex inherited disorders of great clinical impact that present with variable symptoms according to the type of disease. It might occur renal problems of unknown origin, metal retardation with neurological manifestations, retarded

Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.

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Stephen D Bentley

2006-03-01

Full Text Available Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.

Trisubstituted purine inhibitors of PDGFR alpha and their antileukemic activity in the human eosinophilic cell line EOL-1

Czech Academy of Sciences Publication Activity Database

Malínková, Veronika; Řezníčková, Eva; Jorda, Radek; Gucký, T.; Kryštof, Vladimír

2017-01-01

Roč. 25, č. 24 (2017), s. 6523-6535 ISSN 0968-0896 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : dependent kinase inhibitors * src tyrosine kinase * 2,6,9-trisubstituted purines * therapeutic target * potent inhibitor * imatinib * leukemia * mutations * mutant * domain Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Hematology Impact factor: 2.930, year: 2016

Synthesis of purines bearing functionalized C-substituents by the conjugate addition of nucleophiles to 6-vinylpurines and 6-ethynylpurines

Czech Academy of Sciences Publication Activity Database

Kuchař, Martin; Pohl, Radek; Votruba, Ivan; Hocek, Michal

-, č. 22 (2006), s. 5083 -5098 ISSN 1434-193X R&D Projects: GA MŠk(CZ) 1M0508; GA AV ČR(CZ) 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : purines * Michael addition * conjugate additions * cross-coupling Subject RIV: CC - Organic Chemistry Impact factor: 2.769, year: 2006

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Weiler, Monica [Department of Medicine III and Transfusion Medicine, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich (Germany); Schmetzer, Helga [Helmholtz Center Munich (Germany); German Research Center for Environmental Health, Munich (Germany); Braeu, Marion; Buhmann, Raymund [Helmholtz Center Munich (Germany); German Research Center for Environmental Health, Munich (Germany); Department of Medicine III and Transfusion Medicine, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich (Germany)

2016-11-15

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3{sup +}T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

International Nuclear Information System (INIS)

Weiler, Monica; Schmetzer, Helga; Braeu, Marion; Buhmann, Raymund

2016-01-01

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3 + T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

Purine nitrogen index, potentially a new parameter for rapid feed evaluation in ruminants

International Nuclear Information System (INIS)

Chen, X.B.; Oerskov, E.R.; Subba, D.B.; Jayasuriya, M.C.N.

1999-01-01

The concept of a new parameter 'Purine Nitrogen Index (PNI)' for feed evaluation in ruminants is discussed. PNI refers to the ratio of purine derivative (PD) nitrogen to total nitrogen in urine. It is suggested that PNI can potentially be used as an indicator of the efficiency with which degradable dietary nitrogen is converted to microbial protein in the rumen. The excretion of PD in the urine provides an estimation of the intestinal flow of microbial protein, and therefore, PNI effectively corresponds to the amount of microbial protein produced in the rumen relative to the nitrogen loss in the urine. If a diet or a dietary regime has a high conversion efficiency, proportionally more rumen degradable nitrogen is converted to microbial protein and less nitrogen is excreted in the urine, resulting in a high PNI. Conversely, if a diet has a poor conversion efficiency, proportionally less dietary nitrogen is converted to microbial protein and more is excreted in the urine, resulting in a low PNI. Preliminary data from six experiments involving 34 sheep confirmed a positive correlation between PNI and the nitrogen conversion efficiency, and suggested that a dietary regime with a PNI lower than 0.08 for sheep appeared to be a less efficient in the production of microbial protein and have a greater loss of nitrogen in the urine. PNI can theoretically be determined in spot urine samples, and has the potential to serve as a 'dipstick' method for the rapid evaluation of ruminant feeds. However, more research with a mathematical modelling approach is required to evaluate and develop the concept further. (author)

Purine nitrogen index, potentially a new parameter for rapid feed evaluation in ruminants

Energy Technology Data Exchange (ETDEWEB)

Chen, X B; Oerskov, E R [Rowett Research Institute, Bucksburn, Aberdeen (United Kingdom); Subba, D B [Pakhribas Agricultural Centre, Dhankuta, Kathmandu (Nepal); Jayasuriya, M C.N. [International Atomic Energy Agency, Animal Production and Health Section, Vienna (Austria)

1999-06-01

The concept of a new parameter `Purine Nitrogen Index (PNI)` for feed evaluation in ruminants is discussed. PNI refers to the ratio of purine derivative (PD) nitrogen to total nitrogen in urine. It is suggested that PNI can potentially be used as an indicator of the efficiency with which degradable dietary nitrogen is converted to microbial protein in the rumen. The excretion of PD in the urine provides an estimation of the intestinal flow of microbial protein, and therefore, PNI effectively corresponds to the amount of microbial protein produced in the rumen relative to the nitrogen loss in the urine. If a diet or a dietary regime has a high conversion efficiency, proportionally more rumen degradable nitrogen is converted to microbial protein and less nitrogen is excreted in the urine, resulting in a high PNI. Conversely, if a diet has a poor conversion efficiency, proportionally less dietary nitrogen is converted to microbial protein and more is excreted in the urine, resulting in a low PNI. Preliminary data from six experiments involving 34 sheep confirmed a positive correlation between PNI and the nitrogen conversion efficiency, and suggested that a dietary regime with a PNI lower than 0.08 for sheep appeared to be a less efficient in the production of microbial protein and have a greater loss of nitrogen in the urine. PNI can theoretically be determined in spot urine samples, and has the potential to serve as a `dipstick` method for the rapid evaluation of ruminant feeds. However, more research with a mathematical modelling approach is required to evaluate and develop the concept further. (author) 15 refs, 5 figs, 4 tabs

Diversity of Culturable Thermophilic Actinobacteria in Hot Springs in Tengchong, China and Studies of their Biosynthetic Gene Profiles.

Science.gov (United States)

Liu, Lan; Salam, Nimaichand; Jiao, Jian-Yu; Jiang, Hong-Chen; Zhou, En-Min; Yin, Yi-Rui; Ming, Hong; Li, Wen-Jun

2016-07-01

The class Actinobacteria has been a goldmine for the discovery of antibiotics and has attracted interest from both academics and industries. However, an absence of novel approaches during the last few decades has limited the discovery of new microbial natural products useful for industries. Scientists are now focusing on the ecological aspects of diverse environments including unexplored or underexplored habitats and extreme environments in the search for new metabolites. This paper reports on the diversity of culturable actinobacteria associated with hot springs located in Tengchong County, Yunnan Province, southwestern China. A total of 58 thermophilic actinobacterial strains were isolated from the samples collected from ten hot springs distributed over three geothermal fields (e.g., Hehua, Rehai, and Ruidian). Phylogenetic positions and their biosynthetic profiles were analyzed by sequencing 16S rRNA gene and three biosynthetic gene clusters (KS domain of PKS-I, KSα domain of PKS-II and A domain of NRPS). On the basis of 16S rRNA gene phylogenetic analysis, the 58 strains were affiliated with 12 actinobacterial genera: Actinomadura Micromonospora, Microbispora, Micrococcus, Nocardiopsis, Nonomuraea, Promicromonospora, Pseudonocardia, Streptomyces, Thermoactinospora, Thermocatellispora, and Verrucosispora, of which the two novel genera Thermoactinospora and Thermocatellisopora were recently described from among these strains. Considering the biosynthetic potential of these actinobacterial strains, 22 were positive for PCR amplification of at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, and NRPS). These actinobacteria were further subjected to antimicrobial assay against five opportunistic human pathogens (Acinetobacter baumannii, Escherichia coli, Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis). All of the 22 strains that were positive for PCR amplification of at least one of the biosynthetic gene domains exhibited

MiYA, an efficient machine-learning workflow in conjunction with the YeastFab assembly strategy for combinatorial optimization of heterologous metabolic pathways in Saccharomyces cerevisiae.

Science.gov (United States)

Zhou, Yikang; Li, Gang; Dong, Junkai; Xing, Xin-Hui; Dai, Junbiao; Zhang, Chong

2018-05-01

Facing boosting ability to construct combinatorial metabolic pathways, how to search the metabolic sweet spot has become the rate-limiting step. We here reported an efficient Machine-learning workflow in conjunction with YeastFab Assembly strategy (MiYA) for combinatorial optimizing the large biosynthetic genotypic space of heterologous metabolic pathways in Saccharomyces cerevisiae. Using β-carotene biosynthetic pathway as example, we first demonstrated that MiYA has the power to search only a small fraction (2-5%) of combinatorial space to precisely tune the expression level of each gene with a machine-learning algorithm of an artificial neural network (ANN) ensemble to avoid over-fitting problem when dealing with a small number of training samples. We then applied MiYA to improve the biosynthesis of violacein. Feed with initial data from a colorimetric plate-based, pre-screened pool of 24 strains producing violacein, MiYA successfully predicted, and verified experimentally, the existence of a strain that showed a 2.42-fold titer improvement in violacein production among 3125 possible designs. Furthermore, MiYA was able to largely avoid the branch pathway of violacein biosynthesis that makes deoxyviolacein, and produces very pure violacein. Together, MiYA combines the advantages of standardized building blocks and machine learning to accelerate the Design-Build-Test-Learn (DBTL) cycle for combinatorial optimization of metabolic pathways, which could significantly accelerate the development of microbial cell factories. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters

NARCIS (Netherlands)

Cimermancic, P.; Medema, Marnix; Claesen, J.; Kurika, K.; Wieland Brown, L.C.; Mavrommatis, K.; Pati, A.; Godfrey, P.A.; Koehrsen, M.; Clardy, J.; Birren, B. W.; Takano, Eriko; Sali, A.; Linington, R.G.; Fischbach, M.A.

2014-01-01

Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the

Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol

NARCIS (Netherlands)

Cerneus, D. P.; Ueffing, E.; Posthuma, G.; Strous, G. J.; van der Ende, A.

1993-01-01

Alkaline phosphatase is anchored to the outer leaflet of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol anchor. We have studied the biosynthetic transport and endocytosis of alkaline phosphatase in the choriocarcinoma cell line BeWo, which endogenously expresses this

Chromatin landscaping in algae reveals novel regulation pathway for biofuels production

Energy Technology Data Exchange (ETDEWEB)

Ngan, Chew Yee; Wong, Chee-Hong; Choi, Cindy; Pratap, Abhishek; Han, James; Wei, Chia-Lin

2013-02-19

The diminishing reserve of fossil fuels calls for the development of biofuels. Biofuels are produced from renewable resources, including photosynthetic organisms, generating clean energy. Microalgae is one of the potential feedstock for biofuels production. It grows easily even in waste water, and poses no competition to agricultural crops for arable land. However, little is known about the algae lipid biosynthetic regulatory mechanisms. Most studies relied on the homology to other plant model organisms, in particular Arabidopsis or through low coverage expression analysis to identify key enzymes. This limits the discovery of new components in the biosynthetic pathways, particularly the genetic regulators and effort to maximize the production efficiency of algal biofuels. Here we report an unprecedented and de novo approach to dissect the algal lipid pathways through disclosing the temporal regulations of chromatin states during lipid biosynthesis. We have generated genome wide chromatin maps in chlamydomonas genome using ChIP-seq targeting 7 histone modifications and RNA polymerase II in a time-series manner throughout conditions activating lipid biosynthesis. To our surprise, the combinatory profiles of histone codes uncovered new regulatory mechanism in gene expression in algae. Coupled with matched RNA-seq data, chromatin changes revealed potential novel regulators and candidate genes involved in the activation of lipid accumulations. Genetic perturbation on these candidate regulators further demonstrated the potential to manipulate the regulatory cascade for lipid synthesis efficiency. Exploring epigenetic landscape in microalgae shown here provides powerful tools needed in improving biofuel production and new technology platform for renewable energy generation, global carbon management, and environmental survey.

The synthesis of chlorophyll-a biosynthetic precursors and methyl substituted iron porphyrins

International Nuclear Information System (INIS)

Matera, K.M.

1988-01-01

The biosynthetic intermediates were incubated in a plant system. The activity levels calculated show that magnesium 6-acrylate porphyrins and one of the magnesium 6-β-hydroxypropionate porphyrins are not intermediates. In addition, plant systems incubated with 18 O 2 were found to synthesize magnesium 2,4-divinyl pheoporphyrin-a 5 incorporated with 18 O at the 9-carbonyl oxygen. Mass spectroscopy confirmed the presence of the oxygen label, thus eliminating one of two hypothesized pathways to chlorophyll-a. An overall description is given of iron porphyrins and iron porphyrin containing proteins. The function of the propionic side chains of the heme prosthetic group during electron transport reactions will be investigated. The synthesis of a series of iron(III) hexamethyl porphyrins with increasingly longer substituents in the remaining two peripheral positions of the porphyrin is described. Models for NMR studies of iron chlorin containing enzymes are discussed. Iron(III) pyropheophorbide-a and methyl pyropheophorbide-a were synthesized in addition to 5-CD 3 , 10-CD 2 iron(III) pyropheophorbide-a and methyl pyropheophorbide-a. Together, these pyropheophorbides were used to assign NMR resonances and ultimately provide a model for other iron chlorins. The synthesis of nickel(II) anhydro-mesorhodoporphyrin from zinc(III) anhydromesorhodochlorin is described; this nickel porphyrin was used as a standard for ring current calculations of reduced nickel analogs of anhydromesorhodoporphyrin

Exploring metabolic pathway disruption in the subchronic phencyclidine model of schizophrenia with the Generalized Singular Value Decomposition

Directory of Open Access Journals (Sweden)

Morris Brian J

2011-05-01

Encyclopedia of Genes and Genomes (KEGG metabolite pathway database were altered in the PFC of PCP-treated rats. Several significant changes were discovered, notably: 1 neuroactive ligands active at glutamate and GABA receptors are disrupted in the PFC of PCP-treated animals, 2 glutamate dysfunction in these animals was not limited to compromised glutamatergic neurotransmission but also involves the disruption of metabolic pathways linked to glutamate; and 3 a specific series of purine reactions Xanthine ← Hypoxyanthine ↔ Inosine ← IMP → adenylosuccinate is also disrupted in the PFC of PCP-treated animals. Conclusions Network reordering via the GSVD provides a means to discover statistically validated differences in clustering between a pair of networks. In practice this analytical approach, when applied to metabolomic data, allows us to quantify the alterations in metabolic pathways between two experimental groups. With this new computational technique we identified metabolic pathway alterations that are consistent with known results. Furthermore, we discovered disruption in a novel series of purine reactions that may contribute to the PFC dysfunction and cognitive deficits seen in schizophrenia.

Comparison of purine derivatives and creatinine in plasma and urine between local cattle and buffaloes in Vietnam

International Nuclear Information System (INIS)

Vo Thi Kim Thanh; Dao Thi Phuong; Tran Thi Thu Hong; Phung Thi Luu; Ngo Mau Dung; Hoang Quoc Hung; Orskov, E.R.

2004-01-01

In Experiment I, 4 female swamp buffaloes and 4 local cattle fed with the diet based on young maize and rice straw (80/20), and in experiment II, the same number and types of animals as in Experiment I were fed with the diet based on rice straw and rice bran (70/30). The animals were fed twice a day with the diets at 40, 60, 80, 95% of ad libitum intake. The digestibility of nutrients and N excretion were similar for cattle and buffaloes. The purine derivative:creatinine ratio (PDC) index was significantly affected by the level of feed intake (P < 0.001) in both the species. Large differences in urinary purine derivative (PD) excretion were observed, being much lower in buffaloes than in cattle. The regression analysis showed that urinary PD excretion rate per kg of digestible organic matter intake for cattle was higher than that for buffalo. The PDC index also followed the same pattern. The nitrogen retention increased with the supply of energy, both in cattle and buffaloes, indicating that the protein supply was similar. There was no consistent effect of time of day on spot sampling in buffalo and cattle. Buffaloes urinated less frequently than cattle, so sampling time was not really relevant. (author)

Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk.

Science.gov (United States)

Ishikawa, Toshihisa; Aw, Wanping; Kaneko, Kiyoko

2013-11-04

In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid) in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs), i.e., 421C>A (major) and 376C>T (minor), in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

New insights into the organization and regulation of trichothecene biosynthetic genes in Trichoderma

Science.gov (United States)

Collectively, species of the genus Trichoderma can produce numerous structurally diverse secondary metabolites (SM). This ability is conferred by the presence of SM biosynthetic gene clusters in their genomes. Species of Trichoderma in the Brevicompactum clade are able to produce trichothecenes, a f...

Roles of the Amino Group of Purine Bases in the Thermodynamic Stability of DNA Base Pairing

Directory of Open Access Journals (Sweden)

Shu-ichi Nakano

2014-08-01

Full Text Available The energetic aspects of hydrogen-bonded base-pair interactions are important for the design of functional nucleotide analogs and for practical applications of oligonucleotides. The present study investigated the contribution of the 2-amino group of DNA purine bases to the thermodynamic stability of oligonucleotide duplexes under different salt and solvent conditions, using 2'-deoxyriboinosine (I and 2'-deoxyribo-2,6-diaminopurine (D as non-canonical nucleotides. The stability of DNA duplexes was changed by substitution of a single base pair in the following order: G•C > D•T ≈ I•C > A•T > G•T > I•T. The apparent stabilization energy due to the presence of the 2-amino group of G and D varied depending on the salt concentration, and decreased in the water-ethanol mixed solvent. The effects of salt concentration on the thermodynamics of DNA duplexes were found to be partially sequence-dependent, and the 2-amino group of the purine bases might have an influence on the binding of ions to DNA through the formation of a stable base-paired structure. Our results also showed that physiological salt conditions were energetically favorable for complementary base recognition, and conversely, low salt concentration media and ethanol-containing solvents were effective for low stringency oligonucleotide hybridization, in the context of conditions employed in this study.

Use of [75Se]selenomethionine in immunoglobulin biosynthetic studies

International Nuclear Information System (INIS)

Gutman, G.A.; Warner, N.L.; Harris, A.W.; Bowles, A.

1978-01-01

The gamma-emitting amino acid analog, [ 75 Se] selenomethionine, has been used as a biosynthetic label for immunoglobulins secreted by plasmacytomas in tissue culture. The secreted products are structurally intact with respect to their antibody combining sites and their class and allotype antigenic specificities. A component of [ 75 Se] selenomethionine preparations was found to bind to fetal calf serum proteins, in a manner releasable by mercaptoethanol, but not by sodium dodecyl sulfate and urea. Methods for circumventing the problems caused by this binding are described. (Auth.)

Infusion of Sibling Marrow in a Patient with Purine Nucleoside Phosphorylase Deficiency Leads to Split Mixed Donor Chimerism and Normal Immunity

Directory of Open Access Journals (Sweden)

Laura Yeates

2017-06-01

Full Text Available Purine nucleoside phosphorylase (PNP deficiency, a rare autosomal recessive metabolic disease causes combined immunodeficiency and developmental delay, hypotonia, and spasticity. Patients present with recurrent infections associated with T-lymphocytopenia, characteristically presenting later than patients with classical severe combined immunodeficiency (SCID. PNP, with adenosine deaminase (ADA, is part of the purine salvage pathway. The only curative therapy is hematopoietic stem cell transplantation. Myeloablative conditioning is recommended to prevent rejection caused by residual immune function. However, HLA-identical sibling stem cell infusions in ADA-SCID result in some donor stem cell engraftment and long-term thymopoiesis. We report a patient with PNP deficiency, who received HLA-identical sibling marrow without chemotherapy because of disseminated cytomegalovirus (CMV infection. The patient presented at 14 months of age following recurrent infections, from early infancy, with persistent irritability, developmental delay, and hypotonia. She had neutropenia, pan-lymphocytopenia, and hypogammaglobulinemia with low plasma urate and erythrocyte PNP activity. Diagnosis was confirmed with a homozygous mutation in PNP. The patient was viremic with CMV detected in blood and CSF by PCR. Dual antiviral therapy improved the clinical condition and reduced the viral load. In view of the disseminated CMV infection, the decision was made to infuse stem cells without any pre-conditioning chemotherapy. She received a matched sibling donor unconditioned stem cell infusion at 16 months of age. The post-transplant course was uneventful. Blood PCR became negative for CMV. Global hypotonia persisted, although with significant improvement in irritability. At 4 years of age and 29 months post-transplant, the patient demonstrated normal T-lymphocyte and natural killer cell numbers. Recent thymic emigrants represented 12% of the total T

The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

Science.gov (United States)

Brabletz, T; Pietrowski, I; Serfling, E

1991-01-11

Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism.

Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family.

Science.gov (United States)

Pearce, Stephen; Huttly, Alison K; Prosser, Ian M; Li, Yi-dan; Vaughan, Simon P; Gallova, Barbora; Patil, Archana; Coghill, Jane A; Dubcovsky, Jorge; Hedden, Peter; Phillips, Andrew L

2015-06-05

The gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis. The wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp. The comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD

Redox Impact on Starch Biosynthetic Enzymes in Arabidopsis thaliana

DEFF Research Database (Denmark)

Skryhan, Katsiaryna

Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism are coordina......Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism...... are coordinated by the redox state of the cell via post-translational modification of the starch metabolic enzymes containing redox active cysteine residues and these cysteine residues became cross-linked upon oxidation providing a conformational change leading to activity loss; 2) cysteine residues...... of chloroplast enzymes can play a role not only in enzyme activity and redox sensitivity but also in protein folding and stability upon oxidation. Several redox sensitive enzymes identified in this study can serve as potential targets to control the carbon flux to and from starch during the day and night...

Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

Directory of Open Access Journals (Sweden)

Ximena Escalera-Fanjul

2017-06-01

Full Text Available Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s. Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64

The Purine Bias of Coding Sequences is Determined by Physicochemical Constraints on Proteins.

Science.gov (United States)

Ponce de Leon, Miguel; de Miranda, Antonio Basilio; Alvarez-Valin, Fernando; Carels, Nicolas

2014-01-01

For this report, we analyzed protein secondary structures in relation to the statistics of three nucleotide codon positions. The purpose of this investigation was to find which properties of the ribosome, tRNA or protein level, could explain the purine bias (Rrr) as it is observed in coding DNA. We found that the Rrr pattern is the consequence of a regularity (the codon structure) resulting from physicochemical constraints on proteins and thermodynamic constraints on ribosomal machinery. The physicochemical constraints on proteins mainly come from the hydropathy and molecular weight (MW) of secondary structures as well as the energy cost of amino acid synthesis. These constraints appear through a network of statistical correlations, such as (i) the cost of amino acid synthesis, which is in favor of a higher level of guanine in the first codon position, (ii) the constructive contribution of hydropathy alternation in proteins, (iii) the spatial organization of secondary structure in proteins according to solvent accessibility, (iv) the spatial organization of secondary structure according to amino acid hydropathy, (v) the statistical correlation of MW with protein secondary structures and their overall hydropathy, (vi) the statistical correlation of thymine in the second codon position with hydropathy and the energy cost of amino acid synthesis, and (vii) the statistical correlation of adenine in the second codon position with amino acid complexity and the MW of secondary protein structures. Amino acid physicochemical properties and functional constraints on proteins constitute a code that is translated into a purine bias within the coding DNA via tRNAs. In that sense, the Rrr pattern within coding DNA is the effect of information transfer on nucleotide composition from protein to DNA by selection according to the codon positions. Thus, coding DNA structure and ribosomal machinery co-evolved to minimize the energy cost of protein coding given the functional

A pilot study on genetic variation in purine-rich elements in the nephrin gene promoter in type 2 diabetic patients

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RODRIGO GONZÁLEZ

2007-01-01

Full Text Available Diabetic nephropathy (DN is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1 promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls

Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties.

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Hoang, Van L T; Innes, David J; Shaw, P Nicholas; Monteith, Gregory R; Gidley, Michael J; Dietzgen, Ralf G

2015-07-30

Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and

A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae.

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Ren, Hengqian; Hu, Pingfan; Zhao, Huimin

2017-08-01

Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization, and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Here we report a plug-and-play pathway refactoring workflow for high-throughput, flexible pathway construction, and expression in both Escherichia coli and Saccharomyces cerevisiae. Biosynthetic genes were firstly cloned into pre-assembled helper plasmids with promoters and terminators, resulting in a series of expression cassettes. These expression cassettes were further assembled using Golden Gate reaction to generate fully refactored pathways. The inclusion of spacer plasmids in this system would not only increase the flexibility for refactoring pathways with different number of genes, but also facilitate gene deletion and replacement. As proof of concept, a total of 96 pathways for combinatorial carotenoid biosynthesis were built successfully. This workflow should be generally applicable to different classes of natural products produced by various organisms. Biotechnol. Bioeng. 2017;114: 1847-1854. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Interpretation of substituent effects on 13C and 15N NMR chemical shifts in 6-substituted purines

Czech Academy of Sciences Publication Activity Database

Standara, Stanislav; Bouzková, K.; Straka, Michal; Zacharová, Z.; Hocek, Michal; Marek, J.; Marek, R.

2011-01-01

Roč. 13, č. 35 (2011), s. 15854-15864 ISSN 1463-9076 R&D Projects: GA ČR GA203/09/2037 Grant - others:CEITEC(XE) CZ.1.05/1.1.00/02.0068; 7th European Community Framework (XE) 230955; GA MŠk(CZ) LC06030 Program:LC Institutional research plan: CEZ:AV0Z40550506 Keywords : purine * nuclear magnetic shielding * localized molecular orbitals * conformational dependence Subject RIV: CC - Organic Chemistry Impact factor: 3.573, year: 2011

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

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Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

2014-09-01

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

An LL-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants.

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Hudson, André O; Singh, Bijay K; Leustek, Thomas; Gilvarg, Charles

2006-01-01

Although lysine (Lys) biosynthesis in plants is known to occur by way of a pathway that utilizes diaminopimelic acid (DAP) as a central intermediate, the available evidence suggests that none of the known DAP-pathway variants found in nature occur in plants. A new Lys biosynthesis pathway has been identified in Arabidopsis (Arabidopsis thaliana) that utilizes a novel transaminase that specifically catalyzes the interconversion of tetrahydrodipicolinate and LL-diaminopimelate, a reaction requiring three enzymes in the DAP-pathway variant found in Escherichia coli. The LL-DAP aminotransferase encoded by locus At4g33680 was able to complement the dapD and dapE mutants of E. coli. This result, in conjunction with the kinetic properties and substrate specificity of the enzyme, indicated that LL-DAP aminotransferase functions in the Lys biosynthetic direction under in vivo conditions. Orthologs of At4g33680 were identified in all the cyanobacterial species whose genomes have been sequenced. The Synechocystis sp. ortholog encoded by locus sll0480 showed the same functional properties as At4g33680. These results demonstrate that the Lys biosynthesis pathway in plants and cyanobacteria is distinct from the pathways that have so far been defined in microorganisms.

Accumulation of Kaempferitrin and Expression of Phenyl-Propanoid Biosynthetic Genes in Kenaf (Hibiscus cannabinus

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Shicheng Zhao

2014-10-01

Full Text Available Kenaf (Hibiscus cannabinus is cultivated worldwide for its fiber; however, the medicinal properties of this plant are currently attracting increasing attention. In this study, we investigated the expression levels of genes involved in the biosynthesis of kaempferitrin, a compound with many biological functions, in different kenaf organs. We found that phenylalanine ammonia lyase (HcPAL was more highly expressed in stems than in other organs. Expression levels of cinnamate 4-hydroxylase (HcC4H and 4-coumarate-CoA ligase (Hc4CL were highest in mature leaves, followed by stems and young leaves, and lowest in roots and mature flowers. The expression of chalcone synthase (HcCHS, chalcone isomerase (HcCHI, and flavone 3-hydroxylase (HcF3H was highest in young flowers, whereas that of flavone synthase (HcFLS was highest in leaves. An analysis of kaempferitrin accumulation in the different organs of kenaf revealed that the accumulation of this compound was considerably higher (>10-fold in leaves than in other organs. On the basis of a comparison of kaempferitrin contents with the expression levels of different genes in different organs, we speculate that HcFLS plays an important regulatory role in the kaempferitrin biosynthetic pathway in kenaf.

Accumulation of kaempferitrin and expression of phenyl-propanoid biosynthetic genes in kenaf (Hibiscus cannabinus).

Science.gov (United States)

Zhao, Shicheng; Li, Xiaohua; Cho, Dong Ha; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Sang Un

2014-10-23

Kenaf (Hibiscus cannabinus) is cultivated worldwide for its fiber; however, the medicinal properties of this plant are currently attracting increasing attention. In this study, we investigated the expression levels of genes involved in the biosynthesis of kaempferitrin, a compound with many biological functions, in different kenaf organs. We found that phenylalanine ammonia lyase (HcPAL) was more highly expressed in stems than in other organs. Expression levels of cinnamate 4-hydroxylase (HcC4H) and 4-coumarate-CoA ligase (Hc4CL) were highest in mature leaves, followed by stems and young leaves, and lowest in roots and mature flowers. The expression of chalcone synthase (HcCHS), chalcone isomerase (HcCHI), and flavone 3-hydroxylase (HcF3H) was highest in young flowers, whereas that of flavone synthase (HcFLS) was highest in leaves. An analysis of kaempferitrin accumulation in the different organs of kenaf revealed that the accumulation of this compound was considerably higher (>10-fold) in leaves than in other organs. On the basis of a comparison of kaempferitrin contents with the expression levels of different genes in different organs, we speculate that HcFLS plays an important regulatory role in the kaempferitrin biosynthetic pathway in kenaf.

Metabolic and transcriptional elucidation of the carotenoid biosynthesis pathway in peel and flesh tissue of loquat fruit during on-tree development.

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Hadjipieri, Margarita; Georgiadou, Egli C; Marin, Alicia; Diaz-Mula, Huertas M; Goulas, Vlasios; Fotopoulos, Vasileios; Tomás-Barberán, Francisco A; Manganaris, George A

2017-06-14

Carotenoids are the main colouring substances found in orange-fleshed loquat fruits. The aim of this study was to unravel the carotenoid biosynthetic pathway of loquat fruit (cv. 'Obusa') in peel and flesh tissue during distinct on-tree developmental stages through a targeted analytical and molecular approach. Substantial changes regarding colour parameters, both between peel and flesh and among the different developmental stages, were monitored, concomitant with a significant increment in carotenoid content. Key genes and individual compounds that are implicated in the carotenoid biosynthetic pathway were further dissected with the employment of molecular (RT-qPCR) and advanced analytical techniques (LC-MS). Results revealed significant differences in carotenoid composition between peel and flesh. Thirty-two carotenoids were found in the peel, while only eighteen carotenoids were identified in the flesh. Trans-lutein and trans-β-carotene were the major carotenoids in the peel; the content of the former decreased with the progress of ripening, while the latter registered a 7.2-fold increase. However, carotenoid profiling of loquat flesh indicated trans-β-cryptoxanthin, followed by trans-β-carotene and 5,8-epoxy-β-carotene to be the most predominant carotenoids. High amounts of trans-β-carotene in both tissues were supported by significant induction in a chromoplast-specific lycopene β-cyclase (CYCB) transcript levels. PSY1, ZDS, CYCB and BCH were up-regulated and CRTISO, LCYE, ECH and VDE were down-regulated in most of the developmental stages compared with the immature stage in both peel and flesh tissue. Overall, differential regulation of expression levels with the progress of on-tree fruit development was more evident in the middle and downstream genes of carotenoid biosynthetic pathway. Carotenoid composition is greatly affected during on-tree loquat development with striking differences between peel and flesh tissue. A link between gene up- or down

IMG-ABC: An Atlas of Biosynthetic Gene Clusters to Fuel the Discovery of Novel Secondary Metabolites

Energy Technology Data Exchange (ETDEWEB)

Chen, I-Min; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Huang, Jinghua; Reddy, T. B.K.; Cimermancic, Peter; Fischbach, Michael; Ivanova, Natalia; Markowitz, Victor; Kyrpides, Nikos; Pati, Amrita

2014-10-28

In the discovery of secondary metabolites (SMs), large-scale analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of relevant computational resources. We present IMG-ABC (https://img.jgi.doe.gov/abc/) -- An Atlas of Biosynthetic gene Clusters within the Integrated Microbial Genomes (IMG) system1. IMG-ABC is a rich repository of both validated and predicted biosynthetic clusters (BCs) in cultured isolates, single-cells and metagenomes linked with the SM chemicals they produce and enhanced with focused analysis tools within IMG. The underlying scalable framework enables traversal of phylogenetic dark matter and chemical structure space -- serving as a doorway to a new era in the discovery of novel molecules.

Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens

DEFF Research Database (Denmark)

Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin

2014-01-01

Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as stre...

Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

NARCIS (Netherlands)

Elfahmi, [No Value

2006-01-01

In this thesis phytochemical and biosynthetic studies of lignans are described. The focus is on the Indonesian medicinal plants Phyllanthus niruri and Piper cubeba and on two Linum species, Linum flavum and L. leonii, native to European countries. Both Indonesian plants are used in jamu. Jamu is the

Viral exploitation of the MEK/ERK pathway - A tale of vaccinia virus and other viruses.

Science.gov (United States)

Bonjardim, Cláudio A

2017-07-01

The VACV replication cycle is remarkable in the sense that it is performed entirely in the cytoplasmic compartment of vertebrate cells, due to its capability to encode enzymes required either for regulating the macromolecular precursor pool or the biosynthetic processes. Although remarkable, this gene repertoire is not sufficient to confer the status of a free-living microorganism to the virus, and, consequently, the virus relies heavily on the host to successfully generate its progeny. During the complex virus-host interaction, viruses must deal not only with the host pathways to accomplish their temporal demands but also with pathways that counteract viral infection, including the inflammatory, innate and acquired immune responses. This review focuses on VACV and other DNA or RNA viruses that stimulate the MEK (MAPK - Mitogen Activated Protein Kinase)/ERK- Extracellular signal-Regulated Kinase) pathway as part of their replication cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

Wobble↔Watson-Crick tautomeric transitions in the homo-purine DNA mismatches: a key to the intimate mechanisms of the spontaneous transversions.

Science.gov (United States)

Brovarets', Ol'ha O; Hovorun, Dmytro M

2015-01-01

The intrinsic capability of the homo-purine DNA base mispairs to perform wobble↔Watson-Crick/Topal-Fresco tautomeric transitions via the sequential intrapair double proton transfer was discovered for the first time using QM (MP2/DFT) and QTAIM methodologies that are crucial for understanding the microstructural mechanisms of the spontaneous transversions.

Improved L-ornithine production in Corynebacterium crenatum by introducing an artificial linear transacetylation pathway.

Science.gov (United States)

Shu, Qunfeng; Xu, Meijuan; Li, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong; Rao, Zhiming

2018-05-04

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis.

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

Science.gov (United States)

Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

2009-03-04

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

A new method for the construction of a mutant library with a predictable occurrence rate using Poisson distribution.

Science.gov (United States)

Seong, Ki Moon; Park, Hweon; Kim, Seong Jung; Ha, Hyo Nam; Lee, Jae Yung; Kim, Joon

2007-06-01

A yeast transcriptional activator, Gcn4p, induces the expression of genes that are involved in amino acid and purine biosynthetic pathways under amino acid starvation. Gcn4p has an acidic activation domain in the central region and a bZIP domain in the C-terminus that is divided into the DNA-binding motif and dimerization leucine zipper motif. In order to identify amino acids in the DNA-binding motif of Gcn4p which are involved in transcriptional activation, we constructed mutant libraries in the DNA-binding motif through an innovative application of random mutagenesis. Mutant library made by oligonucleotides which were mutated randomly using the Poisson distribution showed that the actual mutation frequency was in good agreement with expected values. This method could save the time and effort to create a mutant library with a predictable mutation frequency. Based on the studies using the mutant libraries constructed by the new method, the specific residues of the DNA-binding domain in Gcn4p appear to be involved in the transcriptional activities on a conserved binding site.

Listeria monocytogenes differential transcriptome analysis reveals temperature-dependent Agr regulation and suggests overlaps with other regulons.

Science.gov (United States)

Garmyn, Dominique; Augagneur, Yoann; Gal, Laurent; Vivant, Anne-Laure; Piveteau, Pascal

2012-01-01

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its ΔagrA mutant at the saprophytic temperature of 25°C and in vivo temperature of 37°C. Variations of transcriptome were higher at 37°C than at 25°C. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and pyrimidine biosynthetic pathways and phage-related functions. Deregulations resulted in a growth advantage at 37°C, but affected salt tolerance. Finally, our results suggest overlaps with PrfA, σB, σH and CodY regulons. These overlaps may suggest that through AgrA, Listeria monocytogenes integrates information on its biotic environment.

Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances

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Anna Ohradanova-Repic

2018-04-01

Full Text Available If misregulated, macrophage (Mϕ–T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA. We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF- and Mϕ colony-stimulating factor (M-CSF-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ, adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX formulation to the immunosuppressive FRβ+CD39+CD73+ Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways.

Science.gov (United States)

Wingler, Laura M; Cornish, Virginia W

2011-09-13

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk

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Kiyoko Kaneko

2013-11-01

Full Text Available In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs, i.e., 421C>A (major and 376C>T (minor, in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

Synthesis of C-Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies-Based Biosynthetic Pathway

DEFF Research Database (Denmark)

Andersen-Ranberg, Johan; Kongstad, Kenneth Thermann; Nafisi, Majse

2017-01-01

unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway...

Ketol-acid reductoisomerase enzymes and methods of use

Science.gov (United States)

Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

2015-10-27

Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

The oxalic acid biosynthetic activity of Burkholderia mallei is encoded by a single locus

Science.gov (United States)

Although it is known that oxalic acid provides a selective advantage to the secreting microbe, our understanding of how this acid is biosynthesized remains incomplete. This study reports the identification, cloning, and partial characterization of the oxalic acid biosynthetic enzyme from the animal ...

An Integrated Metabolomic and Genomic Mining Workflow to Uncover the Biosynthetic Potential of Bacteria

DEFF Research Database (Denmark)

Månsson, Maria; Vynne, Nikolaj Grønnegaard; Klitgaard, Andreas

2016-01-01

Microorganisms are a rich source of bioactives; however, chemical identification is a major bottleneck. Strategies that can prioritize the most prolific microbial strains and novel compounds are of great interest. Here, we present an integrated approach to evaluate the biosynthetic richness in ba...

The PurR regulon in Lactococcus lactis – transcriptional regulation of the purine nucleotide metabolism and translational machinery

DEFF Research Database (Denmark)

Jendresen, Christian Bille; Martinussen, Jan; Kilstrup, Mogens

2012-01-01

Purine nucleotides are either synthesized de novo from 5-phosphoribosyl-1-pyrophosphate (PRPP) or salvaged from the environment. In Lactococcus lactis, transcription of the de novo synthesis operons, purCSQLF and purDEK, has genetically been shown to be activated by the PurR protein when bound to......-related functions. Of special interest is the presence of PurBox motifs in rrn promoters, suggesting a novel connection between nucleotide availability and the translational machinery....

Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

Directory of Open Access Journals (Sweden)

Louw Abraham I

2010-04-01

Full Text Available Abstract Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

Science.gov (United States)

Rabatel, Andréane; Febvay, Gérard; Gaget, Karen; Duport, Gabrielle; Baa-Puyoulet, Patrice; Sapountzis, Panagiotis; Bendridi, Nadia; Rey, Marjolaine; Rahbé, Yvan; Charles, Hubert; Calevro, Federica; Colella, Stefano

2013-04-10

Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch

Señales purinérgicas Purinergic signals

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Eduardo R Lazarowski

2009-04-01

Full Text Available En la última década se ha aportado clara evidencia de que tanto nucleósidos como nucleótidos de adenina y uridina pueden funcionar como factores de señalización extracelular. Su acción es mediada por dos tipos principales de receptores de superficie denominados purinérgicos. Los receptores P1 se activan por adenosina, y son todos metabotrópicos, mientras que los receptores de nucleótidos (ATP, ADP, UTP y UDP y nucleótidos-azúcares (UDP-glucosa y UDP-galactosa pueden ser metabotrópicos (P2Y o ionotrópicos (P2X. La importancia y complejidad de este sistema de señalización se evidencia por la diversidad de mecanismos de liberación de nucleótidos al medio extracelular y por la distribución ubicua de varios grupos de ectonucleotidasas capaces de catalizar la degradación y conversión de nucleótidos. Hasta el momento se han descrito y clonado una veintena de estos receptores que modulan una variedad de respuestas, como el impulso nervioso, la respuesta inflamatoria, la secreción de insulina, la regulación del tono vascular y la percepción del dolor. En la presente revisión se describen las características estructurales y farmacológicas de los receptores purinérgicos y se analiza la interacción dinámica entre estos receptores, los nucleósidos y nucleótidos, y las ectonucleotidasas, con especial atención a la dinámica de la agregación plaquetaria, la respuesta inmune y la hidratación de las mucosas respiratorias.In the last decade evidence accumulated that nucleosides and nucleotides of both uridine and adenine can act as extracellular signaling factors. Their action is mediated by two main types of surface receptors commonly known as purinergic. P1 receptors are metabotropic and activated by adenosine, whereas receptors for nucleotides (ATP, ADP, UTP and UDP and nucleotide-sugars (UDP-glucose and UDP-galactose can be either metabotropic (P2Y or ionotropic (P2X. The importance and complexity of this signaling system

Biosynthetic graft failure to replace infected infrainguinal bypass as developing infection due to Morganella morganii leading to disrupture of the anastomosis. Case report

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Gladiol Zenunaj

Full Text Available Introduction: Biosynthetic prosthesis has become the trend to carry out arterial reconstruction in infected sites since considered to be resistant to infection. Late graft occlusion is the only complication reported in literature so far. We report a case of biosynthetic graft infection which led to early detachment of the femoral anastomosis of a femoral-popliteal above-knee bypass. Material: A 76-year-old man developed groin infection 3 months later after performing an ePTFE femoral-popliteal above-knee bypass for critical limb ischemia. He was re-admitted for groin infection involving the vascular structures. Explantation of the existing bypass and its replacement with a biosynthetic graft (omniflow II was performed. Detachment of the proximal anastomosis occurred 6 days later leading to groin haematoma. Consequently, retroperitoneal access was performed for clamping the external iliac artery so as to control haemorrhage followed by explantation of the biosynthetic graft. An external iliac-popliteal above-knee bypass was tailored in order to save the limb and it was performed using a transobturator approach avoiding the infected site. In both cases bacterial cultures resulted positive for Morganella Morganii. The groin wound was treated separately with negative pressure medication healing definitively within 20 days and after 3-month follow-up the bypass was still patent. Conclusion: This is the first report of biosynthetic graft infection used for infrainguinal reconstruction leading to haemorrhage due to anastomosis disrupture. Using an extra-anatomical access for providing blood inflow to the leg avoiding the infected site and treating safely the groin wound with VAC therapy revealed to be a valid approach. Keywords: Infrainguinal bypass, Graft infection, Biosynthetic material, Graft occlusion, Negative pressure medication, Morganella morgani

Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis.

Science.gov (United States)

Wang, Yaya; Deng, Zixin; Qu, Xudong

2014-01-01

Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5'-fluoro-5'-deoxyadenosine (5'-FDA) from inorganic fluoride and S-adenosyl-l-methionine (SAM). The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of the fl pathway. Kinetic parameters for fluoride ( K m 4153 μM, k cat 0.073 min (-1)) and SAM ( K m 416 μM, k cat 0.139 min (-1)) have been determined, revealing that NobA is slightly (2.3 fold) slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity.

De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-alzheimer's properties.

Science.gov (United States)

Guo, Qianqian; Ma, Xiaojun; Wei, Shugen; Qiu, Deyou; Wilson, Iain W; Wu, Peng; Tang, Qi; Liu, Lijun; Dong, Shoukun; Zu, Wei

2014-08-12

The major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid β protein (a pathological marker of Alzheimer's disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown. In this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis. A combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria, and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.

Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology.

Science.gov (United States)

Pandey, Ramesh Prasad; Parajuli, Prakash; Koffas, Mattheos A G; Sohng, Jae Kyung

2016-01-01

In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described. Copyright © 2016 Elsevier Inc. All rights reserved.

Use of (/sup 75/Se)selenomethionine in immunoglobulin biosynthetic studies

Energy Technology Data Exchange (ETDEWEB)

Gutman, G A; Warner, N L; Harris, A W; Bowles, A [Walter and Elisa Hall Institute of Medical Research, Victoria (Australia). Genetics Unit; Royal Melbourne Hospital, Victoria (Australia))

1978-05-01

The gamma-emitting amino acid analog, (/sup 75/Se) selenomethionine, has been used as a biosynthetic label for immunoglobulins secreted by plasmacytomas in tissue culture. The secreted products are structurally intact with respect to their antibody combining sites and their class and allotype antigenic specificities. A component of (/sup 75/Se) selenomethionine preparations was found to bind to fetal calf serum proteins, in a manner releasable by mercaptoethanol, but not by sodium dodecyl sulfate and urea. Methods for circumventing the problems caused by this binding are described.

Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

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Tozeren Aydin

2006-11-01

Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

Production of manganese peroxidase by white rot fungi from potato ...

African Journals Online (AJOL)

phenylalanine and L-tyrosine blocked the MnP biosynthetic pathway. Ammonium ion released from the L-phenylalanine by the L-phenylalanine ammonia-lyase participated in the repression of the MnP biosynthetic pathway of the strain L-25.

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI.

Science.gov (United States)

Wang, Cheng; Zeng, Jian; Li, Yin; Hu, Wei; Chen, Ling; Miao, Yingjie; Deng, Pengyi; Yuan, Cuihong; Ma, Cheng; Chen, Xi; Zang, Mingli; Wang, Qiong; Li, Kexiu; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2014-06-01

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 μg g(-1) of seed dry weight, a β-carotene increase of 65-fold to 3.21 μg g(-1) of seed dry weight, and a provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold to 3.82 μg g(-1) of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Expression of Terpenoid Biosynthetic Genes and Accumulation of Chemical Constituents in Valeriana fauriei

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Yun Ji Park

2016-05-01

Full Text Available Valeriana fauriei (V. fauriei, which emits a characteristic and unpleasant odor, is important in traditional medicine. In this study, the expression of terpenoid biosynthetic genes was investigated in different organs that were also screened for volatile compounds including valerenic acid and its derivatives. Specific expression patterns from different parts of V. fauriei were observed using quantitative real-time PCR (qRT-PCR. The highest transcript levels of biosynthetic genes involved in mevalonic acid (MVA and methylerythritol phosphate (MEP production were found in the stem. Although the amounts of volatile compounds were varied by organ, most of the volatile terpenoids were accumulated in the root. Gas chromatography mass spectrometry (GC-MS analysis identified 128 volatile compounds, which represented 65.33% to 95.66% of total volatiles. Certain compounds were only found in specific organs. For example, isovalerenic acid and valerenic acid and its derivatives were restricted to the root. Organs with high transcript levels did not necessarily have high levels of the corresponding chemical constituents. According to these results, we hypothesize that translocation may occur between different organs in V. fauriei.

Modification of Purine and Pyrimidine Nucleosides by Direct C-H Bond Activation

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Yong Liang

2015-03-01

Full Text Available Transition metal-catalyzed modifications of the activated heterocyclic bases of nucleosides as well as DNA or RNA fragments employing traditional cross-coupling methods have been well-established in nucleic acid chemistry. This review covers advances in the area of cross-coupling reactions in which nucleosides are functionalized via direct activation of the C8-H bond in purine and the C5-H or C6-H bond in uracil bases. The review focuses on Pd/Cu-catalyzed couplings between unactivated nucleoside bases with aryl halides. It also discusses cross-dehydrogenative arylations and alkenylations as well as other reactions used for modification of nucleoside bases that avoid the use of organometallic precursors and involve direct C-H bond activation in at least one substrate. The scope and efficiency of these coupling reactions along with some mechanistic considerations are discussed.

Flux through the tetrahydrodipicolinate succinylase pathway is dispensable for L-lysine production in Corynebacterium glutamicum.

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Shaw-Reid, C A; McCormick, M M; Sinskey, A J; Stephanopoulos, G

1999-03-01

The N-succinyl-LL-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the L-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. In addition, the dapE- strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE- strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions.

Proteomic analysis of the signaling pathway mediated by the heterotrimeric Gα protein Pga1 of Penicillium chrysogenum.

Science.gov (United States)

Carrasco-Navarro, Ulises; Vera-Estrella, Rosario; Barkla, Bronwyn J; Zúñiga-León, Eduardo; Reyes-Vivas, Horacio; Fernández, Francisco J; Fierro, Francisco

2016-10-06

The heterotrimeric Gα protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways. Penicillium chrysogenum mutants with different levels of activity of the Pga1-mediated signaling pathway were used to perform comparative proteomic analyses by 2D-DIGE and LC-MS/MS. Thirty proteins were identified which showed differences in abundance dependent on Pga1 activity level. By modifying the intracellular levels of cAMP we could establish cAMP-dependent and cAMP-independent pathways in Pga1-mediated signaling. Pga1 was shown to regulate abundance of enzymes in primary metabolic pathways involved in ATP, NADPH and cysteine biosynthesis, compounds that are needed for high levels of penicillin production. An in vivo phosphorylated protein containing a pleckstrin homology domain was identified; this protein is a candidate for signal transduction activity. Proteins with possible roles in purine metabolism, protein folding, stress response and morphogenesis were also identified whose abundance was regulated by Pga1 signaling. Thirty proteins whose abundance was regulated by the Pga1-mediated signaling pathway were identified. These proteins are involved in primary metabolism, stress response, development and signal transduction. A model describing the pathways through which Pga1 signaling regulates different cellular processes is proposed.

Role of Heme and Heme-Proteins in Trypanosomatid Essential Metabolic Pathways

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Karina E. J. Tripodi

2011-01-01

Full Text Available Around the world, trypanosomatids are known for being etiological agents of several highly disabling and often fatal diseases like Chagas disease (Trypanosoma cruzi, leishmaniasis (Leishmania spp., and African trypanosomiasis (Trypanosoma brucei. Throughout their life cycle, they must cope with diverse environmental conditions, and the mechanisms involved in these processes are crucial for their survival. In this review, we describe the role of heme in several essential metabolic pathways of these protozoans. Notwithstanding trypanosomatids lack of the complete heme biosynthetic pathway, we focus our discussion in the metabolic role played for important heme-proteins, like cytochromes. Although several genes for different types of cytochromes, involved in mitochondrial respiration, polyunsaturated fatty acid metabolism, and sterol biosynthesis, are annotated at the Tritryp Genome Project, the encoded proteins have not yet been deeply studied. We pointed our attention into relevant aspects of these protein functions that are amenable to be considered for rational design of trypanocidal agents.

Biosynthetic pathways to delta-aminolevulinic acid induced by blue light in the pigment mutant C-2A' of Scenedesmus obliquus

International Nuclear Information System (INIS)

Klein, O.; Senger, H.

1978-01-01

The X-ray induced mutant C-2A' of Scenedesmus obliquus grows heterotrophically but forms only traces of chlorophyll in the dark. Upon illumination, delta-aminolevulinic acid (ALA) is synthesized and chlorophyll is formed. These processes are blue light dependent and ceased immediately when the cells were transferred back into darkness. Addition of levulinic acid (LA) inhibited the light-dependent formation of chlorophyll and caused accumulation of ALA by competitive inhibition of the ALA dehydratase (EC. 4.2.1.24). By feeding specifically labelled 14 C precursors to the pigment mutant, inhibiting the ALA dehydratase with LA, accumulating, extracting and analyzing the ALA, two pathways leading towards ALA could be established: glycine and succinyl CoA can be condensed to ALA and the 5 carbon skeleton of glutamate can completely be incorporated into ALA via a second pathway. The glycine-succinyl CoA pathway dominated over the glutamate pathway, but both led to chlorophyll formation. (author)

Effects of chicory inulin on serum metabolites of uric acid, lipids, glucose, and abdominal fat deposition in quails induced by purine-rich diets.

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Lin, Zhijian; Zhang, Bing; Liu, Xiaoqing; Jin, Rui; Zhu, Wenjing

2014-11-01

Inulin, a group of dietary fibers, is reported to improve the metabolic disorders. In the present study, we investigated the effects of chicory inulin on serum metabolites of uric acid (UA), lipids, glucose, and abdominal fat deposition in quail model induced by a purine-rich diet. In this study, 60 male French quails were randomly allocated to five groups: CON (control group), MOD (model group), BEN (benzbromarone-treated group), CHI-H (high-dosage chicory inulin-treated group), and CHI-L (low-dosage chicory inulin-treated group). The serum UA level was significantly increased in the model group from days 7 to 28, as well as triglyceride (TG) and free fatty acid (FFA) increased later in the experimental period. The abdominal fat ratio was increased on day 28. Benzbromarone can decrease UA levels on days 14 and 28. The high and low dosage of chicory inulin also decreased serum UA levels on days 7, 14, and 28. The abdominal fat ratio, activity, and protein of acetyl-CoA carboxylase (ACC) were decreased in chicory inulin-treated groups. The activities of xanthine oxidase (XOD) and fatty acid synthase (FAS) were increased in the model group and decreased in the benzbromarone and chicory inulin groups. This study evaluated a quail model of induced hyperuricemia with other metabolic disorders caused by a high-purine diet. The results indicated that a purine-rich diet might contribute to the development of hyperuricemia, hypertriglyceridemia, and abdominal obesity. Chicory inulin decreased serum UA, TG, and abdominal fat deposition in a quail model of hyperuricemia by altering the ACC protein expression and FAS and XOD activities.

Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins

NARCIS (Netherlands)

El Khattabi, Mohamed; van Roosmalen, Maarten L.; Jager, Dennis; Metselaar, Heidi; Permentier, Hjalmar; Leenhouts, Kees; Broos, Jaap

2008-01-01

Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorporation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is

How to prove the existence of metabolons?

DEFF Research Database (Denmark)

Bassard, Jean-Étienne André; Halkier, Barbara Ann

2017-01-01

Sequential enzymes in biosynthetic pathways are organized in metabolons. It is challenging to provide experimental evidence for the existence of metabolons as biosynthetic pathways are composed of highly dynamic protein–protein interactions. Many different methods are being applied, each with str...

Biosynthetic Studies on Water-Soluble Derivative 5c (DTX5c

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José J. Fernández

2012-10-01

Full Text Available The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP. In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of 13C enriched samples obtained by addition of labelled sodium [l-13C], [2-13C] acetate to artificial cultures of this dinoflagellate.

IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites.

Science.gov (United States)

Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Szeto, Ernest; Huang, Jinghua; Reddy, T B K; Cimermančič, Peter; Fischbach, Michael A; Ivanova, Natalia N; Markowitz, Victor M; Kyrpides, Nikos C; Pati, Amrita

2015-07-14

In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of "big" genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to

Development of a neutral network model to predict the excretion of purine derivatives in the urine of cows

International Nuclear Information System (INIS)

Volpe, V.; Stefanon, B.; Moscardini, S.; Susmel, P.; Gruber, L.

1999-01-01

A Neural Network Model to predict the urinary excretion of purine derivative nitrogen (UPDN) in cows is presented. The input variables of the model are dry matter intake (DMINT), NDF intake (NDFINT), total soluble nitrogen (SP), total soluble non-protein dry matter (SNPDM), total degradable nitrogen (DCP), total degradable non-protein dry matter (DNPDM), hourly available CP in the rumen (HACP), hourly available non-protein dry matter (HANPDM), three different gross indexes of synchronization, namely SYNCA (SP/SNPDM), SYNCB (DCP/DNPDM) and SYNCK (HACP/HANPDM) and two variables describing some metabolic aspects of purine derivative excretion such as live weight of the cow (LW) and milk yield (MILKY). The Model developed uses the Multi Layer Perceptron (MLP) utility, with 13 nodes in the input layer, 8 nodes in the hidden layer and 1 node in the output layer. The Model performances have been tested over 24 observations not previously used to train the model. When compared to a linear regression approach, the Neural Network model showed better performance but under predicted the daily excretion of UPDN for values around 20 g/day. When evaluated in terms of behaviour and depicted scenario the model responded to changes of live weight (LW) and milk yield (MILKY) and to modifications of the pattern of nutrients supplied to rumen microbes. (author)

Development of a neutral network model to predict the excretion of purine derivatives in the urine of cows

Energy Technology Data Exchange (ETDEWEB)

Volpe, V; Stefanon, B; Moscardini, S; Susmel, P [University of Udine, Department of Animal Production Science, Pagnacco, UD (Italy); Gruber, L [Federal Research Institute for Agriculture in the Alpine Regions, Irdning (Austria)

1999-06-01

A Neural Network Model to predict the urinary excretion of purine derivative nitrogen (UPDN) in cows is presented. The input variables of the model are dry matter intake (DMINT), NDF intake (NDFINT), total soluble nitrogen (SP), total soluble non-protein dry matter (SNPDM), total degradable nitrogen (DCP), total degradable non-protein dry matter (DNPDM), hourly available CP in the rumen (HACP), hourly available non-protein dry matter (HANPDM), three different gross indexes of synchronization, namely SYNCA (SP/SNPDM), SYNCB (DCP/DNPDM) and SYNCK (HACP/HANPDM) and two variables describing some metabolic aspects of purine derivative excretion such as live weight of the cow (LW) and milk yield (MILKY). The Model developed uses the Multi Layer Perceptron (MLP) utility, with 13 nodes in the input layer, 8 nodes in the hidden layer and 1 node in the output layer. The Model performances have been tested over 24 observations not previously used to train the model. When compared to a linear regression approach, the Neural Network model showed better performance but under predicted the daily excretion of UPDN for values around 20 g/day. When evaluated in terms of behaviour and depicted scenario the model responded to changes of live weight (LW) and milk yield (MILKY) and to modifications of the pattern of nutrients supplied to rumen microbes. (author) 16 refs, 11 figs, 1 tab