Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

Science.gov (United States)

Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

DEFF Research Database (Denmark)

Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

2002-01-01

The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation...... procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit approximately 0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated...... as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore...

Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence

International Nuclear Information System (INIS)

Millan, J.L.; Driscoll, C.E.; LeVan, K.M.; Goldberg, E.

1987-01-01

The sequence and structure of human testis-specific L-lactate dehydrogenase [LDHC 4 , LDHX; (L)-lactate:NAD + oxidoreductase, EC 1.1.1.27] has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC 4 is as different from rodent LDHC 4 (73% homology) as it is from human LDHA 4 (76% homology) and porcine LDHB 4 (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC 4 and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete anti-genic determinants of mouse and human LDHC 4 reveals significant differences. Knowledge of the human LDHC 4 sequence will help design human-specific peptides useful in the development of a contraceptive vaccine

An in-silico investigation of anti-Chagas phytochemicals.

Science.gov (United States)

McCulley, Stephanie F; Setzer, William N

2014-01-01

Over 18 million people in tropical and subtropical America are afflicted by American trypanosomiasis or Chagas disease. In humans, symptoms of the disease include fever, swelling, and heart and brain damage, usually leading to death. There is currently no effective treatment for this disease. Plant products continue to be rich sources of clinically useful drugs, and the biodiversity of the Neotropics suggests great phytomedicinal potential. Screening programs have revealed numerous plant species and phytochemical agents that have shown in-vitro or in-vivo antitrypanosomal activity, but the biochemical targets of these phytochemicals are not known. In this work, we present a molecular docking analysis of Neotropical phytochemicals, which have already demonstrated antiparasitic activity against Trypanosoma cruzi, with potential druggable protein targets of the parasite. Several protein targets showed in-silico selectivity for trypanocidal phytochemicals, including trypanothione reductase, pteridine reductase 2, lipoamide dehydrogenase, glucokinase, dihydroorotate dehydrogenase, cruzain, dihydrofolate-reductase/thymidylate-synthase, and farnesyl diphosphate synthase. Some of the phytochemical ligands showed notable docking preference for trypanothione reductase, including flavonoids, fatty-acid-derived oxygenated hydrocarbons, geranylgeraniol and the lignans ganschisandrine and eupomatenoid-6.

Distribution of the branched-chain α-ketoacid dehydrogenase complex E1α subunit and glutamate dehydrogenase in the human brain and their role in neuro-metabolism.

Science.gov (United States)

Hull, Jonathon; Usmari Moraes, Marcela; Brookes, Emma; Love, Seth; Conway, Myra E

2018-01-01

Glutamate is the major excitatory neurotransmitter of the central nervous system, with the branched-chain amino acids (BCAAs) acting as key nitrogen donors for de novo glutamate synthesis. Despite the importance of these major metabolites, their metabolic pathway in the human brain is still not well characterised. The metabolic pathways that influence the metabolism of BCAAs have been well characterised in rat models. However, the expression of key proteins such as the branched-chain α-ketoacid dehydrogenase (BCKD) complex and glutamate dehydrogenase isozymes (GDH) in the human brain is still not well characterised. We have used specific antibodies to these proteins to analyse their distribution within the human brain and report, for the first time, that the E1α subunit of the BCKD is located in both neurons and vascular endothelial cells. We also demonstrate that GDH is localised to astrocytes, although vascular immunolabelling does occur. The labelling of GDH was most intense in astrocytes adjacent to the hippocampus, in keeping with glutamatergic neurotransmission in this region. GDH was also present in astrocyte processes abutting vascular endothelial cells. Previously, we demonstrated that the branched-chain aminotransferase (hBCAT) proteins were most abundant in vascular cells (hBCATm) and neurons (hBCATc). Present findings are further evidence that BCAAs are metabolised within both the vasculature and neurons in the human brain. We suggest that GDH, hBCAT and the BCKD proteins operate in conjunction with astrocytic glutamate transporters and glutamine synthetase to regulate the availability of glutamate. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions such as Alzheimer's disease. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells.

Science.gov (United States)

Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

2015-01-01

Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.

Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells

Science.gov (United States)

Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

2015-01-01

Background Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). Results/Methodology We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Conclusion Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease. PMID:25826140

An improved method for the assay of platelet pyruvate dehydrogenase

International Nuclear Information System (INIS)

Schofield, P.J.; Griffiths, L.R.; Rogers, S.H.

1980-01-01

An improved method for the assay of human platelet pyruvate dehydrogenase is described. By generating the substrate [1- 14 C]pyruvate in situ from [1- 14 C]lactate plus L-lactate dehydrogenase, the rate of spontaneous decarboxylation is dramatically reduced, allowing far greater sensitivity in the assay of low activities of pyruvate dehydrogenase. In addition, no special precautions are required for the storage and use of [1- 14 C]lactate, in contrast to those for [1- 14 C]pyruvate. These factors allow a 5-10-fold increase in sensitivity compared with current methods. The pyruvate dehydrogenase activity of normal subjects as determined by the [1- 14 C]lactate system was 215+-55 pmol min -1 mg -1 protein (n=18). The advantages of this assay system are discussed. (Auth.)

Dihydrooxonate is a substrate of Dihydroorrotate Dehydrogenase (DHOD) providing evidence for involvement of crsteine and serine residues in base catalysis

DEFF Research Database (Denmark)

Björnberg, Olof; Jordan, Douglas B.; Palfey, Bruce Allan

2001-01-01

pKa of the 5-position in the substrate. Oxonate, the oxidation product of dihydrooxonate, was a competitive inhibitor versus dihydroorotate, and DHODA was the most sensitive of the three enzymes. DHODA was reinvestigated with respect to product inhibition by orotate. The results suggest a classical...... one-site ping-pong mechanism with fumarate as electron acceptor, while the kinetics with ferricyanide is highly dependent on the detailed reaction conditions....

Purification and characterization of glutaryl-CoA dehydrogenase from porcine and human liver

International Nuclear Information System (INIS)

Lenich, A.C.

1985-01-01

Glutaryl-CoA dehydrogenase (GCDH) was purified from porcine liver mitochondria by pH and ammonium sulfate fractionations followed by a series of column chromatographies. The purified porcine enzyme was found by sodium dodecyl-sulfate polyacrylamide gel electrophoresis to have a subunit molecular weight of 47,800 and by gradient polyacrylamide gel electrophoresis (PAGE) to have a native molecular weight of approximately 186,000. The product of the GCDH reaction with its primary substrate, glutaryl-CoA, was investigated by radio-gas chromatography and found to be crotonyl-CoA. Alternate substrates as well as crotonyl-CoA, the glutaryl-CoA reaction end product, demonstrated competitive inhibition when incubated with (1,5- 14 C)-glutaryl-CoA in the presence of porcine GCDH. Kinetic parameters for the interaction of both ETF and glutaryl-CoA with porcine GCDH were determined. Purified porcine GCDH was used to produce an antiserum which cross-reacted with human liver GCDH with a reaction of partial identity, but proved too insensitive to detect GCDH in control human fibroblasts. As a result of these negative findings, GCDH was purified by a series of column chromatographies from human liver. The purified human enzyme was found by SDS-PAGE and gel filtration to have subunit and native molecular weights of 58,800 and 256,000 respectively

Diglycolic acid inhibits succinate dehydrogenase activity in human proximal tubule cells leading to mitochondrial dysfunction and cell death.

Science.gov (United States)

Landry, Greg M; Dunning, Cody L; Conrad, Taylor; Hitt, Mallory J; McMartin, Kenneth E

2013-08-29

Diethylene glycol (DEG) is a solvent used in consumer products allowing the increased risk for consumer exposure. DEG metabolism produces two primary metabolites, 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA). DGA has been shown to be the toxic metabolite responsible for the proximal tubule cell necrosis seen in DEG poisoning. The mechanism of DGA toxicity in the proximal tubule cell is not yet known. The chemical structure of DGA is very similar to citric acid cycle intermediates. Studies were designed to assess whether its mechanism of toxicity involves disruption of cellular metabolic pathways resulting in mitochondrial dysfunction. First, DGA preferentially inhibited succinate dehydrogenase, including human kidney cell enzyme, but had no effect on other citric acid cycle enzyme activities. DGA produces a cellular ATP depletion that precedes cell death. Human proximal tubule (HPT) cells, pre-treated with increasing DGA concentrations, showed significantly decreased oxygen consumption. DGA did not increase lactate levels, indicating no effect on glycolytic activity. DGA increased reactive oxygen species (ROS) production in HPT cells in a concentration and time dependent manner. These results indicate that DGA produced proximal tubule cell dysfunction by specific inhibition of succinate dehydrogenase and oxygen consumption. Disruption of these processes results in decreased energy production and proximal tubule cell death. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase

DEFF Research Database (Denmark)

Yennawar, Hemant; Møller, Magda; Gillilan, Richard

2011-01-01

The X-ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular-replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer...

Structural organization of the human short-chain acyl-CoA dehydrogenase gene

DEFF Research Database (Denmark)

Corydon, M J; Andresen, B S; Bross, P

1997-01-01

Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid beta-oxidation. Defects in the SCAD enzyme are associated with failure to thrive, often with neuromuscular dysfunction and elevated urinary excretion...... shown to be associated with ethylmalonic aciduria. From analysis of 18 unrelated Danish families, we show that the four SCAD gene polymorphisms constitute five allelic variants of the SCAD gene, and that the 625A variant together with the less frequent variant form of the three other polymorphisms (321C....... The evolutionary relationship between SCAD and five other members of the acyl-CoA dehydrogenase family was investigated by two independent approaches that gave similar phylogenetic trees....

Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

Science.gov (United States)

Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M; Di Guilmi, Anne Marie; Frachet, Philippe

2012-12-14

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.

Human and Pneumococcal Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Proteins Are Both Ligands of Human C1q Protein*

Science.gov (United States)

Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M.; Di Guilmi, Anne Marie; Frachet, Philippe

2012-01-01

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (KD = 0.34–2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response. PMID:23086952

Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

Science.gov (United States)

Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

2013-07-01

Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

Science.gov (United States)

Modig, Tobias; Lidén, Gunnar; Taherzadeh, Mohammad J

2002-01-01

The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition. PMID:11964178

Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans

DEFF Research Database (Denmark)

Nguyen, Tien V; Andresen, Brage S; Corydon, Thomas J

2002-01-01

-CoA dehydrogenase. A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine. Amplified ACAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905G>A) in the ACAD8 coding region compared to the sequence from control...... in a patient....

The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases

DEFF Research Database (Denmark)

Zaganas, Ioannis; Pajecka, Kamilla; Nielsen, Camilla Wendel

2013-01-01

Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two...... human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.......0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced...

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

Science.gov (United States)

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-08-01

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

Plant Formate Dehydrogenase

Energy Technology Data Exchange (ETDEWEB)

John Markwell

2005-01-10

The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

Isolation, characterization, and mapping of gene encoding dihydrolipoyl succinyltransferase (E2k) of human [alpha]-ketoglutarate dehydrogenase complex

Energy Technology Data Exchange (ETDEWEB)

Ali, G.; Cai, Xingang; Sheu, Kwan-Fu R.; Blass, J.P. (Cornell Univ. Medical College, White Plains, NY (United States)); Wasco, W.; Gaston, S.M.; Tanzi, R.E.; Cooper, A.J.L.; Gusella, J.F. (Massachusetts General Hospital, Charleston, MA (United States)); Szabo, P. (Cornell Univ. Medical College, New York, NY (United States))

1994-03-01

The authors have isolated and sequenced cDNAs representing the full-length (2987-bp) gene for dihydrolipoyl succinyltransferase (E2k component) of the human [alpha]-ketoglutarate dehydrogenase complex (KHDHC) from a human fetal brain cDNA library. The E2k cDNA was mapped to human chromosome 14 using a somatic cell hybrid panel, and more precisely to band 14q24.3 by in situ hybridization. This cDNA also cross-hybridized to an apparent E2k pseudogene on chromosome 1p31. Northern analysis revealed the E2k gene to be ubiquitously expressed in peripheral tissues and brain. Interestingly, chromosome 14q24.3 has recently been reported to contain gene defects for an early-onset form of familial Alzheimer's disease and for Machado-Joseph disease. Future studies will be necessary to determine whether the E2K gene plays a role in either of these two disorders.

21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages...

Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

DEFF Research Database (Denmark)

Matzen, L E; Kristensen, S R; Kvetny, J

1991-01-01

The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high ...

Identification of the human mitochondrial FAD transporter and its potential role in multiple acyl-CoA dehydrogenase deficiency

NARCIS (Netherlands)

Spaan, András N.; Ijlst, Lodewijk; van Roermund, Carlo W. T.; Wijburg, Frits A.; Wanders, Ronald J. A.; Waterham, Hans R.

2005-01-01

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is most often caused by mutations in the genes encoding the alpha- or beta-subunit of electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETF-DH). Since not all patients have

Kernicterus by glucose-6-phosphate dehydrogenase deficiency: a case report and review of the literature

Directory of Open Access Journals (Sweden)

Cossio de Gurrola Gladys

2008-05-01

Full Text Available Abstract Introduction Glucose-6-phosphate dehydrogenase deficiency is an X-linked recessive disease that causes acute or chronic hemolytic anemia and potentially leads to severe jaundice in response to oxidative agents. This deficiency is the most common human innate error of metabolism, affecting more than 400 million people worldwide. Case presentation Here, we present the first documented case of kernicterus in Panama, in a glucose-6-phosphate dehydrogenase-deficient newborn clothed in naphthalene-impregnated garments, resulting in reduced psychomotor development, neurosensory hypoacousia, absence of speech and poor reflex of the pupil to light. Conclusion Mutational analysis revealed the glucose-6-phosphate dehydrogenase Mediterranean polymorphic variant, which explained the development of kernicterus after exposition of naphthalene. As the use of naphthalene in stored clothes is a common practice, glucose-6-phosphate dehydrogenase testing in neonatal screening could prevent severe clinical consequences.

21 CFR 862.1670 - Sorbitol dehydrogenase test system.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sorbitol dehydrogenase test system. 862.1670... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in serum...

Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

Science.gov (United States)

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy.

Science.gov (United States)

Westermann, C M; Dorland, L; Votion, D M; de Sain-van der Velden, M G M; Wijnberg, I D; Wanders, R J A; Spliet, W G M; Testerink, N; Berger, R; Ruiter, J P N; van der Kolk, J H

2008-05-01

The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity

DEFF Research Database (Denmark)

Nellemann, B.; Vendelbo, M.H.; Nielsen, Thomas Svava

2014-01-01

Insulin resistance induced by growth hormone (GH) is linked to promotion of lipolysis by unknown mechanisms. We hypothesized that suppression of the activity of pyruvate dehydrogenase in the active form (PDHa) underlies GH-induced insulin resistance similar to what is observed during fasting....

Characterization of immunoglobulin A kappa autoantibodies to human lactate dehydrogenase isoenzyme-3

NARCIS (Netherlands)

Weijers, R. N.; Oude Elferink, R. P.; Mulder, J.; Kruijswijk, H.

1987-01-01

We have purified with a cumulative recovery of 48% from the serum of a patient the immunoglobulin A kappa subunit of the lactate dehydrogenase-immunoglobulin A kappa (LD-IgA kappa) complex. It appears that the pI range of the complex is 5.4-5.8. The Ig part of the complex showed a monoclonal

O-Alkyl Hydroxamates as Metaphors of Enzyme-Bound Enolate Intermediates in Hydroxy Acid Dehydrogenases. Inhibitors of Isopropylmalate Dehydrogenase, Isocitrate Dehydrogenase, and Tartrate Dehydrogenase(1).

Science.gov (United States)

Pirrung, Michael C.; Han, Hyunsoo; Chen, Jrlung

1996-07-12

The inhibition of Thermus thermophilus isopropylmalate dehydrogenase by O-methyl oxalohydroxamate was studied for comparison to earlier results of Schloss with the Salmonella enzyme. It is a fairly potent (1.2 &mgr;M), slow-binding, uncompetitive inhibitor against isopropylmalate and is far superior to an oxamide (25 mM K(i) competitive) that is isosteric with the ketoisocaproate product of the enzyme. This improvement in inhibition was attributed to its increased NH acidity, which presumably is due to the inductive effect of the hydroxylamine oxygen. This principle was extended to the structurally homologous enzyme isocitrate dehydrogenase from E. coli, for which the compound O-(carboxymethyl) oxalohydroxamate is a 30 nM inhibitor, uncompetitive against isocitrate. The pH dependence of its inhibition supports the idea that it is bound to the enzyme in the anionic form. Another recently discovered homologous enzyme, tartrate dehydrogenase from Pseudomonas putida, was studied with oxalylhydroxamate. It has a relatively low affinity for the enzyme, though it is superior to tartrate. On the basis of these leads, squaric hydroxamates with increased acidity compared to squaric amides directed toward two of these enzymes were prepared, and they also show increased inhibitory potency, though not approaching the nanomolar levels of the oxalylhydroxamates.

Human liver aldehyde dehydrogenase: coenzyme binding

International Nuclear Information System (INIS)

Kosley, L.L.; Pietruszko, R.

1987-01-01

The binding of [U- 14 C] NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of [U- 14 C] NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

Science.gov (United States)

Hecht, K; Wrba, A; Jaenicke, R

1989-07-15

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

Genetics Home Reference: dihydropyrimidine dehydrogenase deficiency

Science.gov (United States)

... 5-fluorouracil and capecitabine. These drugs are not broken down efficiently by people with dihydropyrimidine dehydrogenase deficiency ... of this enzyme. Because fluoropyrimidine drugs are also broken down by the dihydropyrimidine dehydrogenase enzyme, deficiency of ...

Phosphorylation site on yeast pyruvate dehydrogenase complex

International Nuclear Information System (INIS)

Uhlinger, D.J.

1986-01-01

The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the 32 P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation

Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes

DEFF Research Database (Denmark)

Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw

2017-01-01

A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1...... and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation...

Asexual populations of the human malaria parasite, Plasmodium falciparum, use a two-step genomic strategy to acquire accurate, beneficial DNA amplifications.

Directory of Open Access Journals (Sweden)

Jennifer L Guler

Full Text Available Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes.

Comparative genomics of aldehyde dehydrogenase 5a1 (succinate semialdehyde dehydrogenase and accumulation of gamma-hydroxybutyrate associated with its deficiency

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Malaspina Patrizia

2009-01-01

Full Text Available Abstract Succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5A1 [ALDH5A1]; locus 6p22 occupies a central position in central nervous system (CNS neurotransmitter metabolism as one of two enzymes necessary for γ-aminobutyric acid (GABA recycling from the synaptic cleft. Its importance is highlighted by the neurometabolic disease associated with its inherited deficiency in humans, as well as the severe epileptic phenotype observed in Aldh5a1-/- knockout mice. Expanding evidence now suggests, however, that even subtle decreases in human SSADH activity, associated with rare and common single nucleotide polymorphisms, may produce subclinical pathological effects. SSADH, in conjunction with aldo-keto reductase 7A2 (AKR7A2, represent two neural enzymes responsible for further catabolism of succinic semialdehyde, producing either succinate (SSADH or γ-hydroxybutyrate (GHB; AKR7A2. A GABA analogue, GHB is a short-chain fatty alcohol with unusual properties in the CNS and a long pharmacological history. Moreover, SSADH occupies a further role in the CNS as the enzyme responsible for further metabolism of the lipid peroxidation aldehyde 4-hydroxy-2-nonenal (4-HNE, an intermediate known to induce oxidant stress. Accordingly, subtle decreases in SSADH activity may have the capacity to lead to regional accumulation of neurotoxic intermediates (GHB, 4-HNE. Polymorphisms in SSADH gene structure may also associate with quantitative traits, including intelligence quotient and life expectancy. Further population-based studies of human SSADH activity promise to reveal additional properties of its function and additional roles in CNS tissue.

Radioimmunoassay of lactate dehydrogenase, H forms

International Nuclear Information System (INIS)

Malvano, R.; Massaglia, A.; Zannino, M.; Palmucci, F.; Cali, V.; Zucchelli, G.C.; Consiglio Nazionale delle Ricerche, Pisa

1979-01-01

Antisera to H 4 -lactate dehydrogenase (LDH) were elicited in rabbits, against both human (h) and porcine (p) isoenzymes. 125 I-labelled H 4 -LDH was prepared by electrolytic iodination. A simple and fast procedure (1-h incubation for clinical assays) was set up by using polyethylene glycol for the bound-free separation. The results obtained in the antiserum characterization indicated that the heterologous homotetramer, M 4 was completely discriminated in the porcine system, while a weak cross-reaction with human antisera resulted. In both cases, for the hybrid forms, a cross-reactivity level related to the stoichiometric contents of the H-subunit in the tetramers was observed. The H 4 -LDH from other species was found to be much more effectively distinguished in the procine than in the human system. The assay for human LDH was further validated in terms of analytical suitability and clinical response. For healthy subjects the mean concentration was 0.46 +- 0.19 μg/ml (mean +- SD). Patients with acute myocardial infarction had levels ranging from 1.2 to 5.9 μg/ml. (orig.) [de

Handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins in transgenic mice

DEFF Research Database (Denmark)

Kragh, Peter M; Pedersen, Christina B; Schmidt, Stine P

2007-01-01

Abstract To investigate the in vivo handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins, three transgenic mouse lines were produced by pronuclear injection of cDNA encoding the wild-type, hSCAD-wt, and two disease causing folding variants hSCAD-319C > T and hSCAD-625G > A....... The transgenic mice were mated with an SCAD-deficient mouse strain (BALB/cByJ) and, in the second generation, three mouse lines were obtained without endogenous SCAD expression but harboring hSCAD-wt, hSCAD-319C > T, and hSCAD-625G > A transgenes, respectively. All three lines had expression of the transgene...... developed for any of the lines transgenic for the hSCAD folding variants. The indicated remarkable efficiency of the mouse protein quality control system in the degradation of SCAD folding variants should be further substantiated and investigated, since it might indicate ways to prevent disease...

Purification of methanol dehydrogenase from mouth methylotrophic bacteria of tropical region

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Waturangi, D.

2011-01-01

Full Text Available Aims: Purification of methanol dehydrogenase (MDH from methylotrophic bacteria was conducted to obtain pure enzyme for further research and industrial applications due to the enzyme’s unique activity that catalyzes oxidation of methanol as an important carbon source in methylotrophic bacteria.Methodology and Results: The enzyme was screened from methylotrophic bacteria isolated from human mouth. Purification of this enzyme was conducted using ammonium sulphate precipitation followed by cation exchange chromatography. Two types of media were used to produce the enzymes: luria broth and standard mineral salts media (MSM. MSM produced MDH with higher specific activity than LB. Specific activity was also increased along with the purification steps. Application of ammonium sulphate increased the purity of enzyme and was more effective for the enzyme produced in LB. Using sepharose increased the enzyme activity 10 -57 folds.Conclusion, significant and impact of this study: With this, ammonium sulphate precipitation coupled with single cation exchange chromatographic system has been proved to provide sufficient purified of methanol dehydrogenase from methylotrophic bacteria origin of human mouth with high specific activity for further application.

The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

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Rodrigues Valnês

2009-01-01

Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

Modulation of NADP(+)-dependent isocitrate dehydrogenase in aging.

Science.gov (United States)

Kil, In Sup; Lee, Young Sup; Bae, Young Seuk; Huh, Tae Lin; Park, Jeen-Woo

2004-01-01

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46-48 population doubling level (PDL) and then gradually decreased at later PDL. 2',7'-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.

Shikimate dehydrogenase from Pinu sylvestris L. needles

International Nuclear Information System (INIS)

Osipov, V.I.; Shein, I.V.

1986-01-01

Shikimate dehydrogenase was isolated by extraction from pine needles and partially purified by fractionation with ammonium sulfate. In conifers, in contrast to other plants, all three isoenzymes of shikimate dehydrogenase exhibit activity not only with NADP + , but also with NAD + . The values of K/sub m/ for shikimate, when NADP + and NAD + are used as cofactors, are 0.22 and 1.13 mM, respectively. The enzyme is maximally active at pH 10 with both cofactors. It is suggested that NAD-dependent shikimate dehydrogenase catalyzes the initial reaction of the alternative pathway of the conversion of shikimic acid to hydroxybenzoic acid. The peculiarities of the organization and regulation of the initial reactions of the shikimate pathway in conifers and in plants with shikimate dehydrogenase absolutely specific for NADP are discussed

Triiodothyronine (T3)-associated upregulation and downregulation of nuclear T3 binding in the human fibroblast cell (MRC-5)--stimulation of malic enzyme, glucose-6-phosphate-dehydrogenase, and 6-phosphogluconate-dehydrogenase by insulin, but not by T3

DEFF Research Database (Denmark)

Matzen, L E; Kristensen, S R; Kvetny, J

1991-01-01

The specific nuclear binding of triiodothyronine (T3) (NBT3) and the activity of malic enzyme (ME), glucose-6-phosphate-dehydrogenase (G6PD), and 6-phosphogluconate-dehydrogenase (6PGD) were studied in the human fibroblast cell (MRC-5). The overall apparent binding affinity (Ka) was 2.7 x 10(9) L.......mol-1 estimated from kinetic studies of nuclear T3 binding, and 2.5 x 10(9) L.mol-1 estimated from equilibrium studies. The scatchard plots were curvilinear and composed of a high-affinity binding site with Ka1 3.4 +/- 0.7 x 10(9) L.mol-1 and maximal binding capacity (MBC) MBC1 57.0 +/- 11.9 fmol/mg DNA...... and a low-affinity binding site with Ka2 2.9 +/- 1.1 x 10(8) L.mol-1 and MBC2 124.7 +/- 22.1 fmol/mg DNA (n = 6). Incubation of cells with 6 nmol/L T3 for 20 hours reduced NBT3 to 62.2% +/- 15.7% (P less than .01, n = 11). The Ka estimated from kinetic studies was reduced to 6.7 x 10(7) L.mol-1...

The Effects of Fenarimol and Methyl Parathion on Glucose 6-Phosphate Dehydrogenase Enzyme Activity in Rats

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Ferda ARI

2017-10-01

Full Text Available Fenarimol and methyl parathion are pesticides that have been used in agriculture for several years. These pesticides have significant effects on environmental and human health. Therefore, we investigated the effects of methyl parathion and fenarimol on glucose 6-phosphate dehydrogenase (EC 1.1.1.49 enzyme activity in rats. The glucose 6- phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway and it is important in detoxifying reactions by NADPH generated. In this study, wistar albino rats administrated with methyl parathion (7 mg kg–1 and fenarimol (200 mg kg−1 by intraperitoneally for different periods (2, 4, 8, 16, 32, 64, and 72 h. The glucose 6-phosphate dehydrogenase enzyme activity was assayed in liver, kidney, brain, and small intestine in male and female rats. The exposure of fenarimol and methyl parathion caused increase of glucose 6-phosphate dehydrogenase enzyme activity in rat tissues, especially at last periods. We suggest that this increment of enzyme activity may be the reason of toxic effects of fenarimol and methyl parathion.

INFLUENCE OF SELECTED PHARMACEUTICALS ON ACTIVATED SLUDGE DEHYDROGENASE ACTIVITY

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Agnieszka Tomska

2016-06-01

The aim of this work was to evaluate the effect of selected antibiotics - sulfanilamide and erythromycin on activated sludge dehydrogenase activity with use of trifenyltetrazolinum chloride (TTC test. Dehydrogenases activity is an indicator of biochemical activity of microorganisms present in activated sludge or the ability to degrade organic compounds in waste water. TTC test is particularly useful for the regularity of the course of treatment, in which the presence of inhibitors of biochemical reactions and toxic compounds are present. It was observed that the dehydrogenase activity decreases with the increase of a antibiotics concentration. The lowest value of the dehydrogenase activity equal to 32.4 μmol TF / gMLSS obtained at sulfanilamide concentration 150mg / l. For this sample, an inhibition of dehydrogenase activity was 31%.

Mechanisms underlying metabolic and neural defects in zebrafish and human multiple acyl-CoA dehydrogenase deficiency (MADD.

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Yuanquan Song

2009-12-01

Full Text Available In humans, mutations in electron transfer flavoprotein (ETF or electron transfer flavoprotein dehydrogenase (ETFDH lead to MADD/glutaric aciduria type II, an autosomal recessively inherited disorder characterized by a broad spectrum of devastating neurological, systemic and metabolic symptoms. We show that a zebrafish mutant in ETFDH, xavier, and fibroblast cells from MADD patients demonstrate similar mitochondrial and metabolic abnormalities, including reduced oxidative phosphorylation, increased aerobic glycolysis, and upregulation of the PPARG-ERK pathway. This metabolic dysfunction is associated with aberrant neural proliferation in xav, in addition to other neural phenotypes and paralysis. Strikingly, a PPARG antagonist attenuates aberrant neural proliferation and alleviates paralysis in xav, while PPARG agonists increase neural proliferation in wild type embryos. These results show that mitochondrial dysfunction, leading to an increase in aerobic glycolysis, affects neurogenesis through the PPARG-ERK pathway, a potential target for therapeutic intervention.

Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

Energy Technology Data Exchange (ETDEWEB)

Brautigam, Chad A.; Wynn, R. Max; Chuang, Jacinta L.; Naik, Mandar T.; Young, Brittany B.; Huang, Tai-huang; Chuang, David T. (AS); (UTSMC)

2012-02-27

The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.

Mechanism of flavin reduction in the class 1A dihydroorotate dehydrogenase from Lactococcus lactis

DEFF Research Database (Denmark)

Fagan, Rebecca L; Jensen, Kaj Frank; Björnberg, Olof

2007-01-01

is concerted or stepwise was addressed for the class 1A enzyme from Lactococcus lactis by determining kinetic isotope effects (KIEs) on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined at two pH values. At pH 7.0, KIEs were approximately 2-fold for DHO labeled singly...... at the 5-position or the 6-position and approximately 4-fold for DHO labeled at both the 5- and 6-positions. At pH 8.5, the KIEs observed for DHO labeled at the 5-position, the 6-position, and the 5- and 6-positions were approximately 2-, approximately 3-, and approximately 6-fold, respectively....... These isotope effects are consistent with a concerted oxidation of DHO. The pH dependence of reduction was also determined, and a pKa of 8.3 was found. This pKa can be attributed to the ionization of the active site cysteine which deprotonates C5 of DHO during the reaction. To further investigate the importance...

Reversible inactivation of CO dehydrogenase with thiol compounds

Energy Technology Data Exchange (ETDEWEB)

Kreß, Oliver [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Gnida, Manuel [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Pelzmann, Astrid M. [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Marx, Christian [Institute of Biochemistry and Biophysics, Friedrich-Schiller-University of Jena, 07745 Jena (Germany); Meyer-Klaucke, Wolfram [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Meyer, Ortwin, E-mail: Ortwin.Meyer@uni-bayreuth.de [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany)

2014-05-09

Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in

Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

International Nuclear Information System (INIS)

Ikuta, T.; Szeto, S.; Yoshida, A.

1986-01-01

Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

AAV Gene Therapy for Alcoholism: Inhibition of Mitochondrial Aldehyde Dehydrogenase Enzyme Expression in Hepatoma Cells.

Science.gov (United States)

Sanchez, Anamaria C; Li, Chengwen; Andrews, Barbara; Asenjo, Juan A; Samulski, R Jude

2017-09-01

Most ethanol is broken down in the liver in two steps by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2) enzymes, which metabolize down ethanol into acetaldehyde and then acetate. Some individuals from the Asian population who carry a mutation in the aldehyde dehydrogenase gene (ALDH2*2) cannot metabolize acetaldehyde as efficiently, producing strong effects, including facial flushing, dizziness, hypotension, and palpitations. This results in an aversion to alcohol intake and protection against alcoholism. The large prevalence of this mutation in the human population strongly suggests that modulation of ALDH2 expression by genetic technologies could result in a similar phenotype. scAAV2 vectors encoding ALDH2 small hairpin RNA (shRNA) were utilized to validate this hypothesis by silencing ALDH2 gene expression in human cell lines. Human cell lines HEK-293 and HepG2 were transduced with scAAV2/shRNA, showing a reduction in ALDH2 RNA and protein expression with the two viral concentration assayed (1 × 10 4 and 1 × 10 5 vg/cell) at two different time points. In both cell lines, ALDH2 RNA levels were reduced by 90% and protein expression was inhibited by 90% and 52%, respectively, 5 days post infection. Transduced HepG2 VL17A cells (ADH+) exposed to ethanol resulted in a 50% increase in acetaldehyde levels. These results suggest that gene therapy could be a useful tool for the treatment of alcoholism by knocking down ALDH2 expression using shRNA technology delivered by AAV vectors.

Genetics Home Reference: glucose-6-phosphate dehydrogenase deficiency

Science.gov (United States)

... deficiency Encyclopedia: Glucose-6-phosphate dehydrogenase test Encyclopedia: Hemolytic anemia Encyclopedia: Newborn jaundice Health Topic: Anemia Health Topic: G6PD Deficiency Health Topic: Newborn Screening Genetic and Rare Diseases Information Center (1 link) Glucose-6-phosphate dehydrogenase ...

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

Energy Technology Data Exchange (ETDEWEB)

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.

Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus.

Science.gov (United States)

Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

2017-01-01

Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further studies are needed for introducing aldehyde dehydrogenase as a prognostic

Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus*

Science.gov (United States)

Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

2017-01-01

Background Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. Objective This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Method Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. Results The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. Limitations of the study This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. Conclusions The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further

The Alcohol Dehydrogenase Isoenzyme as a Potential Marker of Pancreatitis.

Science.gov (United States)

Jelski, Wojciech; Piechota, Joanna; Orywal, Karolina; Szmitkowski, Maciej

2018-05-01

Human pancreas parenchyma contains various alcohol dehydrogenase (ADH) isoenzymes and also possesses aldehyde dehydrogenase (ALDH) activity. The altered activities of ADH and ALDH in damaged pancreatic tissue in the course of pancreatitis are reflected in the human serum. The aim of this study was to investigate a potential role of ADH and ALDH as markers for acute (AP) and chronic pancreatitis (CP). Serum samples were collected for routine biochemical investigations from 75 patients suffering from acute pancreatitis and 70 patients with chronic pancreatitis. Fluorometric methods were used to measure the activity of class I and II ADH and ALDH activity. The total ADH activity and activity of class III and IV isoenzymes were measured by a photometric method. There was a significant increase in the activity of ADH III isoenzyme (15.06 mU/l and 14.62 mU/l vs. 11.82 mU/l; ppancreatitis or chronic pancreatitis compared to the control. The diagnostic sensitivity for ADH III was about 84%, specificity was 92 %, positive and negative predictive values were 93% and 87% respectively in acute pancreatitis. Area under the Receiver Operating Curve (ROC) curve for ADH III in AP and CP was 0.88 and 0.86 respectively. ADH III has a potential role as a marker of acute and chronic pancreatitis. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

Directory of Open Access Journals (Sweden)

Margit Winkler

2013-08-01

Full Text Available Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S-selectivity and together with a highly (R-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase.

Science.gov (United States)

Napora-Wijata, Kamila; Strohmeier, Gernot A; Sonavane, Manoj N; Avi, Manuela; Robins, Karen; Winkler, Margit

2013-08-12

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

Cloning and expression analysis of alcohol dehydrogenase ( Adh ...

African Journals Online (AJOL)

Hybrid promoters are created by shuffling of DNA fragments while keeping intact regulatory regions crucial of promoter activity. Two fragments of alcohol dehydrogenase (Adh) promoter from Zea mays were selected to generate hybrid promoter. Sequence analysis of both alcohol dehydrogenase promoter fragments through ...

Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

International Nuclear Information System (INIS)

Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P.

2005-01-01

The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression

The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glucose 6-phosphate dehydrogenase activity

International Nuclear Information System (INIS)

Sahin, Ali; Senturk, Murat; Ciftci, Mehmet; Varoglu, Erhan; Kufrevioglu, Omer Irfan

2010-01-01

Aim: The inhibitory effects of thallium-201 ( 201 Tl) solution on human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) activity were investigated. Methods: For this purpose, erythrocyte G6PD was initially purified 835-fold at a yield of 41.7% using 2',5'-Adenosine diphosphate sepharose 4B affinity gel chromatography. The purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed a single band for the final enzyme preparation. The in vitro and in vivo effects of the 201 Tl solution including Tl + , Fe +3 and Cu +2 metals and the in vitro effects of the radiation effect of the 201 Tl solution and non-radioactive Tl + , Fe +3 and Cu +2 metals on human erythrocyte G6PD enzyme were studied. Enzyme activity was determined with the Beutler method at 340 nm using a spectrophotometer. All purification procedures were carried out at +4 deg. C. Results: 201 Tl solution and radiation exposure had inhibitory effects on the enzyme activity. IC 50 value of 201 Tl solution was 36.86 μl ([Tl + ]: 0.0036 μM, [Cu +2 ]: 0.0116 μM, [Fe +3 ]: 0.0132 μM), of human erythrocytes G6PD. Seven human patients were also used for in vivo studies of 201 Tl solution. Furthermore, non-radioactive Tl + , Fe +3 and Cu +2 were found not to have influenced the enzyme in vitro. Conclusion: Human erythrocyte G6PD activity was inhibited by exposure for up to 10 minutes to 0.057 mCi/kg 201 Tl solution. It was detected in in vitro and in vivo studies that the human erythrocyte G6PD enzyme is inhibited due to the radiation effect of 201 Tl solution.

Study on the triphenyl tetrazolium chloride– dehydrogenase activity ...

African Journals Online (AJOL)

A quick analysis of the sludge activity method based on triphenyltetrazolium chloride-dehydrogenase activity (TTC-DHA) was developed to change the rule and status of the biological activity of the activated sludge in tomato paste wastewater treatment. The results indicate that dehydrogenase activity (DHA) can effectively ...

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

International Nuclear Information System (INIS)

Akduman, Begüm; Uygun, Murat; Uygun, Deniz Aktaş; Akgöl, Sinan; Denizli, Adil

2013-01-01

In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

Energy Technology Data Exchange (ETDEWEB)

Akduman, Begüm [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Uygun, Murat [Koçarlı Vocational and Training School, Adnan Menderes University, Aydın (Turkey); Uygun, Deniz Aktaş, E-mail: daktas@adu.edu.tr [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Akgöl, Sinan [Biochemistry Department, Ege University, İzmir (Turkey); Denizli, Adil [Chemistry Department, Hacettepe University, Ankara (Turkey)

2013-12-01

In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

DEFF Research Database (Denmark)

Gojkovic, Zoran; Knecht, Wolfgang; Warneboldt, J.

2004-01-01

The ability to propagate under anaerobic conditions is an essential and unique trait of brewer's or baker's yeast (Saccharomyces cervisiae). To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic...... activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species. While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen. From....... We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired...

Inducible xylitol dehydrogenases in enteric bacteria.

OpenAIRE

Doten, R C; Mortlock, R P

1985-01-01

Morganella morganii ATCC 25829, Providencia stuartii ATCC 25827, Serratia marcescens ATCC 13880, and Erwinia sp. strain 4D2P were found to induce a xylitol dehydrogenase when grown on a xylitol-containing medium. The xylitol dehydrogenases were partially purified from the four strains, and those from M. morganii ATCC 25829, P. stuartii ATCC 25827, and S. marcescens ATCC 13880 were all found to oxidize xylitol to D-xylulose. These three enzymes had KmS for xylitol of 7.1 to 16.4 mM and molecul...

Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

Directory of Open Access Journals (Sweden)

Sakuko Ueshima

2010-01-01

Full Text Available The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3 were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienylserine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

Enzymatic urea adaptation: lactate and malate dehydrogenase in elasmobranchs

Czech Academy of Sciences Publication Activity Database

Lagana, G.; Bellocco, E.; Mannucci, C.; Leuzzi, U.; Tellone, E.; Kotyk, Arnošt; Galtieri, A.

2006-01-01

Roč. 55, č. 6 (2006), s. 675-688 ISSN 0862-8408 Institutional research plan: CEZ:AV0Z50110509 Keywords : elasmobranchs * lactate dehydrogenase * malate dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 2.093, year: 2006

Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon.

Science.gov (United States)

Lee, Ja-Young; Arai, Hisashi; Nakamura, Yusuke; Fukiya, Satoru; Wada, Masaru; Yokota, Atsushi

2013-11-01

Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90-100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7β-hydroxysteroid dehydrogenase (7β-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7β-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon.

2-Methylbutyryl-coenzyme A dehydrogenase deficiency

DEFF Research Database (Denmark)

Sass, Jörn Oliver; Ensenauer, Regina; Röschinger, Wulf

2008-01-01

2-Methylbutyryl-CoA dehydrogenase (MBD; coded by the ACADSB gene) catalyzes the step in isoleucine metabolism that corresponds to the isovaleryl-CoA dehydrogenase reaction in the degradation of leucine. Deficiencies of both enzymes may be detected by expanded neonatal screening with tandem...... individuals showed clinical symptoms attributable to MBD deficiency although the defect in isoleucine catabolism was demonstrated both in vivo and in vitro. Several mutations in the ACADSB gene were identified, including a novel one. MBD deficiency may be a harmless metabolic variant although significant...

New recombinant bacterium comprises a heterologous gene encoding glycerol dehydrogenase and/or an up-regulated native gene encoding glycerol dehydrogenase, useful for producing ethanol

DEFF Research Database (Denmark)

2010-01-01

dehydrogenase encoding region of the bacterium, or is inserted into a phosphotransacetylase encoding region of the bacterium, or is inserted into an acetate kinase encoding region of the bacterium. It is operably linked to an inducible, a regulated or a constitutive promoter. The up-regulated glycerol......TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preparation (claimed): Producing recombinant bacterium having enhanced ethanol production characteristics when cultivated in growth medium comprising glycerol comprises: (a) transforming a parental bacterium by (i) the insertion of a heterologous gene encoding...... glycerol dehydrogenase; and/or (ii) up-regulating a native gene encoding glycerol dehydrogenase; and (b) obtaining the recombinant bacterium. Preferred Bacterium: In the recombinant bacterium above, the inserted heterologous gene and/or the up-regulated native gene is encoding a glycerol dehydrogenase...

Some Properties of Glutamate Dehydrogenase from the Marine Red ...

African Journals Online (AJOL)

Keywords: ammonia assimilation, glutamate dehydrogenase, GDH, Gracilaria sordida, red alga, enzyme activity. Glutamate dehydrogenases (GDH, EC ... Anabolic functions could be assimilation of ammonia released during photorespiration and synthesis of N-rich transport compounds. Western Indian Ocean Journal of ...

Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D., E-mail: vappanna@laurentian.ca

2014-11-07

Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

Science.gov (United States)

Vogel, O; Hoehn, B; Henning, U

1972-06-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

Determination of Aldehyde Dehydrogenase (ALDH Isozymes in Human Cancer Samples - Comparison of Kinetic and Immunochemical Assays

Directory of Open Access Journals (Sweden)

Dorota Borecka

2002-12-01

Full Text Available A fluorimetric assay of aldehyde dehydrogenase isozymes, based on naphthaldehyde oxidation, is compared with Western Blotting analysis on several clinical samples obtained from surgery. The comparison reveals qualitatively good correlation of ALDH1A1 isozyme detection with two methods and somewhat worse on ALDH3A1 assay.

Pronounced between-subject and circadian variability in thymidylate synthase and dihydropyrimidine dehydrogenase enzyme activity in human volunteers

NARCIS (Netherlands)

Jacobs, Bart A W; Deenen, Maarten J; Pluim, Dick; van Hasselt, J G Coen; Krähenbühl, Martin D; van Geel, Robin M J M; de Vries, Niels; Rosing, Hilde; Meulendijks, Didier; Burylo, Artur M; Cats, Annemieke; Beijnen, Jos H; Huitema, Alwin D R; Schellens, Jan H M

AIMS: The enzymatic activity of dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are important for the tolerability and efficacy of the fluoropyrimidine drugs. In the present study, we explored between-subject variability (BSV) and circadian rhythmicity in DPD and TS activity in

Variation in gastric alcohol dehydrogenase and the risk of alcohol dependence

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Paulina Całka

2017-03-01

Full Text Available Alcohol dependence is both a medical and socioeconomic problem. The disease is multifactorial, i.e. its development is attributable to gene-gene and gene-environment interactions. Multi-centre studies investigating the genetic background of alcoholism stress the role of genes encoding enzymes of the ethanol decomposition pathway in the human body, particularly alcohol dehydrogenase (ADH, in the development of alcohol dependence. Among five classes of alcohol dehydrogenases, class I and IV isoenzymes have been found to be associated with alcohol dependence. Class IV is of particular interest due to its occurrence in the upper gastrointestinal tract, mainly in the stomach. No activity of the enzyme has been demonstrated in the liver. Single nucleotide polymorphism (SNP of the gene encoding ADH class IV (ADH7 affects its ethanol-oxidizing activity in the gastric lumen, thereby influencing the first-pass metabolism (FPM of the substance. The findings published by various research centres have demonstrated that specific SNP changes in the ADH7 gene are of different significance for the risk of alcohol dependence according to the population studied.

Kinetics of soil dehydrogenase in response to exogenous Cd toxicity

Energy Technology Data Exchange (ETDEWEB)

Tan, Xiangping [College of Natural Resources and Environment, Northwest A& F University, Yangling, 712100, Shaanxi (China); Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, CAS 723 Xingke Rd., Tianhe District, Guangzhou 510650 (China); Wang, Ziquan; Lu, Guannan [College of Natural Resources and Environment, Northwest A& F University, Yangling, 712100, Shaanxi (China); He, Wenxiang, E-mail: wenxianghe@nwafu.edu.cn [College of Natural Resources and Environment, Northwest A& F University, Yangling, 712100, Shaanxi (China); Key Laboratory of Plant Nutrition and Agro-environment in Northwest China, Ministry of Agriculture, Northwest A& F University, Yangling, 712100, Shaanxi (China); Wei, Gehong [College of Life Sciences, Northwest A& F University, Yangling, 712100, Shaanxi (China); Huang, Feng; Xu, Xinlan; Shen, Weijun [Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, CAS 723 Xingke Rd., Tianhe District, Guangzhou 510650 (China)

2017-05-05

Highlights: • pH explained 30–45% of the dehydrogenase activity (DHA), V{sub max}, and K{sub m} variations across soils. • Different inhibition mechanism of Cd to DHA varied soil types. • Soil properties and inhibition constant affect the toxicity of Cd. • Reaction constant (k) could indicate sensitively the toxicity of Cd to DHA. - Abstract: Soil dehydrogenase plays a role in the biological oxidation of soil organic matter and can be considered a good measure of the change of microbial oxidative activity under environmental pollutions. However, the kinetic characteristic of soil dehydrogenase under heavy metal stresses has not been investigated thoroughly. In this study, we characterized the kinetic characteristic of soil dehydrogenase in 14 soil types, and investigated how kinetic parameters changed under spiked with different concentrations of cadmium (Cd). The results showed that the K{sub m} and V{sub max} values of soil dehydrogenase was among 1.4–7.3 mM and 15.9–235.2 μM h{sup −1} in uncontaminated soils, respectively. In latosolic red soil and brown soil, the inhibitory kinetic mechanism of Cd to soil dehydrogenase was anticompetitive inhibition with inhibition constants (K{sub i}) of 12 and 4.7 mM, respectively; in other soils belonged to linear mixed inhibition, the values of K{sub i} were between 0.7–4.2 mM. Soil total organic carbon and K{sub i} were the major factors affecting the toxicity of Cd to dehydrogenase activity. In addition, the velocity constant (k) was more sensitive to Cd contamination compared to V{sub max} and K{sub m}, which was established as an early indicator of gross changes in soil microbial oxidative activity caused by Cd contamination.

Mitochondrial type II NAD(PH dehydrogenases in fungal cell death

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A. Pedro Gonçalves

2015-03-01

Full Text Available During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(PH dehydrogenases (also called alternative NAD(PH dehydrogenases are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(PH dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(PH dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF-family.

Pyruvate dehydrogenase complex and lactate dehydrogenase as targets for therapy of acute liver failure.

Science.gov (United States)

Ferriero, Rosa; Nusco, Edoardo; De Cegli, Rossella; Carissimo, Annamaria; Manco, Giuseppe; Brunetti-Pierri, Nicola

2018-03-23

Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate in the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-Ab, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by Gene Ontology Enrichment Analysis. Efficacy of histone acetyltransferase inhibitor garcinol and LDH inhibitor galloflavin at reducing liver damage was evaluated in mice with induced hepatotoxicity. Levels and activities of PDHC and LDH were increased in cytoplasmatic and nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-coA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to response to damage. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus and are targets for therapy of acute liver failure. Acute liver failure is a rapidly progressive and life-threatening deterioration of liver function resulting in high mortality and

Pyruvate dehydrogenase kinase inhibition: Reversing the Warburg effect in cancer therapy

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Hayden Bell

2016-06-01

Full Text Available The poor efficacy of many cancer chemotherapeutics, which are often non-selective and highly toxic, is attributable to the remarkable heterogeneity and adaptability of cancer cells. The Warburg effect describes the up regulation of glycolysis as the main source of adenosine 5’-triphosphate in cancer cells, even under normoxic conditions, and is a unique metabolic phenotype of cancer cells. Mitochondrial suppression is also observed which may be implicated in apoptotic suppression and increased funneling of respiratory substrates to anabolic processes, conferring a survival advantage. The mitochondrial pyruvate dehydrogenase complex is subject to meticulous regulation, chiefly by pyruvate dehydrogenase kinase. At the interface between glycolysis and the tricarboxylic acid cycle, the pyruvate dehydrogenase complex functions as a metabolic gatekeeper in determining the fate of glucose, making pyruvate dehydrogenase kinase an attractive candidate in a bid to reverse the Warburg effect in cancer cells. The small pyruvate dehydrogenase kinase inhibitor dichloroacetate has, historically, been used in conditions associated with lactic acidosis but has since gained substantial interest as a potential cancer chemotherapeutic. This review considers the Warburg effect as a unique phenotype of cancer cells in-line with the history of and current approaches to cancer therapies based on pyruvate dehydrogenase kinase inhibition with particular reference to dichloroacetate and its derivatives.

The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

Energy Technology Data Exchange (ETDEWEB)

MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth (BWH); (Brandeis)

2010-03-29

Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

Energy Technology Data Exchange (ETDEWEB)

Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, University of Leicester, Leicester LE1 9HN (United Kingdom)

2008-08-01

Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

9-Hydroxyprostaglandin dehydrogenase activity in the adult rat kidney. Regional distribution and sub-fractionation.

Science.gov (United States)

Asciak, C P; Domazet, Z

1975-02-20

1. Catabolism of prostaglandin F2alpha in the adult rat kidney takes place by the following sequence of enzymatic steps: (1) 15-hydroxyprostaglandin dehydrogenase; (2) prostaglandin delta13-reductase; and (3) 9-hydroxyprostaglandin dehydrogenase. 2. 9-Hydroxyprostaglandin dehydrogenase activity was highest in the cortex with lesser amounts in the medulla and negligible activity detected in the papilla. A similar distribution was observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 3. Most of the 9-hydroxyprostaglandin dehydrogenase activity in the homogenate was found in the high-speed supernatant as also observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 4. These observations indicate that the rat kidney contains an abundance of prostaglandin-catabolising enzymes which favour formation of metabolites of the E-type.

High-fat diet enhanced retinal dehydrogenase activity, but suppressed retinol dehydrogenase activity in liver of rats

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Mian Zhang

2015-04-01

Full Text Available Evidence has shown that hyperlipidemia is associated with retinoid dyshomeostasis. In liver, retinol is mainly oxidized to retinal by retinol dehydrogenases (RDHs and alcohol dehydrogenases (ADHs, further converted to retinoic acid by retinal dehydrogenases (RALDHs. The aim of this study was to investigate whether high-fat diet (HFD induced hyperlipidemia affected activity and expression of hepatic ADHs/RDHs and RALDHs in rats. Results showed that retinol levels in liver, kidney and adipose tissue of HFD rats were significantly increased, while plasma retinol and hepatic retinal levels were markedly decreased. HFD rats exhibited significantly downregulated hepatic ADHs/RDHs activity and Adh1, Rdh10 and Dhrs9 expression. Oppositely, hepatic RALDHs activity and Raldh1 expression were upregulated in HFD rats. In HepG2 cells, treatment of HFD rat serum inhibited ADHs/RDHs activity and induced RALDHs activity. Among the tested abnormally altered components in HFD rat serum, cholesterol reduced ADHs/RDHs activity and RDH10 expression, while induced RALDHs activity and RALDH1 expression in HepG2 cells. Contrary to the effect of cholesterol, cholesterol-lowering agent pravastatin upregulated ADHs/RDHs activity and RDH10 expression, while suppressed RALDHs activity and RALDH1 expression. In conclusion, hyperlipidemia oppositely altered activity and expression of hepatic ADHs/RDHs and RALDHs, which is partially due to the elevated cholesterol levels.

Alcohol dehydrogenase and aldehyde dehydrogenase gene polymorphisms, alcohol intake and the risk of colorectal cancer in the European Prospective Investigation into Cancer and Nutrition study

DEFF Research Database (Denmark)

Ferrari, P.; McKay, J. D.; Jenab, M.

2012-01-01

BACKGROUND/OBJECTIVES: Heavy alcohol drinking is a risk factor of colorectal cancer (CRC), but little is known on the effect of polymorphisms in the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) on the alcohol-related risk of CRC in Caucasian populati...

ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase

Science.gov (United States)

Giffin, Michelle M.; Modesti, Lucia; Raab, Ronald W.; Wayne, Lawrence G.

2012-01-01

The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown. PMID:22210765

11β-Hydroxysteroid Dehydrogenases: Intracellular Gate-Keepers of Tissue Glucocorticoid Action

Science.gov (United States)

Chapman, Karen; Holmes, Megan

2013-01-01

Glucocorticoid action on target tissues is determined by the density of “nuclear” receptors and intracellular metabolism by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyze interconversion of active cortisol and corticosterone with inert cortisone and 11-dehydrocorticosterone. 11β-HSD type 1, a predominant reductase in most intact cells, catalyzes the regeneration of active glucocorticoids, thus amplifying cellular action. 11β-HSD1 is widely expressed in liver, adipose tissue, muscle, pancreatic islets, adult brain, inflammatory cells, and gonads. 11β-HSD1 is selectively elevated in adipose tissue in obesity where it contributes to metabolic complications. Similarly, 11β-HSD1 is elevated in the ageing brain where it exacerbates glucocorticoid-associated cognitive decline. Deficiency or selective inhibition of 11β-HSD1 improves multiple metabolic syndrome parameters in rodent models and human clinical trials and similarly improves cognitive function with ageing. The efficacy of inhibitors in human therapy remains unclear. 11β-HSD2 is a high-affinity dehydrogenase that inactivates glucocorticoids. In the distal nephron, 11β-HSD2 ensures that only aldosterone is an agonist at mineralocorticoid receptors (MR). 11β-HSD2 inhibition or genetic deficiency causes apparent mineralocorticoid excess and hypertension due to inappropriate glucocorticoid activation of renal MR. The placenta and fetus also highly express 11β-HSD2 which, by inactivating glucocorticoids, prevents premature maturation of fetal tissues and consequent developmental “programming.” The role of 11β-HSD2 as a marker of programming is being explored. The 11β-HSDs thus illuminate the emerging biology of intracrine control, afford important insights into human pathogenesis, and offer new tissue-restricted therapeutic avenues. PMID:23899562

X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase

Energy Technology Data Exchange (ETDEWEB)

Yennawar, Hemant [Pennsylvania State University, 8 Althouse Laboratory, University Park, PA 16802 (United States); Møller, Magda [Cornell High Energy Synchrotron Source, Ithaca, NY 14853 (United States); University of Copenhagen, DK-2100 Copenhagen (Denmark); Gillilan, Richard [Cornell High Energy Synchrotron Source, Ithaca, NY 14853 (United States); Yennawar, Neela, E-mail: nhy1@psu.edu [Pennsylvania State University, 8 Althouse Laboratory, University Park, PA 16802 (United States)

2011-05-01

The X-ray crystal structure and a small-angle X-ray scattering solution structure of sheep liver sorbitol dehydrogenase have been determined. The details of the interactions that enable the tetramer scaffold to be the functional biological unit have been analyzed. The X-ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular-replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer that superposes well with that seen in hSDH (despite belonging to a different space group) and obeying the 222 crystal symmetry is seen in slSDH. An acetate molecule is bound in the active site, coordinating to the active-site zinc through a water molecule. Glycerol, a substrate of slSDH, also occupies the substrate-binding pocket together with the acetate designed by nature to fit large polyol substrates. The substrate-binding pocket is seen to be in close proximity to the tetramer interface, which explains the need for the structural integrity of the tetramer for enzyme activity. Small-angle X-ray scattering was also used to identify the quaternary structure of the tetramer of slSDH in solution.

Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon[S

Science.gov (United States)

Lee, Ja-Young; Arai, Hisashi; Nakamura, Yusuke; Fukiya, Satoru; Wada, Masaru; Yokota, Atsushi

2013-01-01

Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90–100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7β-hydroxysteroid dehydrogenase (7β-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7β-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon. PMID:23729502

Screening of Glucose-6-Phosphate Dehydrogenase Deficiency in Cord Blood

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Can Acipayam

2014-02-01

Aim: Glucose-6-phosphate dehydrogenase deficiency is an important factor in etiology of pathologic neonatal jaundice. The aim of this study was to indicate the significance of screening glucose-6-phosphate dehydrogenase deficiency in the cord blood of neonates and the frequency of this deficiency in the etiology of neonatal hyperbilirubinemia. Material and Method: The study was performed consecutive 1015 neonates were included. Five hundred fifty six (54.8% of them were male and 459 (45.2% were female. The following parameters were recorded: Gender, birth weight, birth height, head circumference and gestational age. The glucose-6-phosphate dehydrogenase level of neonates were measured with quantitative method in cord blood. Also, hemoglobine, hematocrite, red blood cell count and blood group were measured. The following parameters were recorded in cases with jaundice: exchange transfusion, phototherapy, physiologic and pathologic jaundice, peak bilirubin day, maximum bilirubin level, total bilirubin level at the first day of jaundice, beginning time of jaundice. Results: Enzyme deficiency was detected in 133 (13.1% of neonates and 76 (57% of them were male, 57 (43% were female. Significant difference was detected in low glucose-6-phosphate dehydrogenase enzyme level with jaundice group for total bilirubin level at the first day of jaundice, maximum total bilirubin level and pathologic jaundice (p<0.05. Discussion: The ratio of glucose-6-phosphate dehydrogenase deficiency was found in Edirne in this study and this ratio was higher than other studies conducted in our country. For this reason, glucose-6-phosphate dehydrogenase enzyme level in cord blood of neonates should be measured routinely and high risk neonates should be followed up for hyperbilirubinemia and parents should be informed in our region.

Leflunomide/teriflunomide inhibit Epstein-Barr virus (EBV)- induced lymphoproliferative disease and lytic viral replication.

Science.gov (United States)

Bilger, Andrea; Plowshay, Julie; Ma, Shidong; Nawandar, Dhananjay; Barlow, Elizabeth A; Romero-Masters, James C; Bristol, Jillian A; Li, Zhe; Tsai, Ming-Han; Delecluse, Henri-Jacques; Kenney, Shannon C

2017-07-04

EBV infection causes mononucleosis and is associated with specific subsets of B cell lymphomas. Immunosuppressed patients such as organ transplant recipients are particularly susceptible to EBV-induced lymphoproliferative disease (LPD), which can be fatal. Leflunomide (a drug used to treat rheumatoid arthritis) and its active metabolite teriflunomide (used to treat multiple sclerosis) inhibit de novo pyrimidine synthesis by targeting the cellular dihydroorotate dehydrogenase, thereby decreasing T cell proliferation. Leflunomide also inhibits the replication of cytomegalovirus and BK virus via both "on target" and "off target" mechanisms and is increasingly used to treat these viruses in organ transplant recipients. However, whether leflunomide/teriflunomide block EBV replication or inhibit EBV-mediated B cell transformation is currently unknown. We show that teriflunomide inhibits cellular proliferation, and promotes apoptosis, in EBV-transformed B cells in vitro at a clinically relevant dose. In addition, teriflunomide prevents the development of EBV-induced lymphomas in both a humanized mouse model and a xenograft model. Furthermore, teriflunomide inhibits lytic EBV infection in vitro both by preventing the initial steps of lytic viral reactivation, and by blocking lytic viral DNA replication. Leflunomide/teriflunomide might therefore be clinically useful for preventing EBV-induced LPD in patients who have high EBV loads yet require continued immunosuppression.

Histochemical localization of cytokinin oxidase/dehydrogenase ...

African Journals Online (AJOL)

Jane

2011-08-15

dehydrogenase, Withania somnifera, CKX localization. INTRODUCTION. Cytokinin (Ck) is a plant hormone that plays a crucial role in many fundamental processes of plant development throughout the life cycle. These include ...

The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey.

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Thomas Gronemeyer

Full Text Available The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

Evidence of redox imbalance in a patient with succinic semialdehyde dehydrogenase deficiency

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Anna-Kaisa Niemi

2014-01-01

Full Text Available The pathophysiology of succinic semialdehyde dehydrogenase (SSADH deficiency is not completely understood. Oxidative stress, mitochondrial pathology, and low reduced glutathione levels have been demonstrated in mice, but no studies have been reported in humans. We report on a patient with SSADH deficiency in whom we found low levels of blood reduced glutathione (GSH, and elevations of dicarboxylic acids in urine, suggestive of possible redox imbalance and/or mitochondrial dysfunction. Thus, targeting the oxidative stress axis may be a potential therapeutic approach if our findings are confirmed in other patients.

Toxicity of Nitrification Inhibitors on Dehydrogenase Activity in Soils

OpenAIRE

Ferisman Tindaon; Gero Benckiser; Johannes C. G. Ottow

2011-01-01

The objective of this research was to determine the effects of nitrification inhibitors (NIs) such as 3,4-dimethylpyrazolephosphate=DMPP, 4-Chlor-methylpyrazole phosphate=ClMPP and dicyandiamide,DCD) which might be expected to inhibit microbial activity, on dehydrogenase activity (DRA),in three different soils in laboratory conditions. Dehydrogenase activity were assessed via reduction of 2-p-Iodophenyl-3-p-nitrophenyl-5-phenyltetrazoliumchloride (INT). The toxicity and dose response curve of...

Lactate dehydrogenase-B is silenced by promoter methylation in a high frequency of human breast cancers.

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Nicola J Brown

Full Text Available Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.Lactate Dehydrogenase (LDH isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/25 cases of breast cancer tissues, but not in 5/5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained, was seen in 23/26 (88% breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O(2, for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002, and T-47D cells (2.9 fold, p = 0.009, but not in MDA-MB-436 (-0.9 fold, p = 0.229, or MCF10AT (1.2 fold, p = 0.09 cells.Loss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.

Effects of sh-reagents on rat hepatic aldehyde dehydrogenase activity

Energy Technology Data Exchange (ETDEWEB)

Konoplitskaya, K.L.; Kuz' mina, G.I.; Grigor' yeva, M.V.; Poznyakova, T.N.

The liver serves as the primary organ for the oxidation of ingested ethanol via a pathway involving alcohol- and aldehyde dehydrogenase. In view of the problem of alcoholism, three enzymes are of particular interest in understanding the biochemical mechanism that may be involved in alcohol addiction and in the formulation of therapeutic approaches. While alcohol dehydrogenase has been studied in considerable detail, current attention is centered on aldehyde dehydrogenase. A comparative analysis of the effects of a series of SH-active reagents - tetraethylthiuram disulfide (TETD), 5,5-dithiobisnitrobenzoic acid (DTNB), p-chloromercurybenzoate (PCMB), and N-ethylmaleimide (NEM) - were tested for their effects on the activity of aldehyde dehydrogenase of the hepatic mitochondrial (isozymes I and II) and microsomal (isozyme II) fractions of outbred albino rats. DTNB was found to be inhibited by 100 and 50% mitochondrial isozymes I and II, respectively, and by 20%, the microsomal enzyme under the conditions employed. DTNB and NEM inhibited by 30 and 50% isozymes I and II of the mitochondria, but had no effect on the microsomal isozyme. 24 references, 3 figures.

Multiple states of the Tyr318Leu mutant of dihydroorotate dehydrogenase revealed by single molecule kinetics

DEFF Research Database (Denmark)

Shi, J.; Palfey, B.A.; Dertouzos, J.

2004-01-01

, with some molecules going through the on-off cycles 5-fold faster than others, however, there is no detectable dynamic disorder in DHOD turnover. When 0.1% reduced Triton X-100, a detergent that more closely simulates the natural membrane environment, is added, our data suggest the degree of static...

Kinetic and biophysical investigation of the inhibitory effect of caffeine on human salivary aldehyde dehydrogenase: Implications in oral health and chemotherapy

Science.gov (United States)

Laskar, Amaj Ahmed; Alam, Md Fazle; Ahmad, Mohammad; Younus, Hina

2018-04-01

Human salivary aldehyde dehydrogenase (hsALDH) is primarily a class 3 ALDH (ALDH3A1), and is an important antioxidant enzyme present in the saliva which maintains healthy oral cavity. It detoxifies toxic aldehydes into non-toxic carboxylic acids in the oral cavity. Reduced level of hsALDH activity is a risk factor for oral cancer development. It is involved in the resistance of certain chemotherapeutic drugs. Coffee has been reported to affect the activity of salivary ALDH. In this study, the effect of caffeine on the activity (dehydrogenase and esterase) of hsALDH was investigated. The binding of caffeine to hsALDH was studied using different biophysical methods and molecular docking analysis. Caffeine was found to inhibit both crude and purified hsALDH. The Km increased and the Vmax decreased showing a mixed type of inhibition. Caffeine decreased the nucleophilicity of the catalytic cysteine residue. It binds to the active site of ALDH3A1 by forming a complex through non-covalent interactions with some highly conserved amino acid residues. It partially alters the secondary structure of the enzyme. Therefore, it is very likely that caffeine binds and inhibits the activity of hsALDH by decreasing substrate binding affinity and the catalytic efficiency of the enzyme. The study indicates that oral intake of caffeine may have a harmful effect on the oral health and may increase the risk of carcinogenesis through the inhibition of this important enzyme. Further, the inactivation of oxazaphosphorine based chemotherapeutic drugs by ALDH3A1 may be prevented by using caffeine as an adjuvant during medication which is expected to increase the sensitivity of these drugs through its inhibitory effect on the enzyme.

Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci

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Guillermo Hugo Peralta

Full Text Available ABSTRACT Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

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Wang, Jizhong; Yang, Chengli; Chen, Xing; Bao, Bingxin; Zhang, Xuan; Li, Dali; Du, Xingfan; Shi, Ruofu; Yang, Junfang; Zhu, Ronghui

2016-08-01

To find an efficient and cheap system for NAD(+) regeneration A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

OpenAIRE

Napora-Wijata, Kamila; Strohmeier, Gernot A.; Sonavane, Manoj N.; Avi, Manuela; Robins, Karen; Winkler, Margit

2013-01-01

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisia...

Enhancement of Nucleoside Production in Hirsutella sinensis Based on Biosynthetic Pathway Analysis

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Liu, Zhi-Qiang; Zhang, Bo; Lin, Shan; Baker, Peter James; Chen, Mao-Sheng; Xue, Ya-Ping; Wu, Hui; Xu, Feng; Yuan, Shui-Jin; Teng, Yi; Wu, Ling-Fang

2017-01-01

To enhance nucleoside production in Hirsutella sinensis, the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase, inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase, uridine nucleosidase, uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis. PMID:29333435

Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

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Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-11-02

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

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Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-01-01

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

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Wakamatsu, Taisuke; Sakuraba, Haruhiko; Kitamura, Megumi; Hakumai, Yuichi; Fukui, Kenji; Ohnishi, Kouhei; Ashiuchi, Makoto; Ohshima, Toshihisa

2017-01-15

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P) + -dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD + Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD + /NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the

The Effect of Direct Current Transthoracic Countershock on Human Myocardial Cells Evidenced by Creatine Kinase and Lactic Dehydrogenase Isoenzymes.

Science.gov (United States)

1986-05-01

however, fractionation of these enzymes will identify their specific source. Plasma levels of CK isoenzymes (CKMB) and LDH isoenzymes ( LDHI ) are most...damage--inferred by isoenzyme levels of CKMB and/or LDHI in the serum above normal levels (see definitions of creatine kinase and lactic dehydrogenase

The Role of Pyruvate Dehydrogenase Kinase in Diabetes and Obesity

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In-Kyu Lee

2014-06-01

Full Text Available The pyruvate dehydrogenase complex (PDC is an emerging target for the treatment of metabolic syndrome. To maintain a steady-state concentration of adenosine triphosphate during the feed-fast cycle, cells require efficient utilization of fatty acid and glucose, which is controlled by the PDC. The PDC converts pyruvate, coenzyme A (CoA, and oxidized nicotinamide adenine dinucleotide (NAD+ into acetyl-CoA, reduced form of nicotinamide adenine dinucleotide (NADH, and carbon dioxide. The activity of the PDC is up- and down-regulated by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase, respectively. In addition, pyruvate is a key intermediate of glucose oxidation and an important precursor for the synthesis of glucose, glycerol, fatty acids, and nonessential amino acids.

Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization.

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Shimojo, M; Ricketts, M L; Petrelli, M D; Moradi, P; Johnson, G D; Bradwell, A R; Hewison, M; Howie, A J; Stewart, P M

1997-03-01

11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the

Kinetic studies of the inhibition of a human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme by bile acids and anti-inflammatory drugs.

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Miyabe, Y; Amano, T; Deyashiki, Y; Hara, A; Tsukada, F

1995-01-01

We have investigated the steady-state kinetics for a cytosolic 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme of human liver and its inhibition by several bile acids and anti-inflammatory drugs such as indomethacin, flufemanic acid and naproxen. Initial velocity and product inhibition studies performed in the NADP(+)-linked (S)-1-indanol oxidation at pH 7.4 were consistent with a sequential ordered mechanism in which NADP+ binds first and leaves last. The bile acids and drugs, competitive inhibitors with respect to the alcohol substrate, exhibited uncompetitive inhibition with respect to the coenzyme, with Ki values less than 1 microM, whereas indomethacin exhibited noncompetitive inhibition (Ki < 24 microM). The kinetics of the inhibition by a mixture of the two inhibitors suggests that bile acids and drugs, except indomethacin, bind to overlapping sites at the active center of the enzyme-coenzyme binary complex.

Soil dehydrogenase activity of natural macro aggregates in a toposequence of forest soil

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Maira Kussainova

2013-01-01

Full Text Available The main objective of this study was to determine changes in soil dehydrogenase activity in natural macro aggregates development along a slope in forest soils. This study was carried out in Kocadag, Samsun, Turkey. Four landscape positions i.e., summit, shoulder backslope and footslope, were selected. For each landseape position, soil macro aggregates were separated into six aggregate size classes using a dry sieving method and then dehydrogenase activity was analyzed. In this research, topography influenced the macroaggregate size and dehydrogenase activity within the aggregates. At all landscape positions, the contents of macro aggregates (especially > 6.3 mm and 2.00–4.75 mm in all soil samples were higher than other macro aggregate contents. In footslope position, the soils had generally the higher dehydrogenase activity than the other positions at all landscape positions. In all positions, except for shoulder, dehydrogenase activity was greater macro aggregates of <1 mm than in the other macro aggregate size.

Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase Expression in Non Small Cell Lung Cancer and Tumor-Associated Stroma

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Michael I. Koukourakis

2005-01-01

Full Text Available Pyruvate dehydrogenase (PDH catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs. Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5. In cancer cells, however, pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect. Although hypoxic intratumoral conditions account for HIFia stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIFia stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIFia stabilization and “aerobic glycolysis.” However, about half of PDHdeficient carcinomas are not able to switch on the HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor-supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time.

Characterization of the L-lactate dehydrogenase from Aggregatibacter actinomycetemcomitans.

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Stacie A Brown

Full Text Available Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a K(m of approximately 150 microM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans.

Molecular Basis for Converting (2S-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

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Simon Burgener

2017-12-01

Full Text Available Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

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Koji Sode

2012-11-01

Full Text Available Mutagenesis studies on glucose oxidases (GOxs were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe and Aspergillus niger GOx (PDB ID; 1cf3. We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

Structural studies of MFE-1: the 1.9 A crystal structure of the dehydrogenase part of rat peroxisomal MFE-1.

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Taskinen, Jukka P; Kiema, Tiila R; Hiltunen, J Kalervo; Wierenga, Rik K

2006-01-27

The 1.9 A structure of the C-terminal dehydrogenase part of the rat peroxisomal monomeric multifunctional enzyme type 1 (MFE-1) has been determined. In this construct (residues 260-722 and referred to as MFE1-DH) the N-terminal hydratase part of MFE-1 has been deleted. The structure of MFE1-DH shows that it consists of an N-terminal helix, followed by a Rossmann-fold domain (domain C), followed by two tightly associated helical domains (domains D and E), which have similar topology. The structure of MFE1-DH is compared with the two known homologous structures: human mitochondrial 3-hydroxyacyl-CoA dehydrogenase (HAD; sequence identity is 33%) (which is dimeric and monofunctional) and with the dimeric multifunctional alpha-chain (alphaFOM; sequence identity is 28%) of the bacterial fatty acid beta-oxidation alpha2beta2-multienzyme complex. Like MFE-1, alphaFOM has an N-terminal hydratase part and a C-terminal dehydrogenase part, and the structure comparisons show that the N-terminal helix of MFE1-DH corresponds to the alphaFOM linker helix, located between its hydratase and dehydrogenase part. It is also shown that this helix corresponds to the C-terminal helix-10 of the hydratase/isomerase superfamily, suggesting that functionally it belongs to the N-terminal hydratase part of MFE-1.

Molecular diagnosis and characterization of medium-chain acyl-CoA dehydrogenase deficiency

DEFF Research Database (Denmark)

Andresen, B S; Bross, P; Jensen, T G

1995-01-01

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in mitochondrial beta-oxidation in humans. It is an autosomal recessive disorder which usually presents in infancy. The disease manifests itself in periods of metabolic stress to the beta-oxidation system and may...... of correct enzyme structure, and does not directly affect the catalytically active regions of the enzyme. We find that our diagnostic set up, consisting of an initial testing by the G985 assay, followed by semi-automated sequencing of DNA from those patients who were indicated to be compound heterozygous...

Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

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Archana B Siva

Full Text Available BACKGROUND/AIMS: The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc and its E3 subunit, dihydrolipoamide dehydrogenase (DLD in hamster in vitro fertilization (IVF via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. METHODOLOGY AND PRINCIPAL FINDINGS: Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid. Oocytes fertilized with MICA-treated (MT [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. CONCLUSIONS: This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In

Newborn screening for dihydrolipoamide dehydrogenase deficiency: Citrulline as a useful analyte

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Shane C. Quinonez

2014-01-01

Full Text Available Dihydrolipoamide dehydrogenase deficiency, also known as maple syrup urine disease (MSUD type III, is caused by the deficiency of the E3 subunit of branched chain alpha-ketoacid dehydrogenase (BCKDH, α-ketoglutarate dehydrogenase (αKGDH, and pyruvate dehydrogenase (PDH. DLD deficiency variably presents with either a severe neonatal encephalopathic phenotype or a primarily hepatic phenotype. As a variant form of MSUD, it is considered a core condition recommended for newborn screening. The detection of variant MSUD forms has proven difficult in the past with no asymptomatic DLD deficiency patients identified by current newborn screening strategies. Citrulline has recently been identified as an elevated dried blood spot (DBS metabolite in symptomatic patients affected with DLD deficiency. Here we report the retrospective DBS analysis and second-tier allo-isoleucine testing of 2 DLD deficiency patients. We show that an elevated citrulline and an elevated allo-isoleucine on second-tier testing can be used to successfully detect DLD deficiency. We additionally recommend that DLD deficiency be included in the “citrullinemia/elevated citrulline” ACMG Act Sheet and Algorithm.

What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

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Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M S; Engel, Paul C

2008-08-01

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

Exercise training induces similar elevations in the activity of oxoglutarate dehydrogenase and peak oxygen uptake in the human quadriceps muscle

DEFF Research Database (Denmark)

Blomstrand, Eva; Krustrup, Peter; Søndergaard, Hans

2011-01-01

During exercise involving a small muscle mass, peak oxygen uptake is thought to be limited by peripheral factors, such as the degree of oxygen extraction from the blood and/or mitochondrial oxidative capacity. Previously, the maximal activity of the Krebs cycle enzyme oxoglutarate dehydrogenase has...

Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

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Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

2016-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site. PMID:27213370

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

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Broadbent, Jeffery R; Gummalla, Sanjay; Hughes, Joanne E; Johnson, Mark E; Rankin, Scott A; Drake, Mary Anne

2004-08-01

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

Identification and Overexpression of a Bifunctional Aldehyde/Alcohol Dehydrogenase Responsible for Ethanol Production in Thermoanaerobacter mathranii

DEFF Research Database (Denmark)

Yao, Shuo; Just Mikkelsen, Marie

2010-01-01

Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (Adh....... Overexpressions of AdhE in strain BG1E1 with xylose as a substrate facilitate the production of ethanol at an increased yield. Copyright © 2010 S. Karger AG, Basel...

Peroxyl radical- and photo-oxidation of glucose 6- phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues

DEFF Research Database (Denmark)

Leinisch, Fabian; Mariotti, Michele; Rykær, Martin

2017-01-01

indicate that pathophysiological processes and multiple human diseases are associated with the accumulation of damaged proteins. In this study we investigated the mechanisms and consequences of exposure of the key metabolic enzyme glucose-6-phosphate dehydrogenase (G6PDH) to peroxyl radicals (ROO...

Engineering of Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

DEFF Research Database (Denmark)

Ortiz, Roberto; Rahman, Mahbubur; Zangrilli, Beatrice

2017-01-01

Cellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome capable of direct electron transfer (DET). Unlike other CDHs, the pH optimum for CDHs from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) is close to the human physiological pH in blood (7.4). These are......, therefore, interesting candidates for glucose measurements in human blood and the application in enzymatic fuel cells is, however, limited by their relatively low activity with this substrate. In this work, the substrate specificities of CtCDH and HiCDH have been altered by a single cysteine to tyrosine...... substitution in the active sites of CtCDH (position 291) and HiCDH (position 285), which resulted in improved kinetic constants with glucose while decreasing the activity with several disaccharides, including maltose. The DET properties of the generated CDH variants were tested in the absence...

Effect of Punica granatum fruit peel on glucose-6-phosphate dehydrogenase and malate dehydrogenase in amphistome Gastrothylax indicus.

Science.gov (United States)

Aggarwal, Rama; Bagai, Upma

2017-03-01

Increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against helminth parasite in sheep. Important lipogenic enzymes like glucose-6-phosphate dehydrogenase (G-6-PDH) and malate dehydrogenase (MDH) show subcellular distribution pattern. Activity of G-6-PDH was largely restricted to cytosolic fraction while MDH was found in both cytosolic and mitochondrial fraction in Gastrothylax indicus. Following in vitro treatment with ethanolic and aqueous extracts of Punica granatum fruit peel and commercial anthelmintic, albendazole G-6-PDH activity was decreased by 19-32 %, whereas MDH was suppressed by 24-41 %, compared to the respective control. Albendazole was quite effective when compared with negative control and both the extracts. The results indicate that phytochemicals of plant may act as potential vermifuge or vermicide.

High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

Science.gov (United States)

Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

2017-06-01

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix

Homology modelling and docking analysis of L-lactate dehydrogenase from Streptococcus thermopilus

Directory of Open Access Journals (Sweden)

Vukić Vladimir R.

2016-01-01

Full Text Available The aim of this research was to create a three-dimensional model of L-lactate dehydrogenase from the main yoghurt starter culture - Streptococcus thermopilus, to analyse its structural features and investigate substrate binding in the active site. NCBI BlastP was used against the Protein Data Bank database in order to identify the template for construction of homology models. Multiple sequence alignment was performed using the program MUSCULE within the UGENE 1.11.3 program. Homology models were constructed using the program Modeller v. 9.17. The obtained 3D model was verified by Ramachandran plots. Molecular docking simulations were performed using the program Surflex-Dock. The highest sequence similarity was observed with L-lactate dehydrogenase from Lactobacillus casei subsp. casei, with 69% identity. Therefore, its structure (PDB ID: 2ZQY:A was selected as a modelling template for homology modelling. Active residues are by sequence similarity predicted: S. thermophilus - HIS181 and S. aureus - HIS179. Binding energy of pyruvate to L-lactate dehydrogenase of S. thermopilus was - 7.874 kcal/mol. Pyruvate in L-lactate dehydrogenase of S. thermopilus makes H bonds with catalytic HIS181 (1.9 Å, as well as with THR235 (3.6 Å. Although our results indicate similar position of substrates between L-lactate dehydrogenase of S. thermopilus and S. aureus, differences in substrate distances and binding energy values could influence the reaction rate. Based on these results, the L-lactate dehydrogenase model proposed here could be used as a guide for further research, such as transition states of the reaction through molecular dynamics. [Projekat Ministarstva nauke Republike Srbije, br. III 46009

The Diagnostic Significance of Serum Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase Activity in Urinary Bladder Cancer Patients.

Science.gov (United States)

Orywal, Karolina; Jelski, Wojciech; Werel, Tadeusz; Szmitkowski, Maciej

2017-07-01

The aim of this study was to investigate a potential role of alcohol dehydrogenase and aldehyde dehydrogenase as tumor markers for urinary bladder cancer. Serum samples were obtained from 41 patients with bladder cancer and 52 healthy individuals. Class III and IV of ADH and total ADH activity were measured by the photometric method. For measurement of class I and II ADH and ALDH activity, the fluorometric method was employed. Significantly higher total activity of ADH was found in sera of both, low-grade and high-grade bladder cancer patients. The diagnostic sensitivity for total ADH activity was 81.5%, specificity 98.1%, positive (PPV) and negative (NPV) predictive values were 97.4% and 92.3% respectively. Area under ROC curve for total ADH activity was 0.848. A potential role of total ADH activity as a marker for bladder cancer, is herein proposed. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Energy Technology Data Exchange (ETDEWEB)

Begley, Darren W.; Davies, Douglas R.; Hartley, Robert C.; Hewitt, Stephen N.; Rychel, Amanda L.; Myler, Peter J.; Van Voorhis, Wesley C.; Staker, Bart L.; Stewart, Lance J. (Emerald)

2014-10-02

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.

Efficiency of superoxide anions in the inactivation of selected dehydrogenases

International Nuclear Information System (INIS)

Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

2010-01-01

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as · OH and ONOO - . In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

Efficiency of superoxide anions in the inactivation of selected dehydrogenases

Energy Technology Data Exchange (ETDEWEB)

Rodacka, Aleksandra, E-mail: olakow@biol.uni.lodz.p [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Serafin, Eligiusz, E-mail: serafin@biol.uni.lodz.p [Laboratory of Computer and Analytical Techniques, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Puchala, Mieczyslaw, E-mail: puchala@biol.uni.lodz.p [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland)

2010-09-15

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as {sup {center_dot}}OH and ONOO{sup -}. In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

Retinol Dehydrogenases Regulate Vitamin A Metabolism for Visual Function

Directory of Open Access Journals (Sweden)

Bhubanananda Sahu

2016-11-01

Full Text Available The visual system produces visual chromophore, 11-cis-retinal from dietary vitamin A, all-trans-retinol making this vitamin essential for retinal health and function. These metabolic events are mediated by a sequential biochemical process called the visual cycle. Retinol dehydrogenases (RDHs are responsible for two reactions in the visual cycle performed in retinal pigmented epithelial (RPE cells, photoreceptor cells and Müller cells in the retina. RDHs in the RPE function as 11-cis-RDHs, which oxidize 11-cis-retinol to 11-cis-retinal in vivo. RDHs in rod photoreceptor cells in the retina work as all-trans-RDHs, which reduce all-trans-retinal to all-trans-retinol. Dysfunction of RDHs can cause inherited retinal diseases in humans. To facilitate further understanding of human diseases, mouse models of RDHs-related diseases have been carefully examined and have revealed the physiological contribution of specific RDHs to visual cycle function and overall retinal health. Herein we describe the function of RDHs in the RPE and the retina, particularly in rod photoreceptor cells, their regulatory properties for retinoid homeostasis and future therapeutic strategy for treatment of retinal diseases.

21 CFR 862.1500 - Malic dehydrogenase test system.

Science.gov (United States)

2010-04-01

... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver... marrow) leukemia. (b) Classification. Class I (general controls). The device is exempt from the premarket...

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

Science.gov (United States)

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

International Nuclear Information System (INIS)

Harris, R.A.; Powell, S.M.; Paxton, R.; Gillim, S.E.; Nagae, H.

1985-01-01

A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

Science.gov (United States)

Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2016-04-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

Optimization of Adsorptive Immobilization of Alcohol Dehydrogenases

NARCIS (Netherlands)

Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C.; Daussmann, Thomas; Büchs, Jochen

2005-01-01

In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently

Glucose-6-phosphate dehydrogenase deficiency and Alzheimer's disease: Partners in crime? The hypothesis.

Science.gov (United States)

Ulusu, N Nuray

2015-08-01

Alzheimer's disease is a multifaceted brain disorder which involves various coupled irreversible, progressive biochemical reactions that significantly reduce quality of life as well as the actual life expectancy. Aging, genetic predispositions, head trauma, diabetes, cardiovascular disease, deficiencies in insulin signaling, dysfunction of mitochondria-associated membranes, cerebrovascular changes, high cholesterol level, increased oxidative stress and free radical formation, DNA damage, disturbed energy metabolism, and synaptic dysfunction, high blood pressure, obesity, dietary habits, exercise, social engagement, and mental stress are noted among the risk factors of this disease. In this hypothesis review I would like to draw the attention on glucose-6-phosphate dehydrogenase deficiency and its relationship with Alzheimer's disease. This enzymopathy is the most common human congenital defect of metabolism and defined by decrease in NADPH+H(+) and reduced form of glutathione concentration and that might in turn, amplify oxidative stress due to essentiality of the enzyme. This most common enzymopathy may manifest itself in severe forms, however most of the individuals with this deficiency are not essentially symptomatic. To understand the sporadic Alzheimer's disease, the writer of this paper thinks that, looking into a crystal ball might not yield much of a benefit but glucose-6-phosphate dehydrogenase deficiency could effortlessly give some clues. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of two mutations detected in medium chain acyl-CoA dehydrogenase (MCAD)-deficient patients on folding, oligomer assembly, and stability of MCAD enzyme

DEFF Research Database (Denmark)

Bross, P; Jespersen, C; Jensen, T G

1995-01-01

We have used expression of human medium chain acyl-CoA dehydrogenase (MCAD) in Escherichia coli as a model system for dissecting the molecular effects of two mutations detected in patients with MCAD deficiency. We demonstrate that the R28C mutation predominantly affects polypeptide folding...

Medium-chain acyl-CoA dehydrogenase deficiency

DEFF Research Database (Denmark)

Waddell, Leigh; Wiley, Veronica; Carpenter, Kevin

2006-01-01

The fatty acid oxidation disorder most commonly identified by tandem mass spectrometry newborn screening is the potentially fatal medium-chain acyl-CoA dehydrogenase deficiency (MCAD). In clinically presenting cases, 80% are homozygous for the common mutation, c.985A > G and 18% heterozygous. We ...

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

Science.gov (United States)

Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

1998-01-01

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum

OpenAIRE

Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

2016-01-01

Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from thes...

Kinetic alteration of a human dihydrodiol/3alpha-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine.

Science.gov (United States)

Ohta, T; Ishikura, S; Shintani, S; Usami, N; Hara, A

2000-01-01

Human dihydrodiol dehydrogenase with 3alpha-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1-1C4) that belong to the aldo-keto reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1-1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the K(m) for NADP(+) and differently influenced the K(m) and k(cat) for substrates depending on their structures. The kinetic constants for bile acids with a 12alpha-hydroxy group were decreased 1.5-7-fold and those for the other substrates were increased 1.3-9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17beta-oestradiol, phenolphthalein and flufenamic acid and 3,5,3', 5'-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the K(m) for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators. PMID:11104674

Inhibition of dehydrogenase activity in petroleum refinery wastewater bacteria by phenolic compounds

Directory of Open Access Journals (Sweden)

Gideon C. Okpokwasili

2010-04-01

Full Text Available The toxicity of phenol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol on Pseudomonas, Bacillus and Escherichia species isolated from petroleum refinery wastewater was assessed via inhibition of dehydrogenase enzyme activity. At low concentrations, 2-nitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol stimulated dehydrogenase activity and at sufficient concentrations, phenolic compounds inhibited dehydrogenase activities. Generally, phenol is less toxic than substituted phenols. Estimations of the degree of inhibition/stimulation of dehydrogenase activities showed significant dose-dependent responses that are describable by logistic functions. The toxicity thresholds varied significantly (P < 0.05 among the bacterial strains and phenolic compounds. The median inhibitory concentrations (IC50s ranged from 4.118 ± 0.097 mg.L-1 for 4-nitrophenol against Pseudomonas sp. DAF1 to 1407.997 ± 7.091 mg.L-1 for phenol against Bacillus sp. DISK1. This study suggested that the organisms have moderate sensitivity to phenols and have the potential to be used as indicators for assessment of chemical toxicity. They could also be used as catalysts for degradation of phenols in effluents.

Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

Science.gov (United States)

Hurd, Thomas R; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T; Baranowski, Bartlomiej; Fearnley, Ian M; Prime, Tracy A; Murphy, Michael P; James, Andrew M

2012-10-12

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.

11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue and prospective changes in body weight and insulin resistance

DEFF Research Database (Denmark)

Koska, Juraj; de Courten, Barbora; Wake, Deborah J

2006-01-01

Increased mRNA and activity levels of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) in human adipose tissue (AT) are associated with obesity and insulin resistance. The aim of our study was to investigate whether 11betaHSD1 expression or activity in abdominal subcutaneous AT of non-diab......-diabetic subjects are associated with subsequent changes in body weight and insulin resistance [homeostasis model assessment of insulin resistance (HOMA-IR)]....

Inhibition of dehydrogenase activity in petroleum refinery wastewater bacteria by phenolic compounds

OpenAIRE

Gideon C. Okpokwasili; Christian Okechukwu Nweke

2010-01-01

The toxicity of phenol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol on Pseudomonas, Bacillus and Escherichia species isolated from petroleum refinery wastewater was assessed via inhibition of dehydrogenase enzyme activity. At low concentrations, 2-nitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol stimulated dehydrogenase activity and at sufficient concentrations, phenolic compounds inhibi...

A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol

Science.gov (United States)

2009-01-01

Background L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity. Results Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant. Conclusion These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH. PMID:19674460

Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method.

OpenAIRE

Stanley, C J; Perham, R N

1980-01-01

A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate...

Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

International Nuclear Information System (INIS)

White, Tommi A.; Tanner, John J.

2005-01-01

Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2 1 2 1 2 1 , with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells

Directory of Open Access Journals (Sweden)

Paolo Bollella

2018-04-01

Full Text Available Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared.

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

Science.gov (United States)

Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

2015-01-01

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1 in Neisseria meningitidis adherence to human cells

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Wooldridge Karl G

2010-11-01

Full Text Available Abstract Background Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms; where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1 to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2 to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3 to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion. Results In this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an

Genetics Home Reference: 3-beta-hydroxysteroid dehydrogenase deficiency

Science.gov (United States)

... for This Page Lutfallah C, Wang W, Mason JI, Chang YT, Haider A, Rich B, Castro-Magana ... A, Copeland KC, Chang YT, Lutfallah C, Mason JI. Carriers for type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) ...

Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene

DEFF Research Database (Denmark)

Andresen, B S; Bross, P; Vianey-Saban, C

1996-01-01

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE...

Discovery of nonsteroidal 17beta-hydroxysteroid dehydrogenase 1 inhibitors by pharmacophore-based screening of virtual compound libraries.

Science.gov (United States)

Schuster, Daniela; Nashev, Lyubomir G; Kirchmair, Johannes; Laggner, Christian; Wolber, Gerhard; Langer, Thierry; Odermatt, Alex

2008-07-24

17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) plays a pivotal role in the local synthesis of the most potent estrogen estradiol. Its expression is a prognostic marker for the outcome of patients with breast cancer and inhibition of 17beta-HSD1 is currently under consideration for breast cancer prevention and treatment. We aimed to identify nonsteroidal 17beta-HSD1 inhibitor scaffolds by virtual screening with pharmacophore models built from crystal structures containing steroidal compounds. The most promising model was validated by comparing predicted and experimentally determined inhibitory activities of several flavonoids. Subsequently, a virtual library of nonsteroidal compounds was screened against the 3D pharmacophore. Analysis of 14 selected compounds yielded four that inhibited the activity of human 17beta-HSD1 (IC 50 below 50 microM). Specificity assessment of identified 17beta-HSD1 inhibitors emphasized the importance of including related short-chain dehydrogenase/reductase (SDR) members to analyze off-target effects. Compound 29 displayed at least 10-fold selectivity over the related SDR enzymes tested.

Novel 11β-hydroxysteroid dehydrogenase 1 inhibitors reduce cortisol levels in keratinocytes and improve dermal collagen content in human ex vivo skin after exposure to cortisone and UV.

Directory of Open Access Journals (Sweden)

Stéphanie M Boudon

Full Text Available Activity and selectivity assessment of new bi-aryl amide 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1 inhibitors, prepared in a modular manner via Suzuki cross-coupling, are described. Several compounds inhibiting 11β-HSD1 at nanomolar concentrations were identified. Compounds 2b, 3e, 7b and 12e were shown to selectively inhibit 11β-HSD1 over 11β-HSD2, 17β-HSD1 and 17β-HSD2. These inhibitors also potently inhibited 11β-HSD1 activity in intact HEK-293 cells expressing the recombinant enzyme and in intact primary human keratinocytes expressing endogenous 11β-HSD1. Moreover, compounds 2b, 3e and 12e were tested for their activity in human skin biopsies. They were able to prevent, at least in part, both the cortisone- and the UV-mediated decreases in collagen content. Thus, inhibition of 11β-HSD1 by these compounds can be further investigated to delay or prevent UV-mediated skin damage and skin aging.

Crystallization behaviour of glyceraldehyde dehydrogenase from Thermoplasma acidophilum

Czech Academy of Sciences Publication Activity Database

Lermark, L.; Degtjarik, Oksana; Steffler, F.; Sieber, V.; Kutá-Smatanová, Ivana

2015-01-01

Roč. 71, č. 12 (2015), s. 1475-1480 ISSN 2053-230X Institutional support: RVO:67179843 Keywords : TaAlDH * Thermoplasma acidophilum * bioproduction * cell-free enzyme cascade * glyceraldehyde dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 0.647, year: 2015

Serum creatine kinase and lactate dehydrogenase activities in ...

African Journals Online (AJOL)

... in thyroid function are common endocrine disorders affecting 5-10% of individuals over ... Key words: Hyperthyroidism, hypothyroidism, lactate dehydrogenase, serum creatine kinase ... individuals depends on age, race, lean body mass and physical activity. ... measured by radioimmunoassay on AXSYM System (Abbott.

Glucose-6-phosphate dehydrogenase deficiency in Singapore.

Science.gov (United States)

Quak, S H; Saha, N; Tay, J S

1996-01-01

Glucose-6-phosphate dehydrogenase (G6PD) in man is an X-linked enzyme. The deficiency of this enzyme is one of the most common inherited metabolic disorders in man. In Singapore, three clinical syndromes associated with G6PD deficiency had been described: severe haemolysis in neonates with kernicterus, haemoglobinuria and "viral hepatitis"-like syndrome. The human G6PD monomer consists of 515 amino acids. Only the tetrameric or dimeric forms composed of a single type subunit are catylitically active. The complete amino acid sequence of G6PD had been elucidated in man and various other animals. The region of high homology among the enzymes of various animals is presumably functionally active. Among the Chinese in Singapore, three common molecular variants had been identified: Canton (nt 1376 G --> T), Kaiping (nt 1388 G --> A) and Mediterranean (nt 563 C --> T) in frequencies of 24%, 21% and 10% respectively. In addition, two common mutants (Gaozhou, nt 95 A --> G and Chinese 5, nt 1024 C --> T) have been detected in Singapore Chinese in low frequencies. In Malays, 6 different deficient variants are known in Singapore (3 new, 1 Mahidol, 1 Indonesian and 1 Mediterranean).

Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

OpenAIRE

Lee, Su Jeong; Park, Jeen-Woo

2014-01-01

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocyte...

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

Science.gov (United States)

Logemann, E; Reinold, S; Somssich, I E; Hahlbrock, K

1997-08-01

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

Physiological regulation of isocitrate dehydrogenase and the role of 2-oxoglutarate in Prochlorococcus sp. strain PCC 9511.

Directory of Open Access Journals (Sweden)

María Agustina Domínguez-Martín

Full Text Available The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42 catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.

Rapid synthesis of triazine inhibitors of inosine monophosphate dehydrogenase.

Science.gov (United States)

Pitts, William J; Guo, Junqing; Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Watterson, Scott H; Bednarz, Mark S; Chen, Bang Chi; Barrish, Joel C; Bassolino, Donna; Cheney, Daniel; Fleener, Catherine A; Rouleau, Katherine A; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-08-19

A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.

Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase.

Science.gov (United States)

Watterson, Scott H; Liu, Chunjian; Dhar, T G Murali; Gu, Henry H; Pitts, William J; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-10-21

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

Phosphorylation of formate dehydrogenase in potato tuber mitochondria

DEFF Research Database (Denmark)

Bykova, N.V.; Stensballe, A.; Egsgaard, H.

2003-01-01

Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha...

Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

Science.gov (United States)

NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

Energy Technology Data Exchange (ETDEWEB)

White, Tommi A.; Tanner, John J., E-mail: tannerjj@missouri.edu [Departments of Chemistry and Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States)

2005-08-01

Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

Structural characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from Lactobacillus delbrueckii ssp. bulgaricus.

Science.gov (United States)

Holton, Simon J; Anandhakrishnan, Madhankumar; Geerlof, Arie; Wilmanns, Matthias

2013-02-01

Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD⁺ complexed crystal structures of the L. bulgaricusD-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme. Copyright © 2012 Elsevier Inc. All rights reserved.

Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

Science.gov (United States)

Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

2015-12-01

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Very long chain acyl-coenzyme A dehydrogenase deficiency with adult onset

DEFF Research Database (Denmark)

Smelt, A H; Poorthuis, B J; Onkenhout, W

1998-01-01

Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9), tetrade......Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9......), tetradecadienoic acid, 14:2(n-6), and hexadecadienoic acid, 16:2(n-6). Palmitoyl-CoA and behenoyl-CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may...... be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L-carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis....

Prevalence of glucose-6-phosphate dehydrogenase deficiency in ...

African Journals Online (AJOL)

Background: Glucose-6-phosphate dehydrogenase (G6PD) is a house keeping enzyme which catalyzes the first step in the hexose monophosphate pathway of glucose metabolism. G6PD deficiency is the commonest hemolytic X-linked genetic disease, which affects approximately 400 million people worldwide.

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

Science.gov (United States)

Vasilchenko, Liliya G; Ludwig, Roland; Yershevich, Olga P; Haltrich, Dietmar; Rabinovich, Mikhail L

2012-07-01

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetics Home Reference: 17-beta hydroxysteroid dehydrogenase 3 deficiency

Science.gov (United States)

... 000 newborns. It is more common in the Arab population of Gaza, where it affects 1 in ... fetus, resulting in the abnormalities in the external sex organs that occur in 17-beta hydroxysteroid dehydrogenase ...

Thioredoxin, thioredoxin reductase, and alpha-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts.

Science.gov (United States)

Yan, Hong; Lou, Marjorie F; Fernando, M Rohan; Harding, John J

2006-10-02

To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses. The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-crystallin was investigated. GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, alpha(L)-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus. This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and alpha-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by

Role and structural characterization of plant aldehyde dehydrogenases from family 2 and family 7

Czech Academy of Sciences Publication Activity Database

Končitíková, R.; Vigouroux, A.; Kopečná, M.; Andree, T.; Bartoš, Jan; Šebela, M.; Moréra, S.; Kopečný, D.

2015-01-01

Roč. 468, Part: 1 (2015), s. 109-123 ISSN 0264-6021 R&D Projects: GA ČR GA15-22322S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : aldehyde dehydrogenase 2 (ALDH2) * aldehyde dehydrogenase 7 (ALDH7) * benzaldehyde Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.562, year: 2015

Identification of glucose 6 phosphate dehydrogenase mutations by ...

African Journals Online (AJOL)

Identification of glucose 6 phosphate dehydrogenase mutations by single strand conformation polymorphism and gene sequencing analysis. ... Subject: Six DNA samples from Turkish males confirmed to have G-6-PD deficiency where available for the study. Results: One subject was found to have an abnormal mobility shift ...

Assessment of creatine kinase and lactate dehydrogenase activities ...

African Journals Online (AJOL)

Ina bid to investigate the influence of menopausal on coronary heart disease, plasma creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes were analysed on a prospective cohort of 100 women attending Irrua Specialist Teaching Hospital (ISTH), Irrua, Edo state-Nigeria. They were divided into two groups; ...

Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass.

Science.gov (United States)

Spinelli, Jessica B; Yoon, Haejin; Ringel, Alison E; Jeanfavre, Sarah; Clish, Clary B; Haigis, Marcia C

2017-11-17

Ammonia is a ubiquitous by-product of cellular metabolism; however, the biological consequences of ammonia production are not fully understood, especially in cancer. We found that ammonia is not merely a toxic waste product but is recycled into central amino acid metabolism to maximize nitrogen utilization. In our experiments, human breast cancer cells primarily assimilated ammonia through reductive amination catalyzed by glutamate dehydrogenase (GDH); secondary reactions enabled other amino acids, such as proline and aspartate, to directly acquire this nitrogen. Metabolic recycling of ammonia accelerated proliferation of breast cancer. In mice, ammonia accumulated in the tumor microenvironment and was used directly to generate amino acids through GDH activity. These data show that ammonia is not only a secreted waste product but also a fundamental nitrogen source that can support tumor biomass. Copyright © 2017, American Association for the Advancement of Science.

Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/paragangliomas due to succinate dehydrogenase deficiency

NARCIS (Netherlands)

Richter, S; Peitzsch, M.; Rapizzi, E.; Lenders, J.W.M.; Qin, N.; Cubas, A.A. de; Schiavi, F.; Rao, J.U.; Beuschlein, F.; Quinkler, M.; Timmers, H.J.L.M.; Opocher, G.; Mannelli, M.; Pacak, K.; Robledo, M.; Eisenhofer, G.

2014-01-01

CONTEXT: Mutations of succinate dehydrogenase A/B/C/D genes (SDHx) increase susceptibility to development of pheochromocytomas and paragangliomas (PPGLs), with particularly high rates of malignancy associated with SDHB mutations. OBJECTIVE: We assessed whether altered succinate dehydrogenase

Preadipocyte 11beta-hydroxysteroid dehydrogenase type 1 is a keto-reductase and contributes to diet-induced visceral obesity in vivo.

Science.gov (United States)

De Sousa Peixoto, R A; Turban, S; Battle, J H; Chapman, K E; Seckl, J R; Morton, N M

2008-04-01

Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Although elevated activity of the glucocorticoid-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) within adipocytes might explain this paradox, the potential role of 11beta-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11beta-dehydrogenase activity (inactivation of glucocorticoids) probably due to the absence of cofactor provision by hexose-6-phosphate dehydrogenase. To clarify the depot-specific impact of 11beta-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and sc tissue displayed 11beta-HSD1 activity in mice. 11beta-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11beta-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11beta-HSD1 was an 11beta-reductase, thus reactivating glucocorticoids in ASV cells, consistent with hexose-6-phosphate dehydrogenase mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV cells despite a lower expression of 11beta-HSD1 mRNA and protein (homogenate activity) levels than sc ASV cells. This suggests a novel depot-specific control over 11beta-HSD1 enzyme activity. In vivo, high-fat diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11beta-HSD1(-/-) mice. The results suggest that 11beta-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.

Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

Science.gov (United States)

Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

2016-11-01

Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.

Science.gov (United States)

Barnard, Dale L; Day, Craig W; Bailey, Kevin; Heiner, Matthew; Montgomery, Robert; Lauridsen, Larry; Winslow, Scott; Hoopes, Justin; Li, Joseph K-K; Lee, Jongdae; Carson, Dennis A; Cottam, Howard B; Sidwell, Robert W

2006-08-01

thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.

Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

Science.gov (United States)

Santoso, D; Thornburg, R

2000-08-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

Fluoroorotic Acid-Selected Nicotiana plumbaginifolia Cell Lines with a Stable Thymine Starvation Phenotype Have Lost the Thymine-Regulated Transcriptional Program1

Science.gov (United States)

Santoso, Djoko; Thornburg, Robert

2000-01-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines. PMID:10938367

2-methylbutyryl-CoA dehydrogenase deficiency associated with autism and mental retardation

DEFF Research Database (Denmark)

Kanavin, Oivind J; Woldseth, Berit; Jellum, Egil

2007-01-01

BACKGROUND: 2-methylbutyryl-CoA dehydrogenase deficiency or short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) is caused by a defect in the degradation pathway of the amino acid L-isoleucine. METHODS: We report a four-year-old mentally retarded Somali boy with autism and a history...... cases with SBCADD, both originating from Somalia and Eritrea, indicating that it is relatively prevalent in this population. Autism has not previously been described with mutations in this gene, thus expanding the clinical spectrum of SBCADD....

Abnormal expression of 11 beta-hydroxysteroid dehydrogenase type 2 in human pituitary adenomas: a prereceptor determinant of pituitary cell proliferation.

Science.gov (United States)

Rabbitt, E H; Ayuk, J; Boelaert, K; Sheppard, M C; Hewison, M; Stewart, P M; Gittoes, N J L

2003-03-20

The physiological effects of glucocorticoids (GCs) are, at least in part, mediated by inhibition of cell proliferation. Two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) interconvert cortisol (F) and inactive cortisone (E), and are thus able to modulate GC action at an autocrine level. Previously, we have demonstrated absent expression of 11 beta-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11 beta-HSD2 was readily demonstrable. Here we have used real-time RT-PCR to quantify expression of mRNA for 11 beta-HSD1 and 2 in 105 human pituitary tumors and have performed enzyme expression and activity studies in primary pituitary cultures. Overall, pituitary tumors expressed lower levels of 11 beta-HSDl mRNA compared with normals (0.2-fold, Pprotein (mean+/-s.d.)) but no detectable 11 beta-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11 beta-HSD2 inhibitor) resulted in a 30.3+/-7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11 beta-HSDl to 11 beta-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11 beta-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.

Glucose-6-phosphate dehydrogenase deficiency; the single most ...

African Journals Online (AJOL)

Introduction: Glucose- 6-phosphate dehydrogenase deficiency is the most common enzymatic disorder of the red cell and an important risk factor for neonatal jaundice. Methodology: The aim of the study was to determine the incidence of G-6-PD deficiency among jaundiced neonates, and describe the associated morbidity ...

Interaction between alcohol dehydrogenase II gene, alcohol consumption, and risk for breast cancer

OpenAIRE

St?rmer, T; Wang-Gohrke, S; Arndt, V; Boeing, H; Kong, X; Kreienberg, R; Brenner, H

2002-01-01

MaeIII Restriction Fragment Length Polymorphism in exon 3 of the alcohol dehydrogenase II was assessed in serum from 467 randomly selected German women and 278 women with invasive breast cancer to evaluate the interaction between a polymorphism of the alcohol dehydrogenase II gene, alcohol consumption and risk for breast cancer. In both groups, usual consumption of different alcoholic beverages was asked for using semiquantitative food frequency questionnaires. We used multivariable logistic ...

Properties of glucoside 3-dehydrogenase and its potential applications

African Journals Online (AJOL)

STORAGESEVER

2008-12-29

Dec 29, 2008 ... dehydrogenase has attracted considerable attention in recent years due to broad substrate specificity and excellent ... site-selective oxidation of the C-3 hydroxyl group. .... single peptide with a molecular mass of 67 kDa in.

Glucose-6-phosphate dehydrogenase deficiency: an unusual cause of acute jaundice after paracetamol overdose.

Science.gov (United States)

Phillpotts, Simon; Tash, Elliot; Sen, Sambit

2014-11-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest human enzyme defect causing haemolytic anaemia after exposure to specific triggers. Paracetamol-induced haemolysis in G6PD deficiency is a rare complication and mostly reported in children. We report the first case (to the best of our knowledge) of acute jaundice without overt clinical features of a haemolytic crisis, in an otherwise healthy adult female following paracetamol overdose, due to previously undiagnosed G6PD deficiency. It is important that clinicians consider this condition when a patient presents following a paracetamol overdose with significant and disproportionate jaundice, without transaminitis or coagulopathy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nicotinoprotein methanol dehydrogenase enzymes in Gram-positive methylotrophic bacteria

NARCIS (Netherlands)

Hektor, Harm J.; Kloosterman, Harm; Dijkhuizen, Lubbert

2000-01-01

A novel type of alcohol dehydrogenase enzyme has been characterized from Gram-positive methylotrophic (Bacillus methanolicus, the actinomycetes Amycolatopsis methanolica and Mycobacterium gastri) and non-methylotrophic bacteria (Rhodococcus strains). Its in vivo role is in oxidation of methanol and

Modeling of NAD+ analogues in horse liver alcohol dehydrogenase

NARCIS (Netherlands)

Beijer, N.A.; Buck, H.M.; Sluyterman, L.A.A.E.; Meijer, E.M.

1990-01-01

So far, the interactions of nicotinamide adenine dinucleotide (NAD+) derivatives with dehydrogenases are not very well understood. This hampers the introduction of NAD+ analogues with improved characteristics concerning industrial application. We have developed an AMBER molecular mechanics model in

New enzymatic assay, parasite lactate dehydrogenase in diagnosis ...

African Journals Online (AJOL)

Background: The unique ability of plasmodial lactate dehydrogenase p(LDH) to utilise 3-acetyl pyridine dinucleotide (APAD) in lieu of NAD as a coenzyme in the conversion of pyruvate to lactate, led to the development of a biochemical assay for the detection of plasmodial parasitaemia. Researchers have reported that ...

Intravenous immunoglobulin to treat hyperbilirubinemia in neonates with isolated Glucose-6-Phosphate dehydrogenase deficiency

Directory of Open Access Journals (Sweden)

Wadah Khriesat

2017-04-01

Full Text Available Background Glucose-6-phosphate dehydrogenase deficiency alone or concomitant with ABO isoimmunisation is a widespread indication for neonatal exchange transfusion. Aims To evaluate the effectiveness of Intravenous Immunoglobulin in the treatment of neonatal hyperbilirubinemia due to glucose-6-phosphate dehydrogenase deficiency. Methods A retrospective cohort study was conducted between 2006 and 2014 at the Jordan University of Science and technology. The medical records of 43 infants admitted to the neonatal intensive care unit for isolated glucose-6- phosphate dehydrogenase deficiency hemolytic disease of the newborns were reviewed. Patients were divided into two groups. Group I, a historical cohort, included newborns born between 2006 and 2010, Treatment included phototherapy and exchange transfusion. Group II included newborns born between 2011 and 2014, where, in addition to phototherapy, intravenous immunoglobulin was administered. The duration of phototherapy and number of exchange transfusions were evaluated. Results Of 412 newborns that were admitted with neonatal hyperbilirubinemia, Glucose-6-phosphate dehydrogenase deficiency was present in 43. Of these, 22, did not receive intravenous immunoglobulin and served as a control group. The other 21 newborns received intravenous immunoglobulin. There was no difference in the demographic characteristics between the two groups. Infants in the control group were significantly more likely to receive exchange blood transfusion than infants in the immunoglobulin treatment group, but were significantly less likely to need phototherapy. Conclusion Intravenous immunoglobulin is an effective alternative to exchange transfusion in infants with glucose-6-phosphate dehydrogenase deficiency hemolytic disease of the newborn. It is suggested that intravenous immunoglobulin may be beneficial as a prophylaxis for infants with hyperbilirubinemia.

Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

Directory of Open Access Journals (Sweden)

Barretto O.C. de O.

2006-01-01

Full Text Available In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa. The Michaelis-Menten constants (Km: 55 µM for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively. A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves.

Science.gov (United States)

Ahmad-Sohdi, Nor-Ain-Shahajar; Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Hassan, Maizom

2015-01-01

Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control.

Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

Science.gov (United States)

Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

2015-12-01

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

15-hydroxyprostaglandin dehydrogenase activity in vitro in lung and kidney of essential fatty acid-deficient rats

DEFF Research Database (Denmark)

Hansen, Harald S.; Toft, B.S.

1978-01-01

Weanling rats were fed for 6 months on a diet deficient in essential fatty acids: either fat-free, or with 28% (w/w) partially hydrogenated fish oil. Control rats were fed a diet with 28% (w/w) arachis oil for 6 months. 15-Hydroxyprostaglandin dehydrogenase activity was determined as initial rates...... of the two groups on diets deficient in essential fatty acids as compared to the control group. No difference was observed in dehydrogenase activity in the kidneys. The dehydrogenase may be of importance for the regulation of the level of endogenous prostaglandins and, thus, a decrease in activity could...

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue.

Science.gov (United States)

Parker, Antony R

2003-10-01

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.

Cloning and expression of chicken 20-hydroxysteroid dehydrogenase

Czech Academy of Sciences Publication Activity Database

Bryndová, Jana; Klusoňová, Petra; Kučka, Marek; Vagnerová, Karla; Mikšík, Ivan; Pácha, Jiří

2006-01-01

Roč. 37, č. 3 (2006), s. 453-462 ISSN 0952-5041 R&D Projects: GA AV ČR(CZ) IAA6011201 Grant - others:GA UK(CZ) 216/2004 Institutional research plan: CEZ:AV0Z50110509 Keywords : 20-hydroxysteroid dehydrogenase * SDR family Subject RIV: CE - Biochemistry Impact factor: 2.988, year: 2006

Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase

Czech Academy of Sciences Publication Activity Database

Nisler, Jaroslav; Kopečný, D.; Končitíková, R.; Zatloukal, Marek; Bazgier, Václav; Berka, K.; Zalabák, D.; Briozzo, P.; Strnad, Miroslav; Spíchal, Lukáš

2016-01-01

Roč. 92, 1-2 (2016), s. 235-248 ISSN 0167-4412 R&D Projects: GA MŠk(CZ) LO1204; GA ČR GA15-22322S Institutional support: RVO:61389030 Keywords : Cytokinin oxidase/dehydrogenase * Crystal structure * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.356, year: 2016

Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein ...

African Journals Online (AJOL)

Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein Expression Defines the Proliferative Nature of Cervical Cancer Stem Cells. ... of cervical cancer stem cells and also to validate them in initial and advanced stages of cervical cancer. Keywords: Cervical cancer, ALDH1, BALB/c-nu/nu, HeLa cells, RKIP, Sox2 ...

Natural history of succinic semialdehyde dehydrogenase deficiency through adulthood

NARCIS (Netherlands)

Lapalme-Remis, S.; Lewis, E.C.; De Meulemeester, C.; Chakraborty, P.; Gibson, K.M.; Torres, C.; Guberman, A.; Salomons, G.; Jakobs, C.; Ali-Ridha, A.; Parviz, M.; Pearl, P.L.

2015-01-01

Objective: The natural history of succinic semialdehyde dehydrogenase (SSADH) deficiency in adulthood is unknown; we elucidate the clinical manifestations of the disease later in life. Methods: A 63-year-old man with long-standing intellectual disability was diagnosed with SSADH deficiency following

Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives.

Science.gov (United States)

Sanchez-Moreno, M; Ortega, J E; Valero, A

1989-12-01

High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat (Capra hircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

Increased superoxide accumulation in pyruvate dehydrogenase complex deficient fibroblasts.

Science.gov (United States)

Glushakova, Lyudmyla G; Judge, Sharon; Cruz, Alex; Pourang, Deena; Mathews, Clayton E; Stacpoole, Peter W

2011-11-01

The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA and is critically important in maintaining normal cellular energy homeostasis. Loss-of-function mutations in PDC give rise to congenital lactic acidosis and to progressive cellular energy failure. However, the subsequent biochemical consequences of PDC deficiency that may contribute to the clinical manifestations of the disorder are poorly understood. We postulated that altered flux through PDC would disrupt mitochondrial electron transport, resulting in oxidative stress. Compared to cells from 4 healthy subjects, primary cultures of skin fibroblasts from 9 patients with variable mutations in the gene encoding the alpha subunit (E1α) of pyruvate dehydrogenase (PDA1) demonstrated reduced growth and viability. Superoxide (O(2)(.-)) from the Qo site of complex III of the electron transport chain accumulated in these cells and was associated with decreased activity of manganese superoxide dismutase. The expression of uncoupling protein 2 was also decreased in patient cells, but there were no significant changes in the expression of cellular markers of protein or DNA oxidative damage. The expression of hypoxia transcription factor 1 alpha (HIF1α) also increased in PDC deficient fibroblasts. We conclude that PDC deficiency is associated with an increase in O(2)(.-) accumulation coupled to a decrease in mechanisms responsible for its removal. Increased HIF1α expression may contribute to the increase in glycolytic flux and lactate production in PDC deficiency and, by trans-activating pyruvate dehydrogenase kinase, may further suppress residual PDC activity through phosphorylation of the E1α subunit. Copyright © 2011 Elsevier Inc. All rights reserved.

Coulometric bioelectrocatalytic reactions based on NAD-dependent dehydrogenases in tricarboxylic acid cycle

Energy Technology Data Exchange (ETDEWEB)

Fukuda, Jun [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan); Tsujimura, Seiya [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: seiya@kais.kyoto-u.ac.jp; Kano, Kenji [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: kkano@kais.kyoto-u.ac.jp

2008-12-30

This paper describes the characterization of mediated electro-enzymatic electrolysis systems based on NAD-dependent dehydrogenase reactions in the tricarboxylic acid (TCA) cycle. A micro-bulk electrolysis system with a carbon felt anode immersed in an electrolysis solution with a value of about 10 {mu}L was constructed for coulometric analysis of the substrate oxidation. Diaphorase (DI) was used to couple the NAD-dependent dehydrogenase reaction with the anode reaction of a suitable redox mediator. We focused on three types of NAD-dependant dehydrogenases reactions in this research: (1) isocitrate oxidation, in which the standard Gibbs energy change ({delta}G{sup o}') is negative; (2) {alpha}-ketoglutarate oxidation, which involves an electrochemically active coenzyme A (CoA); and (3) malate oxidation, which is thermodynamically unfavorable because of a large positive {delta}G{sup o}' value. The complete electrolysis of isocitrate was easily achieved, supporting the effective re-oxidation of NADH in the diaphorase-catalyzed electrochemical reaction. CoA was unfavorably oxidized at the electrodes in the presence of some mediators. The electrocatalytic oxidation of CoA was suppressed and the quantitative electrochemical oxidation of {alpha}-ketoglutarate was achieved by selecting a suitable mediator with negligibly slow electron transfer kinetics with CoA. The uphill malate oxidation was susceptible to product inhibition in the bioelectrochemical system, although NADH generated in the malate dehydrogenase reaction was immediately oxidized in the electrochemical system. The inhibition was successfully suppressed by linking citrate synthase to quench oxaloacetate and to make the total {delta}G{sup o}' value negative.

Coulometric bioelectrocatalytic reactions based on NAD-dependent dehydrogenases in tricarboxylic acid cycle

International Nuclear Information System (INIS)

Fukuda, Jun; Tsujimura, Seiya; Kano, Kenji

2008-01-01

This paper describes the characterization of mediated electro-enzymatic electrolysis systems based on NAD-dependent dehydrogenase reactions in the tricarboxylic acid (TCA) cycle. A micro-bulk electrolysis system with a carbon felt anode immersed in an electrolysis solution with a value of about 10 μL was constructed for coulometric analysis of the substrate oxidation. Diaphorase (DI) was used to couple the NAD-dependent dehydrogenase reaction with the anode reaction of a suitable redox mediator. We focused on three types of NAD-dependant dehydrogenases reactions in this research: (1) isocitrate oxidation, in which the standard Gibbs energy change (ΔG o ') is negative; (2) α-ketoglutarate oxidation, which involves an electrochemically active coenzyme A (CoA); and (3) malate oxidation, which is thermodynamically unfavorable because of a large positive ΔG o ' value. The complete electrolysis of isocitrate was easily achieved, supporting the effective re-oxidation of NADH in the diaphorase-catalyzed electrochemical reaction. CoA was unfavorably oxidized at the electrodes in the presence of some mediators. The electrocatalytic oxidation of CoA was suppressed and the quantitative electrochemical oxidation of α-ketoglutarate was achieved by selecting a suitable mediator with negligibly slow electron transfer kinetics with CoA. The uphill malate oxidation was susceptible to product inhibition in the bioelectrochemical system, although NADH generated in the malate dehydrogenase reaction was immediately oxidized in the electrochemical system. The inhibition was successfully suppressed by linking citrate synthase to quench oxaloacetate and to make the total ΔG o ' value negative

Alcohol consumption, alcohol dehydrogenase 3 polymorphism, and colorectal adenomas

NARCIS (Netherlands)

Tiemersma, E.W.; Wark, P.A.; Ocké, M.C.; Bunschoten, A.; Otten, M.H.; Kok, F.J.; Kampman, E.

2003-01-01

Alcohol is a probable risk factor with regard to colorectal neoplasm and is metabolized to the carcinogen acetaldehyde by the genetically polymorphic alcohol dehydrogenase 3 (ADH3) enzyme. We evaluated whether the association between alcohol and colorectal adenomas is modified by ADH3 polymorphism.

Expanding the clinical spectrum of 3-phosphoglycerate dehydrogenase deficiency

NARCIS (Netherlands)

Tabatabaie, L; Klomp, L W J; Rubio-Gozalbo, M E; Spaapen, L J M; Haagen, A A M; Dorland, L; de Koning, T J

UNLABELLED: 3-Phosphoglycerate dehydrogenase (3-PGDH) deficiency is considered to be a rare cause of congenital microcephaly, infantile onset of intractable seizures and severe psychomotor retardation. Here, we report for the first time a very mild form of genetically confirmed 3-PGDH deficiency in

Kinetic and modelling studies of NAD+ and poly(ethylene glycol)-bound NAD+ in horse liver alcohol dehydrogenase

NARCIS (Netherlands)

Vanhommerig, S.A.M.; Sluyterman, L.A.A.E.; Meijer, E.M.

1996-01-01

Poly(ethylene glycol)-bound nicotinamide adenine dinucleotide (PEG-NAD+) has been successfully employed in the continuous production of L-amino acids from the corresponding alpha-keto acids by stereospecific reductive amination. Like many other dehydrogenases also horse liver alcohol dehydrogenase

Assay of partially purified glutamate dehydrogenase isolated from ...

African Journals Online (AJOL)

Glutamate dehydrogenase (E C 1.4.1.1) isolated from the seeds of asparagus beans was partially purified to a factor of 22 by dialysis after fractional precipitation with solid ammonium sulphate at 40 and 60% saturation. A specific activity of 11.78μmol min-1 mg-1 protein was calculated for the partially purified enzyme when ...

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

Science.gov (United States)

Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

2002-10-21

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

The retinoid X receptor response element in the human aldehyde dehydrogenase 2 promoter is antagonized by the chicken ovalbumin upstream promoter family of orphan receptors

NARCIS (Netherlands)

Pinaire, J; Hasanadka, R; Fang, M; Chou, WY; Stewart, MJ; Kruijer, W; Crabb, D

2000-01-01

Two tandem sites in the aldehyde dehydrogenase 2 promoter (designated FP330-5' and FP330-3') that bind members of the nuclear receptor superfamily mere recently identified. Antibodies against apolipoprotein regulatory protein (ARP-1) altered DNA-protein interactions in electrophoretic mobility shift

Glutamate dehydrogenase affects resistance to cell wall antibiotics in Bacillus subtilis.

Science.gov (United States)

Lee, Yong Heon; Kingston, Anthony W; Helmann, John D

2012-03-01

The glutamate dehydrogenase RocG of Bacillus subtilis is a bifunctional protein with both enzymatic and regulatory functions. Here we show that the rocG null mutant is sensitive to β-lactams, including cefuroxime (CEF), and to fosfomycin but that resistant mutants arise due to gain-of-function mutations in gudB, which encodes an otherwise inactive glutamate dehydrogenase. In the presence of CEF, ΔrocG ΔgudB mutant cells exhibit growth arrest when they reach mid-exponential phase. Using microarray-based transcriptional profiling, we found that the σ(W) regulon was downregulated in the ΔrocG ΔgudB null mutant. A survey of σ(W)-controlled genes for effects on CEF resistance identified both the NfeD protein YuaF and the flotillin homologue YuaG (FloT). Notably, overexpression of yuaFG in the rocG null mutant prevents the growth arrest induced by CEF. The YuaG flotillin has been shown previously to localize to defined lipid microdomains, and we show here that the yuaFGI operon contributes to a σ(W)-dependent decrease in membrane fluidity. We conclude that glutamate dehydrogenase activity affects the expression of the σ(W) regulon, by pathways that are yet unclear, and thereby influences resistance to CEF and other antibiotics.

Application of a radioimmunoassay to the induction of the 20β hydroxy steroid dehydrogenases with streptomyces hydrogenans

International Nuclear Information System (INIS)

Lotz, B.

1978-01-01

An antiserum has been prepared against crystallized 20β-hydroxysteroid dehydrogenate of streptomyces hydrogenous and used for different immunodiffusion and immunoprecipitation tests. A de novo synthesis of the 20β-hydroxysteroid dehydrogenase with streptomyces hydrogenous after cultivation of the cells in the presence of diene diol was hence found. The halflife of the 20β-hydroxysteroid dehydrogenase synthetizing mRNA in induced cells and that of the total mRNA in non-induced cells were calculated to be 126 sec and 66 sec respectively. The 20β-hydroxysteroid dehydrogenase in vivo appears to consist of four identical subunits. The monomers with a molecular weight of 27 350 exhibited a strong tendency to form diners and tetrameric complexes in the absence of dissociation agents. The synthesis rates of the 20β-hydroxysteroid dehydrogenase under induction conditions was 8.33%, the percentage of the total protein after induction 1.6%. (orig.) [de

Exploring the Molecular Basis for Selective Binding of Homoserine Dehydrogenase from Mycobacterium leprae TN toward Inhibitors: A Virtual Screening Study

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Dongling Zhan

2014-01-01

Full Text Available Homoserine dehydrogenase (HSD from Mycobacterium leprae TN is an antifungal target for antifungal properties including efficacy against the human pathogen. The 3D structure of HSD has been firmly established by homology modeling methods. Using the template, homoserine dehydrogenase from Thiobacillus denitrificans (PDB Id 3MTJ, a sequence identity of 40% was found and molecular dynamics simulation was used to optimize a reliable structure. The substrate and co-factor-binding regions in HSD were identified. In order to determine the important residues of the substrate (l-aspartate semialdehyde (l-ASA binding, the ASA was docked to the protein; Thr163, Asp198, and Glu192 may be important because they form a hydrogen bond with HSD through AutoDock 4.2 software. neuraminidaseAfter use of a virtual screening technique of HSD, the four top-scoring docking hits all seemed to cation–π ion pair with the key recognition residue Lys107, and Lys207. These ligands therefore seemed to be new chemotypes for HSD. Our results may be helpful for further experimental investigations.

Zinc and glutamate dehydrogenase in putative glutamatergic brain structures.

Science.gov (United States)

Wolf, G; Schmidt, W

1983-01-01

A certain topographic parallelism between the distribution of histochemically (TIMM staining) identified zinc and putative glutamatergic structures in the rat brain was demonstrated. Glutamate dehydrogenase as a zinc containing protein is in consideration to be an enzyme synthesizing transmitter glutamate. In a low concentration range externally added zinc ions (10(-9) to 10(-7) M) induced an increase in the activity of glutamate dehydrogenase (GDH) originating from rat hippocampal formation, neocortex, and cerebellum up to 142.4%. With rising molarity of Zn(II) in the incubation medium, the enzyme of hippocampal formation and cerebellum showed a biphasic course of activation. Zinc ions of a concentration higher than 10(-6) M caused a strong inhibition of GDH. The effect of Zn(II) on GDH originating from spinal ganglia and liver led only to a decrease of enzyme activity. These results are discussed in connection with a functional correlation between zinc and putatively glutamatergic system.

Fecal hydroxysteroid dehydrogenase activities in vegetarian Seventh-Day Adventists, control subjects, and bowel cancer patients.

Science.gov (United States)

Macdonald, I A; Webb, G R; Mahony, D E

1978-10-01

Cell-free extracts were prepared from mixed fecal anaerobic bacteria grown from stools of 14 vegetarian Seventh-Day Adventists, 16 omnivorous control subjects, and eight patients recently diagnosed with cancer of the large bowel. Preparations were assayed for NAD- and NADP-dependent 3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenases with bile salts and androsterone as substrates (eight substrate-cofactor combinations were tested). A significant intergroup difference was observed in the amounts of NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenase produced: bowel cancer patients exceeded controls, and controls exceeded Seventh-Day Adventists. Other enzyme activity comparisons were not significant. The pH values of the stools were significantly higher in cancer patients compared to Seventh-Day Adventists; values were 7.03 +/- 0.60 and 6.46 +/- 0.58 respectively. The pH value for controls was 6.66 +/- 0.62. A plot of pH value versus NADP-dependent 7alpha-hydroxysteroid dehydrogenase tended to separate the cancer patients from the other groups. Comparative data suggest that much of the 3alpha-hydroxysteroid dehydrogenase active against bile salt is also active against androsterone.

High energy electron beam inactivation of lactate dehydrogenase suspended in different aqueous media

International Nuclear Information System (INIS)

Hategan, A.; Oproiu, C.; Popescu, A.; Hategan, D.; Morariu, V.V.

1998-01-01

The direct and indirect effects of 5 MeV electron beam irradiation at various low temperatures, as well as the influence of the presence or absence of deuterium ions in the suspending medium of the enzyme, on the global enzymatic activity of lactate dehydrogenase have been studied. Frozen lactate dehydrogenase suspensions at 0 degC, -3 degC and -196 degC temperatures have been irradiated with the 5 MeV electron beam of a linear accelerator in the dose range 0-400 Gy. Liquid lactate dehydrogenase suspensions in D 2 O (99.98 %) and ultrapure water (17 ppm) at 0 degC have been irradiated in the dose range 0 -15 Gy. An exponential decrease was found in the enzymatic activity of irradiated lactate dehydrogenase, at all irradiation temperatures. The drastic decrease in the activity for the enzyme irradiated at 0 degC (total inhibition for a final dose of 100 Gy) indicate that at this temperature the indirect effects of radiation (due to the water radicals induced by radiation in the samples) are predominant. At -3 degC irradiation temperature the indirect effects of radiation are smaller but still present (a total decrease in the enzymatic activity for a dose of 250 Gy), while at -196 degC they are orders of magnitude reduced and the decrease in the enzymatic activity is due almost to the direct interaction of electrons with the macromolecules (70 % for a dose of 400 Gy)

Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus.

Science.gov (United States)

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Pyrococcus furiosus (Pf), a hyperthermophillic archeon. Sequence analysis of the Pf gene indicated an open reading frame specifying a protein of 485 amino acids (aa) with a calculated M(r) of 52900. Canonical Archaea promoter elements, Box A and Box B, are located -49 and -17 nucleotides (nt), respectively, upstream of the putative start codon. The sequence of the putative active-site region conforms to the IMPDH signature motif and contains a putative active-site cysteine. Phylogenetic relationships derived by using all available IMPDH sequences are consistent with trees developed for other molecules; they do not precisely resolve the history of Pf IMPDH but indicate a close similarity to bacterial IMPDH proteins. The phylogenetic analysis indicates that a gene duplication occurred prior to the division between rodents and humans, accounting for the Type I and II isoforms identified in mice and humans.

Inosine monophosphate dehydrogenase messenger RNA expression is correlated to clinical outcomes in mycophenolate mofetil-treated kidney transplant patients, whereas inosine monophosphate dehydrogenase activity is not

NARCIS (Netherlands)

Sombogaard, Ferdi; Peeters, Annemiek M. A.; Baan, Carla C.; Mathot, Ron A. A.; Quaedackers, Monique E.; Vulto, Arnold G.; Weimar, Willem; van Gelder, Teun

2009-01-01

Measurement of the pharmacodynamic biomarker inosine monophosphate dehydrogenase (IMPDH) activity in renal transplant recipients has been proposed to reflect the biological effect better than using pharmacokinetic parameters to monitor mycophenolate mofetil therapy. The IMPDH assays are however

Myopathy in very-long-chain acyl-CoA dehydrogenase deficiency

DEFF Research Database (Denmark)

Scholte, H R; Van Coster, R N; de Jonge, P C

1999-01-01

was deficient in muscle and fibroblasts, consistent with deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD). The gene of this enzyme had a homozygous deletion of three base pairs in exon 9, skipping lysine residue 238. Fibroblasts oxidised myristate, palmitate and oleate at a rate of 129, 62 and 38......A 30-year-old man suffered since the age of 13 years from exercise induced episodes of intense generalised muscle pain, weakness and myoglobinuria. Fasting ketogenesis was low, while blood glucose remained normal. Muscle mitochondria failed to oxidise palmitoylcarnitine. Palmitoyl-CoA dehydrogenase......% of controls. In contrast to patients with cardiac VLCAD deficiency, our patient had no lipid storage, a normal heart function, a higher rate of oleate oxidation in fibroblasts and normal free carnitine in plasma and fibroblasts. 31P-nuclear magnetic resonance spectroscopy of muscle showed a normal oxidative...

The ORF slr0091 of Synechocystis sp. PCC6803 encodes a high-light induced aldehyde dehydrogenase converting apocarotenals and alkanals

KAUST Repository

Trautmann, Danika

2013-07-05

Oxidative cleavage of carotenoids and peroxidation of lipids lead to apocarotenals and aliphatic aldehydes called alkanals, which react with vitally important compounds, promoting cytotoxicity. Although many enzymes have been reported to deactivate alkanals by converting them into fatty acids, little is known about the mechanisms used to detoxify apocarotenals or the enzymes acting on them. Cyanobacteria and other photosynthetic organisms must cope with both classes of aldehydes. Here we report that the Synechocystis enzyme SynAlh1, encoded by the ORF slr0091, is an aldehyde dehydrogenase that mediates oxidation of both apocarotenals and alkanals into the corresponding acids. Using a crude lysate of SynAlh1-expressing Escherichia coli cells, we show that SynAlh1 converts a wide range of apocarotenals and alkanals, with a preference for apocarotenals with defined chain lengths. As suggested by in vitro incubations and using engineered retinal-forming E. coli cells, we found that retinal is not a substrate for SynAlh1, making involvement in Synechocystis retinoid metabolism unlikely. The transcript level of SynAlh1 is induced by high light and cold treatment, indicating a role in the stress response, and the corresponding gene is a constituent of a stress-related operon. The assumptions regarding the function of SynAlh are further supported by the surprisingly high homology to human and plant aldehyde dehydrogenase that have been assigned to aldehyde detoxification. SynAlh1 is the first aldehyde dehydrogenase that has been shown to form both apocarotenoic and fatty acids. This dual function suggests that its eukaryotic homologs may also be involved in apocarotenal metabolism, a function that has not been considered so far. Aldehyde dehydrogenases play an important role in detoxification of reactive aldehydes. Here, we report on a cyanbacterial enzyme capable in converting two classes of lipid-derived aldehydes, apocaotenals and alkanals. The corresponding gene is a

Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

NARCIS (Netherlands)

Machielsen, M.P.; Looger, L.L.; Raedts, J.G.J.; Dijkhuizen, S.; Hummel, W.; Henneman, H.G.; Daussmann, T.; Oost, van der J.

2009-01-01

The R-specific alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for

The radiation inactivation of glutamate and isocitrate dehydrogenases

International Nuclear Information System (INIS)

El Failat, R.R.A.

1980-12-01

The reaction of free radicals produced by ionizing radiation with the enzymes glutamate dehydrogenase (GDH) and NADP + -specific isocitrate dehydrogenase (ICDH) have been studied by steady-state and pulse radiolysis techniques. In de-aerated GDH solutions, hydroxyl radicals have been found to be the most efficient of the primary radicals generated from water in causing inactivation. The effect of reaction with the enzyme of selective free radicals (SCN) 2 - , (Br) 2 - and (I) 2 - on its activity has also been studied. In neutral solutions, the order of inactivating effectiveness is (I) 2 - > (Br) 2 - > (SCN) 2 - . In the case of the thiocyanate radical anion (SCN) 2 - , the inactivation efficiency is found to depend on KSCN concentration. The radiation inactivation of GDH at both neutral and alkaline pH is accompanied by the loss of sulphydryl groups. Pulse radiolysis was also used to determine the rate constants and the transient absorption spectra following the reaction of the free radicals with GDH. 60 Co-γ-radiolysis and pulse radiolysis were also used to study the effect of ionizing radiation on the activity of ICDH. The results obtained were similar to those of GDH. (author)

Evidence for catabolite degradation in the glucose-dependent inactivation of yeast cytoplasmic malate dehydrogenase

International Nuclear Information System (INIS)

Neeff, J.; Haegele, E.; Nauhaus, J.; Heer, U.; Mecke, D.

1978-01-01

The cytoplasmic malate dehydrogenase of Saccharomyces cerevisiae was radioactively labeled during its synthesis on a glucose-free derepression medium. After purification a sensitive radioimmunoassay for this enzyme could be developed. The assay showed that after the physiological, glucose-dependent 'catabolite inactivation' of cytoplasmic malate dehydrogenase an inactive enzyme protein is immunologically not detectable. Together with the irreversibility of this reaction in vivo this finding strongly suggests a proteolytic mechanism of enzyme inactivation. For this process the term 'catabolite degradation' is used. (orig.) [de

In vitro modeling of experimental succinic semialdehyde dehydrogenase deficiency (SSADHD using brain-derived neural stem cells.

Directory of Open Access Journals (Sweden)

Kara R Vogel

Full Text Available We explored the utility of neural stem cells (NSCs as an in vitro model for evaluating preclinical therapeutics in succinic semialdehyde dehydrogenase-deficient (SSADHD mice. NSCs were obtained from aldh5a1+/+ and aldh5a1-/- mice (aldh5a1 = aldehyde dehydrogenase 5a1 = SSADH. Multiple parameters were evaluated including: (1 production of GHB (γ-hydroxybutyrate, the biochemical hallmark of SSADHD; (2 rescue from cell death with the dual mTOR (mechanistic target of rapamycin inhibitor, XL-765, an agent previously shown to rescue aldh5a1-/- mice from premature lethality; (3 mitochondrial number, total reactive oxygen species, and mitochondrial superoxide production, all previously documented as abnormal in aldh5a1-/- mice; (4 total ATP levels and ATP consumption; and (5 selected gene expression profiles associated with epilepsy, a prominent feature in both experimental and human SSADHD. Patterns of dysfunction were observed in all of these parameters and mirrored earlier findings in aldh5a1-/- mice. Patterns of dysregulated gene expression between hypothalamus and NSCs centered on ion channels, GABAergic receptors, and inflammation, suggesting novel pathomechanisms as well as a developmental ontogeny for gene expression potentially associated with the murine epileptic phenotype. The NSC model of SSADHD will be valuable in providing a first-tier screen for centrally-acting therapeutics and prioritizing therapeutic concepts of preclinical animal studies applicable to SSADHD.

Efficient production of (R-2-hydroxy-4-phenylbutyric acid by using a coupled reconstructed D-lactate dehydrogenase and formate dehydrogenase system.

Directory of Open Access Journals (Sweden)

Binbin Sheng

Full Text Available (R-2-hydroxy-4-phenylbutyric acid [(R-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R-HPBA synthetic processes remain unsatisfactory.The Y52L/F299Y mutant of NAD-dependent D-lactate dehydrogenase (D-nLDH in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA. The mutant D-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3 to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R-HPBA from OPBA. The biocatalysis conditions were then optimized.Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R-HPBA in 90 min. Given its high product enantiomeric excess (>99% and productivity (47.9 mM h(-1, the constructed coupling biocatalysis system is a promising alternative for (R-HPBA production.

Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells

NARCIS (Netherlands)

van Noorden, C. J.; Tas, J.; Vogels, I. M.

1983-01-01

With the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into

Regulation of human cerebrospinal fluid malate dehydrogenase 1 in sporadic Creutzfeldt-Jakob disease patients.

Science.gov (United States)

Schmitz, Matthias; Llorens, Franc; Pracht, Alexander; Thom, Tobias; Correia, Ângela; Zafar, Saima; Ferrer, Isidre; Zerr, Inga

2016-11-14

The identification of reliable diagnostic biomarkers in differential diagnosis of neurodegenerative diseases is an ongoing topic. A previous two-dimensional proteomic study on cerebrospinal fluid (CSF) revealed an elevated level of an enzyme, mitochondrial malate dehydrogenase 1 (MDH1), in sporadic Creutzfeldt-Jakob disease (sCJD) patients. Here, we could demonstrate the expression of MDH1 in neurons as well as in the neuropil. Its levels are lower in sCJD brains than in control brains. An examination of CSF-MDH1 in sCJD patients by ELISA revealed a significant elevation of CSF-MDH1 levels in sCJD patients (independently from the PRNP codon 129 MV genotype or the prion protein scrapie (PrP Sc ) type) in comparison to controls. In combination with total tau (tau), CSF-MDH1 detection exhibited a high diagnostic accuracy for sCJD diagnosis with a sensitivity of 97.5% and a specificity of 95.6%. A correlation study of MDH1 level in CSF with other neurodegenerative marker proteins revealed a significant positive correlation between MDH1 concentration with tau, 14-3-3 and neuron specific enolase level. In conclusion, our study indicated the potential of MDH1 in combination with tau as an additional biomarker in sCJD improving diagnostic accuracy of tau markedly.

Identification of the 2-hydroxyglutarate and isovaleryl-CoA dehydrogenases as alternative electron donors linking lysine catabolism to the electron transport chain of Arabidopsis mitochondria.

Science.gov (United States)

Araújo, Wagner L; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Larson, Tony R; Tohge, Takayuki; Krahnert, Ina; Witt, Sandra; Obata, Toshihiro; Schauer, Nicolas; Graham, Ian A; Leaver, Christopher J; Fernie, Alisdair R

2010-05-01

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route.

Purification and characterization of the amine dehydrogenase from a facultative methylotroph.

Science.gov (United States)

Coleman, J P; Perry, J J

1984-01-01

Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.

[Genetic variations in alcohol dehydrogenase, drinking habits and alcoholism

DEFF Research Database (Denmark)

Tolstrup, J.S.; Rasmussen, S.; Tybjaerg-Hansen, A.

2008-01-01

Alcohol is degraded primarily by alcohol dehydrogenase (ADH), and genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. By genotyping 9,080 white men and women from the general population, we found that men and women with ADH1B slow versus fast alcohol degrad...

Clinical aspects of short-chain acyl-CoA dehydrogenase deficiency

NARCIS (Netherlands)

Maldegem, B.T.; Wanders, R.J.A.; Wijburg, F.A.

2010-01-01

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation. SCADD is biochemically characterized by increased C4-carnitine in plasma and ethylmalonic acid in urine. The diagnosis of SCADD is confirmed by DNA analysis showing

Coenzyme- and His-tag-induced crystallization of octopine dehydrogenase

International Nuclear Information System (INIS)

Smits, Sander H. J.; Mueller, Andre; Grieshaber, Manfred K.; Schmitt, Lutz

2008-01-01

The crystal structure of octopine dehydrogenase revealed a specific role of the His 5 tag in inducing the crystal contacts required for successful crystallization. Over the last decade, protein purification has become more efficient and standardized through the introduction of affinity tags. The choice and position of the tag, however, can directly influence the process of protein crystallization. Octopine dehydrogenase (OcDH) without a His tag and tagged protein constructs such as OcDH-His 5 and OcDH-LEHis 6 have been investigated for their crystallizability. Only OcDH-His 5 yielded crystals; however, they were multiple. To improve crystal quality, the cofactor NADH was added, resulting in single crystals that were suitable for structure determination. As shown by the structure, the His 5 tag protrudes into the cleft between the NADH and l-arginine-binding domains and is mainly fixed in place by water molecules. The protein is thereby stabilized to such an extent that the formation of crystal contacts can proceed. Together with NADH, the His 5 tag obviously locks the enzyme into a specific conformation which induces crystal growth

Purification and characterization of xylitol dehydrogenase from Fusarium oxysporum

DEFF Research Database (Denmark)

Panagiotou, Gianni; Kekos, D.; Macris, B.J.

2002-01-01

An NAD(+)-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M-r 48 000, and pI 3.6. It was optimally active at 45degreesC and pH 9-10. It was fully...

Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

International Nuclear Information System (INIS)

Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

2009-01-01

Potassium tellurite (K 2 TeO 3 ) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

Purification, crystallization and preliminary X-ray analysis of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli

International Nuclear Information System (INIS)

Zheng, Jimin; Lee, Daniel C.; Jia, Zongchao

2009-01-01

Isocitrate dehydrogenase kinase/phosphatase has been crystallized in three different crystal forms. Data were collected from each crystal form for structure determination. The Escherichia coli aceK gene encodes isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5), a bifunctional protein that phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH), resulting in its inactivation and activation, respectively. This reversible (de)phosphorylation directs isocitrate, an intermediate of the citric acid cycle, to either go through the full cycle or to enter the glyoxylate bypass. In the present study, the AceK protein from E. coli has been purified and crystallized. Three crystal forms were obtained from very similar crystallization conditions. The crystals belong to space groups P4 1 2 1 2, P3 2 21 and P2 1 2 1 2 1 and diffracted X-rays to resolutions of 2.9, 3.0 and 2.7 Å, respectively

Determination of dehydrogenase activities involved in D-glucose oxidation in Gluconobacter and Acetobacter strains

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Florencia Sainz

2016-08-01

Full Text Available Acetic acid bacteria (AAB are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane bound dehydrogenases. In the present study, the enzyme activity of the membrane bound dehydrogenases (membrane-bound PQQ-glucose dehydrogenase (mGDH, D-gluconate dehydrogenase (GADH and membrane-bound glycerol dehydrogenase (GLDH involved in the oxidation of D-glucose and D-gluconic acid (GA was determined in six strains of three different species of AAB (three natural and three type strains. Moreover, the effect of these activities on the production of related metabolites (GA, 2-keto-D-gluconic acid (2KGA and 5-keto-D-gluconic acid (5KGA was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the A. malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h, which coincided with glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of G. oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition.Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were

Orthodontic Force Application in Correlation with Salivary Lactate Dehydrogenase Activity

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Erik Husin

2013-07-01

Full Text Available Orthodontic tooth movement generate mechanical forces to periodontal ligament and alveolar bone. The forces correlate with initial responses of periodontal tissues and involving many metabolic changes. One of the metabolic changes detected in saliva is lactate dehydrogenase (LDH activity. Objectives: To evaluate the correlation between orthodontic interrupted force application, lactate dehydrogenase activity and the distance of tooth movement. Methods: upper premolar, pre-retraction of upper canine and 1, 7, 14, 21 and 28 days post-retraction of upper canine with 100g interrupted orthodontic force. Results: duration of force (F=11.926 p 14 and 28 days post-retraction of canine. The region of retraction correlated with the distance of tooth movement (F=7.377 p=0.007. The duration of force correlated with the distance of tooth movement (F=66.554 p=0.000. retraction of canine. Conclusion: This study concluded that orthodontic interrupted force application on canine could increase the distance of tooth movement and LDH activity in saliva.

Purification and properties of a 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs.

OpenAIRE

Penning, T M; Mukharji, I; Barrows, S; Talalay, P

1984-01-01

An NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) was purified to homogeneity from rat liver cytosol, where it is responsible for most if not all of the capacity for the oxidation of androsterone, 1-acenaphthenol and benzenedihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene). The dehydrogenase has many properties (substrate specificity, pI, Mr, amino acid composition) in common with the dihydrodiol dehydrogenase (EC 1.3.1.20) purified from the same source [Vogel, Bentley...

Anticancer Properties of Distinct Antimalarial Drug Classes

Science.gov (United States)

Hooft van Huijsduijnen, Rob; Guy, R. Kiplin; Chibale, Kelly; Haynes, Richard K.; Peitz, Ingmar; Kelter, Gerhard; Phillips, Margaret A.; Vennerstrom, Jonathan L.; Yuthavong, Yongyuth; Wells, Timothy N. C.

2013-01-01

We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC50s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC50s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings. PMID:24391728

Anticancer properties of distinct antimalarial drug classes.

Directory of Open Access Journals (Sweden)

Rob Hooft van Huijsduijnen

Full Text Available We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase inhibitors effected potent inhibition of proliferation with IC50s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor, emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC50s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings.

A novel 3-hydroxysteroid dehydrogenase that regulates reproductive development and longevity.

Directory of Open Access Journals (Sweden)

Joshua Wollam

Full Text Available Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans.

Solution structures of lipoyl domains of the 2-oxo acid dehydrogenase complexes from Azotobacter vinelandii : implications for molecular recognition

NARCIS (Netherlands)

Berg, A.

1997-01-01

The 2-oxo acid dehydrogenase complexes are large multienzyme complexes that catalyse the irreversible oxidative decarboxylation of a specific 2-oxo acid to the corresponding acyl-CoA derivative. The pyruvate dehydrogenase complex (PDHC) converts the product of the glycolysis, pyruvate, to

Alcoholism and alcohol drinking habits predicted from alcohol dehydrogenase genes

DEFF Research Database (Denmark)

Tolstrup, J.S.; Nordestgaard, Børge; Rasmussen, S.

2008-01-01

Alcohol is degraded primarily by alcohol dehydrogenase (ADH) wherein genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. It is biologically plausible that these variations may be associated with alcohol drinking habits and alcoholism. By genotyping 9080 whi...

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

Science.gov (United States)

Munawar, Nayla; Engel, Paul C

2013-01-01

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

Vitality Improvement of the Mediterranean Fruit Fly, Ceratitis capitata Wied 1- Measured by using dehydrogenase Enzyme Activities

International Nuclear Information System (INIS)

Salama, M.S.; Shoman, A.A.; Elbermawy, S.M.; Abul Yazid, I.

2000-01-01

The present study searches for the improvement vitality of the Mediterranean fruit fly, Ceratitis capitata Wied. Through the induction of a specific variance (mutation) in the genetic material. Several types of treatments that were thought to cause this mutation were used, as IGR's, temperature, formaldehyde, colchicine, alcohols, several types of larval rearing media and gamma-rays. Generally, the activities of the energy enzymes alpha-glycerophosphate dehydrogenase (alpha-GPDH) enzyme lactate dehydrogenase (LDH) enzyme and malate dehydrogenase (MDH) enzyme, when used as a direct measure for the fly vitality, increased due to treatments of the egg stage by the previously mentioned treatments specially by the usage of rice hulls in the larval rearing medium alone or followed by irradiation of the pupal stage with 90 Gy

A single arginine residue is required for the interaction of the electron transferring flavoprotein (ETF) with three of its dehydrogenase partners.

Science.gov (United States)

Parker, Antony R

2003-12-01

The interaction of several dehydrogenases with the electron transferring flavoprotein (ETF) is a crucial step required for the successful transfer of electrons into the electron transport chain. The exact determinants regarding the interaction of ETF with its dehydrogenase partners are still unknown. Chemical modification of ETF with arginine-specific reagents resulted in the loss, to varying degrees, of activity with medium chain acyl-coenzyme A dehydrogenase (MCAD). The kinetic profiles showed the inactivations followed pseudo-first-order kinetics for all reagents used. For activity with MCAD, maximum inactivation of ETF was accomplished by 2,3-butanedione (4% residual activity after 120 min) and it was shown that modification of one arginine residue was responsible for the inactivation. Almost 100% restoration of this ETF activity was achieved upon incubation with free arginine. However, the same 2,3-butanedione modified ETF only possessed decreased activity with dimethylglycine-(DMGDH, 44%) and sarcosine- (SDH, 27%) dehydrogenases unlike the abolition with MCAD. Full protection of ETF from arginine modification by 2,3-butanedione was achieved using substrate-protected DMGDH, MCAD and SDH respectively. Cross-protection studies of ETF with the three dehydrogenases implied use of the same single arginine residue in the binding of all three dehydrogenases. These results lead us to conclude that this single arginine residue is essential in the binding of the ETF to MCAD, but only contributes partially to the binding of ETF to SDH and DMGDH and thus, the determinants of the dehydrogenase binding sites overlap but are not identical.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

Energy Technology Data Exchange (ETDEWEB)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742 (Korea, Republic of)

2006-02-01

Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B{sub 6} (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V{sub M}) of 3.64 Å{sup 3} Da{sup −1} and a solvent content of 66%.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-01-01

Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B 6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M ) of 3.64 Å 3 Da −1 and a solvent content of 66%

Construction of an integrated enzyme system consisting azoreductase and glucose 1-dehydrogenase for dye removal.

Science.gov (United States)

Yang, Yuyi; Wei, Buqing; Zhao, Yuhua; Wang, Jun

2013-02-01

Azo dyes are toxic and carcinogenic and are often present in industrial effluents. In this research, azoreductase and glucose 1-dehydrogenase were coupled for both continuous generation of the cofactor NADH and azo dye removal. The results show that 85% maximum relative activity of azoreductase in an integrated enzyme system was obtained at the conditions: 1U azoreductase:10U glucose 1-dehydrogenase, 250mM glucose, 1.0mM NAD(+) and 150μM methyl red. Sensitivity analysis of the factors in the enzyme system affecting dye removal examined by an artificial neural network model shows that the relative importance of enzyme ratio between azoreductase and glucose 1-dehydrogenase was 22%, followed by dye concentration (27%), NAD(+) concentration (23%) and glucose concentration (22%), indicating none of the variables could be ignored in the enzyme system. Batch results show that the enzyme system has application potential for dye removal. Copyright © 2012 Elsevier Ltd. All rights reserved.

Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

International Nuclear Information System (INIS)

D'Ardenne, S.C.; Edmondson, D.E.

1990-01-01

The effect of isotopic substitution of the 8-H of xanthine (with 2 H and 3 H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T (V/K) values was observed between xanthine oxidase and xanthine dehydrogenase. Xanthine dehydrogenase exhibited a larger T/D (V/K) value than that observed for xanthine oxidase. Observed H/T (V/K) values for either enzyme are less than those H/T (V/K) values calculated with D/T (V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8- 3 H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (C r ) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect ( D k) for xanthine oxidation is calculated. By the use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be ∼75-fold faster than k cat for xanthine oxidase and ∼10-fold faster than k cat for xanthine dehydrogenase. Values calculated for each enzyme were found to be identical within experimental uncertainty

A new dawn for plant mitochondrial NAD(P)H dehydrogenases

DEFF Research Database (Denmark)

Møller, I.M.

2002-01-01

The expression of complex I and two homologues of bacterial and yeast NADH dehydrogenases, NDA and NDB, have been studied in potato leaf mitochondria. The mRNA level of NDA is completely light dependent and shows a diurnal rhythm with a sharp maximum just after dawn. NDA protein quantity and inte...

Neonatal jaundice and glucose-6-phosphate dehydrogenase

OpenAIRE

Leite, Amauri Antiquera [UNESP

2010-01-01

A deficiência de glicose-6-fosfato desidrogenase em neonatos pode ser a responsável pela icterícia neonatal. Este comentário científico é decorrente do relato sobre o tema publicado neste fascículo e que preocupa diversos autores de outros países em relação às complicações em neonatos de hiperbilirrubinemia, existindo inclusive proposições de alguns autores em incluir o teste para identificar a deficiência de glicose-6-fosfato desidrogenase nos recém-nascidos.Glucose-6-phosphate dehydrogenase...

Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells.

Science.gov (United States)

Yang, Eun Sun; Park, Jeen-Woo

2011-05-01

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.

A novel mutation in the succinate dehydrogenase subunit D gene in siblings with the hereditary paraganglioma–pheochromocytoma syndrome

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Chaithra Prasad

2014-10-01

Full Text Available Germline mutations in the succinate dehydrogenase complex subunit D gene are now known to be associated with hereditary paraganglioma–pheochromocytoma syndromes. Since the initial succinate dehydrogenase complex subunit D gene mutation was identified about a decade ago, more than 131 unique variants have been reported. We report the case of two siblings presenting with multiple paragangliomas and pheochromocytomas; they were both found to carry a mutation in the succinate dehydrogenase complex subunit D gene involving a substitution of thymine to guanine at nucleotide 236 in exon 3. This particular mutation of the succinate dehydrogenase complex subunit D gene has only been reported in one previous patient in Japan; this is, therefore, the first report of this pathogenic mutation in siblings and the first report of this mutation in North America. With continued screening of more individuals, we will be able to create a robust mutation database that can help us understand disease patterns associated with particular variants and may be a starting point in the development of new therapies for familial paraganglioma syndromes.

Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols.

Science.gov (United States)

Yamamoto, Hiroaki; Kudoh, Masatake

2013-09-01

A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity.

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins.

Science.gov (United States)

Lapierre, Catherine; Pilate, Gilles; Pollet, Brigitte; Mila, Isabelle; Leplé, Jean-Charles; Jouanin, Lise; Kim, Hoon; Ralph, John

2004-02-01

A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.

Dihydropyrimidine Dehydrogenase Deficiency in Two Malaysian Siblings with Abnormal MRI Findings

NARCIS (Netherlands)

Chen, Bee Chin; Mohd Rawi, Rowani; Meinsma, Rutger; Meijer, Judith; Hennekam, Raoul C. M.; van Kuilenburg, André B. P.

2014-01-01

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine metabolism. Deficiency of this enzyme leads to an accumulation of thymine and uracil and a deficiency of metabolites distal to the catabolic enzyme. The disorder presents with a wide clinical

Glu504Lys Single Nucleotide Polymorphism of Aldehyde Dehydrogenase 2 Gene and the Risk of Human Diseases

Directory of Open Access Journals (Sweden)

Yan Zhao

2015-01-01

Full Text Available Aldehyde dehydrogenase (ALDH 2 is a mitochondrial enzyme that is known for its important role in oxidation and detoxification of ethanol metabolite acetaldehyde. ALDH2 also metabolizes other reactive aldehydes such as 4-hydroxy-2-nonenal and acrolein. The Glu504Lys single nucleotide polymorphism (SNP of ALDH2 gene, which is found in approximately 40% of the East Asian populations, causes defect in the enzyme activity of ALDH2, leading to alterations in acetaldehyde metabolism and alcohol-induced “flushing” syndrome. Evidence suggests that ALDH2 Glu504Lys SNP is a potential candidate genetic risk factor for a variety of chronic diseases such as cardiovascular disease, cancer, and late-onset Alzheimer’s disease. In addition, the association between ALDH2 Glu504Lys SNP and the development of these chronic diseases appears to be affected by the interaction between the SNP and lifestyle factors such as alcohol consumption as well as by the presence of other genetic variations.

Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

Czech Academy of Sciences Publication Activity Database

Kopečný, D.; Šebela, M.; Briozzo, P.; Spíchal, Lukáš; Houba-Hérin, N.; Mašek, V.; Joly, N.; Madzak, C.; Anzenbacher, P.; Laloue, M.

2008-01-01

Roč. 380, č. 5 (2008), s. 886-899 ISSN 0022-2836 R&D Projects: GA ČR(CZ) GP522/08/P113 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin oxidase/dehydrogenase * cytokinin signaling * protein structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.146, year: 2008

Effects of accelerated electrons and microwaves on frozen enzyme lactate dehydrogenase

International Nuclear Information System (INIS)

Hategan, A.; Martin, D.; Popescu, L.M.; Butan, C.

2000-01-01

Results on the influence of 6 MeV electron beam irradiation and 2.45 GHz 565 W microwaves as well as the effects of the combined electron and microwave irradiation, at - 21 deg. C, on enzyme lactate dehydrogenase are presented. The microwave irradiated macromolecules exhibited a non-linear behaviour (successive activation and inactivation of the enzyme molecules) suggesting the major influence of the nonthermal component of microwave radiation. The combined electron and microwave irradiation lead to a similar decrease of the activity as the electron beam irradiation, the microwave influence being apparently insignificant in the dose, power and time ranges used. Radiation target analysis of the enzymatic decrease due to electron irradiation indicated very large aggregation of the enzyme molecules. Our data suggest that radiation target analysis is not suitable to measure the molecular mass of lactate dehydrogenase, when irradiating frozen enzyme suspensions. (authors)

Identification of the 2-Hydroxyglutarate and Isovaleryl-CoA Dehydrogenases as Alternative Electron Donors Linking Lysine Catabolism to the Electron Transport Chain of Arabidopsis Mitochondria[W][OA

Science.gov (United States)

Araújo, Wagner L.; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Larson, Tony R.; Tohge, Takayuki; Krahnert, Ina; Witt, Sandra; Obata, Toshihiro; Schauer, Nicolas; Graham, Ian A.; Leaver, Christopher J.; Fernie, Alisdair R.

2010-01-01

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route. PMID:20501910

Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

NARCIS (Netherlands)

Wisselink, H.W.; Mars, A.E.; Meer, van der P.; Eggink, G.; Hugenholtz, J.

2004-01-01

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance

Conversion of xanthine dehydrogenase into xanthine oxidase in rat liver and plasma at the onset of reperfusion after ischemia

NARCIS (Netherlands)

Kooij, A.; Schiller, H. J.; Schijns, M.; van Noorden, C. J.; Frederiks, W. M.

1994-01-01

The aim of this study was to test whether conversion of xanthine dehydrogenase into xanthine oxidase as induced by fasting, ischemia of the liver or both is an in vivo process or only occurs in vitro in homogenates. For this purpose, the conversion rate of xanthine dehydrogenase into xanthine

Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion

Directory of Open Access Journals (Sweden)

Pei-Wen Tsai

2017-12-01

Full Text Available Background/Purpose: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. Methods: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. Results: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Conclusion: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs. Keywords: Candida albicans, cell adhesion, Hom6, homoserine dehydrogenase, protein synthesis

Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

Energy Technology Data Exchange (ETDEWEB)

Small, Juan E. [Massachusetts General Hospital and Harvard Medical School, Department of Radiology, Boston, MA (United States); Gonzalez, Guido E. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States); Clinica Alemana de Santiago, Departmento de Imagenes, Santiago (Chile); Nagao, Karina E.; Walton, David S. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Ophthalmology, Boston, MA (United States); Caruso, Paul A. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States)

2009-10-15

Pyruvate dehydrogenase (PDH) deficiency is a genetic disorder of mitochondrial metabolism. The clinical manifestations range from severe neonatal lactic acidosis to chronic neurodegeneration. Optic neuropathy is an uncommon clinical sequela and the imaging findings of optic neuropathy in these patients have not previously been described. We present a patient with PDH deficiency with bilateral decreased vision in whom MRI demonstrated bilateral optic neuropathy and chiasmopathy. (orig.)

Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

International Nuclear Information System (INIS)

Small, Juan E.; Gonzalez, Guido E.; Nagao, Karina E.; Walton, David S.; Caruso, Paul A.

2009-01-01

Pyruvate dehydrogenase (PDH) deficiency is a genetic disorder of mitochondrial metabolism. The clinical manifestations range from severe neonatal lactic acidosis to chronic neurodegeneration. Optic neuropathy is an uncommon clinical sequela and the imaging findings of optic neuropathy in these patients have not previously been described. We present a patient with PDH deficiency with bilateral decreased vision in whom MRI demonstrated bilateral optic neuropathy and chiasmopathy. (orig.)

2-methylbutyryl-CoA dehydrogenase deficiency associated with autism and mental retardation: a case report

DEFF Research Database (Denmark)

Kanavin, Øjvind; Woldseth, Berit; Jellum, Egil

2007-01-01

previously reported cases with SBCADD, both originating from Somalia and Eritrea, indicating that it is relatively prevalent in this population. Autism has not previously been described with mutations in this gene, thus expanding the clinical spectrum of SBCADD. PMID: 17883863 [PubMed - in process]......ABSTRACT: BACKGROUND: 2-methylbutyryl-CoA dehydrogenase deficiency or short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) is caused by a defect in the degradation pathway of the amino acid L-isoleucine. METHODS: We report a four-year-old mentally retarded Somali boy with autism...

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Directory of Open Access Journals (Sweden)

Hahn-Hägerdal Bärbel

2008-10-01

Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

Hydroxysteroid dehydrogenase HSD1L is localised to the pituitary–gonadal axis of primates

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A Daniel Bird

2017-09-01

Full Text Available Steroid hormones play clinically important and specific regulatory roles in the development, growth, metabolism, reproduction and brain function in human. The type 1 and 2 11-beta hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 2 have key roles in the pre-receptor modification of glucocorticoids allowing aldosterone regulation of blood pressure, control of systemic fluid and electrolyte homeostasis and modulation of integrated metabolism and brain function. Although the activity and function of 11β-HSDs is thought to be understood, there exists an open reading frame for a distinct 11βHSD-like gene; HSD11B1L, which is present in human, non-human primate, sheep, pig and many other higher organisms, whereas an orthologue is absent in the genomes of mouse, rat and rabbit. We have now characterised this novel HSD11B1L gene as encoded by 9 exons and analysis of EST library transcripts indicated the use of two alternate ATG start sites in exons 2 and 3, and alternate splicing in exon 9. Relatively strong HSD11B1L gene expression was detected in human, non-human primate and sheep tissue samples from the brain, ovary and testis. Analysis in non-human primates and sheep by immunohistochemistry localised HSD11B1L protein to the cytoplasm of ovarian granulosa cells, testis Leydig cells, and gonadatroph cells in the anterior pituitary. Intracellular localisation analysis in transfected human HEK293 cells showed HSD1L protein within the endoplasmic reticulum and sequence analysis suggests that similar to 11βHSD1 it is membrane bound. The endogenous substrate of this third HSD enzyme remains elusive with localisation and expression data suggesting a reproductive hormone as a likely substrate.

Novel approaches for using dehydrogenases and ene-reductases for organic synthesis

NARCIS (Netherlands)

Gargiulo, S.

2015-01-01

Oxidation of alcohols is a reaction of major interest for organic chemistry. However, the most common chemical routes developed so far involve the use of toxic or hazardous reagents or catalysts that often lack good chemoselectivity. In this respect, alcohol dehydrogenases (ADHs) represent a very

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency in patients ...

African Journals Online (AJOL)

This is a study of Glucose-6-phosphate dehydrogenase(G6PD) deficiency in sickle cell anaemia patients attending the haematology clinic of the Jos University Teaching Hospital (JUTH), Jos- Nigeria. The prevalence of G6PD deficiency among the 130 sickle cell anaemia patients studied was found to be 18.5%. G6PD ...

Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

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Gabriele Möller

Full Text Available Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1 for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17beta-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17beta-HSDs analyzed were observed. Especially, the rodent 17beta-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17beta-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.

Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

Science.gov (United States)

Murakami, Taro; Matsuo, Masayuki; Shimizu, Ayako; Shimomura, Yoshiharu

2005-02-01

Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice.

Science.gov (United States)

Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

2006-03-01

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

Science.gov (United States)

Santiago, Rocío; Alarcón, Borja; de Armas, Roberto; Vicente, Carlos; Legaz, María Estrella

2012-06-01

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD. Copyright © Physiologia Plantarum 2012.

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver

NARCIS (Netherlands)

Griffini, P.; Vigorelli, E.; Bertone, V.; Freitas, I.; van Noorden, C. J.

1994-01-01

The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate

Clinical variability in 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency

NARCIS (Netherlands)

Ensenauer, Regina; Niederhoff, Helmut; Ruiter, Jos P. N.; Wanders, Ronald J. A.; Schwab, K. Otfried; Brandis, Matthias; Lehnert, Willy

2002-01-01

We report the identification of two new 7-year-old patients with 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency, a recently described inborn error of isoleucine metabolism. The defect is localized one step above 3-ketothiolase, resulting in a urinary metabolite pattern similar to that seen

XoxF Is Required for Expression of Methanol Dehydrogenase in Methylobacterium extorquens AM1 ▿

Science.gov (United States)

Skovran, Elizabeth; Palmer, Alexander D.; Rountree, Austin M.; Good, Nathan M.; Lidstrom, Mary E.

2011-01-01

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ∼50% identical to MxaF and ∼90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes. PMID:21873495

5,5'-Dithiobis-(2-nitrobenzoic acid) as a probe for a non-essential cysteine residue at the medium chain acyl-coenzyme A dehydrogenase binding site of the human 'electron transferring flavoprotein' (ETF).

Science.gov (United States)

Parker, A; Engel, P C

1999-01-01

Human 'electron transferring flavoprotein' (ETF) was inactivated by the thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency

DEFF Research Database (Denmark)

Andresen, B S; Olpin, S; Poorthuis, B J

1999-01-01

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyop......Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence...... of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting......-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established....

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

Science.gov (United States)

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-02-27

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

Science.gov (United States)

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-01-01

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

Cytokinin oxidase/dehydrogenase genes in barley and wheat. Cloning and heterologous expression

Czech Academy of Sciences Publication Activity Database

Galuszka, P.; Frébortová, Jitka; Werner, T.; Yamada, M.; Strnad, Miroslav; Schmülling, T.; Frébort, I.

2004-01-01

Roč. 271, č. 20 (2004), s. 3990-4002 ISSN 0014-2956 Institutional research plan: CEZ:AV0Z5038910 Keywords : cereals * cloning * cytokinin oxidase/dehydrogenase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.260, year: 2004

STUDIES ON THE DYNAMICS OF DEHYDROGENASES KREBS CYCLE ACTIVITY AT MONILINIA LAXA (ADERH. & RUHL. HONEY FUNGUS GROWN ON MEDIA WITH DIFFERENT CARBOHYDRATES

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Elena Ciornea

2010-09-01

Full Text Available As ubiquitous organisms, fungi grow on a large number of organic substrate, alive or dead, confronting therefore with a wide variety of carbohydrates and various physical factors, and their versatility to adapt and be able to use a large number of these compounds could provide them the chance to survive. Given that, these fungi have a rich enzyme equipment that allows them to operate on different metabolic pathways, this study aims to monitor the dynamics activity of some Krebs cycle dehydrogenases in Monilinia laxa (Aderh & Ruhl. Honey species parasitic on various species of plum trees. To this end, the fungus was cultivated in vitro on media enriched with different carbohydrates and the isocitrate dehydrogenase, �-cetoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activity in the fungus mycelium was followed, at 7, respectively, 14 days after the inoculation of the culture medium and determined using the spectrophotometric Sîsoev and Krasna method (Cojocaru, D.C., 2009. Data revealed obvious differences depending on the type of carbohydrate introduced into the medium and the age of the culture mycelia.

STUDIES ON THE DYNAMICS OF DEHYDROGENASES KREBS CYCLE ACTIVITY AT MONILINIA LAXA (ADERH. & RUHL. HONEY FUNGUS GROWN ON MEDIA WITH DIFFERENT CARBOHYDRATES

Directory of Open Access Journals (Sweden)

Elena Ciornea

2011-11-01

Full Text Available As ubiquitous organisms, fungi grow on a large number of organic substrate, alive or dead, confronting therefore with a wide variety of carbohydrates and various physical factors, and their versatility to adapt and be able to use a large number of these compounds could provide them the chance to survive. Given that, these fungi have a rich enzyme equipment that allows them to operate on different metabolic pathways, this study aims to monitor the dynamics activity of some Krebs cycle dehydrogenases in Monilinia laxa (Aderh & Ruhl. Honey species parasitic on various species of plum trees. To this end, the fungus was cultivated in vitro on media enriched with different carbohydrates and the isocitrate dehydrogenase, �-cetoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activity in the fungus mycelium was followed, at 7, respectively, 14 days after the inoculation of the culture medium and determined using the spectrophotometric Sîsoev and Krasna method (Cojocaru, D.C., 2009. Data revealed obvious differences depending on the type of carbohydrate introduced into the medium and the age of the culture mycelia.

Alcohol drinking habits, alcohol dehydrogenase genotypes and risk of acute coronary syndrome

DEFF Research Database (Denmark)

Tolstrup, J.S.; Hansen, J.L.; Gronbaek, M.

2010-01-01

Aims: The risk of myocardial infarction is lower among light-to-moderate drinkers compared with abstainers. Results from some previous studies, but not all, suggest that this association is modified by variations in genes coding for alcohol dehydrogenase (ADH). We aimed to test this hypothesis......, including alcohol as both the amount of alcohol and the frequency of drinking. Methods: we conducted a nested case-cohort study within the Danish Diet, Cancer and Health study, including 1,645 men (770 incident cases of acute coronary syndrome from 1993-1997 through 2004 and 875 randomly selected controls......). Results: Higher alcohol intake (measured as amount or drinking frequency) was associated with lower risk of acute coronary syndrome; however, there was no evidence that these finding were modified by ADH1B or ADH1C genotypes. Conclusions: The importance of functional variation in alcohol dehydrogenase...

Human 3α-hydroxysteroid dehydrogenase type 3: structural clues of 5α-DHT reverse binding and enzyme down-regulation decreasing MCF7 cell growth.

Science.gov (United States)

Zhang, Bo; Hu, Xiao-Jian; Wang, Xiao-Qiang; Thériault, Jean-François; Zhu, Dao-Wei; Shang, Peng; Labrie, Fernand; Lin, Sheng-Xiang

2016-04-15

Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP(+)·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP(+) Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP(+)·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

Energy Technology Data Exchange (ETDEWEB)

Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, Department of Biochemistry, University of Leicester, Leicester LE1 9HN (United Kingdom)

2008-08-01

Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

International Nuclear Information System (INIS)

Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

2008-01-01

Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2 1 ; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3 2 . The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP

Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

Science.gov (United States)

Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

2010-04-01

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium

NARCIS (Netherlands)

Biagiotti, E.; Bosch, K. S.; Ninfali, P.; Frederiks, W. M.; van Noorden, C. J.

2000-01-01

Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown, it may be related;either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized

D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens

International Nuclear Information System (INIS)

Lessmann, D.; Schimz, K.L.; Kurz, G.

1975-01-01

The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300μmol NADH formed per min per mg protein, was shown to be homogeneous. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220,000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265,000. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal acivity at pH 8.9. The Entner-Doudoroff enzyme showed specificity for NAD + as well as for NADP + and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of β-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase. (orig.) [de

Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal

NARCIS (Netherlands)

Keller, Markus A.; Watschinger, Katrin; Golderer, Georg; Maglione, Manuel; Sarg, Bettina; Lindner, Herbert H.; Werner-Felmayer, Gabriele; Terrinoni, Alessandro; Wanders, Ronald J. A.; Werner, Ernst R.

2010-01-01

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde,

2-ethylhydracrylic aciduria in short/branched-chain acyl-CoA dehydrogenase deficiency

DEFF Research Database (Denmark)

Korman, Stanley H; Andresen, Brage S; Zeharia, Avraham

2005-01-01

BACKGROUND: Isolated excretion of 2-methylbutyrylglycine (2-MBG) is the hallmark of short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD), a recently identified defect in the proximal pathway of L-isoleucine oxidation. SBCADD might be underdiagnosed because detection and recognition...

Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria

NARCIS (Netherlands)

Feldman-Salit, A.; Hering, S.; Messiha, H.L.; Veith, N.; Cojocaru, V.; Sieg, A.; Westerhoff, H.V.; Kreikemeyer, B.; Wade, R.C.; Fiedler, T.

2013-01-01

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the

High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase

International Nuclear Information System (INIS)

Nagae, Takayuki; Kawamura, Takashi; Chavas, Leonard M. G.; Niwa, Ken; Hasegawa, Masashi; Kato, Chiaki; Watanabe, Nobuhisa

2012-01-01

Structures of 3-isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration. Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH

Discovery of Clinical Candidate 2-((2S,6 S)-2-Phenyl-6-hydroxyadamantan-2-yl)-1-(3'-hydroxyazetidin-1-yl)ethanone [BMS-816336], an Orally Active Novel Selective 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor

Energy Technology Data Exchange (ETDEWEB)

Ye, Xiang-Yang; Chen, Stephanie Y.; Wu, Shung; Yoon, David S.; Wang, Haixia; Hong, Zhenqiu; O’Connor, Stephen P.; Li, Jun; Li, James J.; Kennedy, Lawrence J.; Walker, Steven J.; Nayeem, Akbar; Sheriff, Steven; Camac, Daniel M.; Ramamurthy, Vidyhashankar; Morin, Paul E.; Zebo, Rachel; Taylor, Joseph R.; Morgan, Nathan N.; Ponticiello, Randolph P.; Harrity, Thomas; Apedo, Atsu; Golla, Rajasree; Seethala, Ramakrishna; Wang, Mengmeng; Harper, Timothy W.; Sleczka, Bogdan G.; He, Bin; Kirby, Mark; Leahy, David K.; Li, Jianqing; Hanson, Ronald L.; Guo, Zhiwei; Li, Yi-Xin; DiMarco, John D.; Scaringe, Raymond; Maxwell, Brad; Moulin, Frederick; Barrish, Joel C.; Gordon, David A.; Robl, Jeffrey A.

2017-06-05

BMS-816336 (6n-2), a hydroxy-substituted adamantyl acetamide, has been identified as a novel, potent inhibitor against human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme (IC₅₀ 3.0 nM) with >10000-fold selectivity over human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). 6n-2 exhibits a robust acute pharmacodynamic effect in cynomolgus monkeys (ED₅₀ 0.12 mg/kg) and in DIO mice. It is orally bioavailable (%F ranges from 20 to 72% in preclinical species) and has a predicted pharmacokinetic profile of a high peak to trough ratio and short half-life in humans. This ADME profile met our selection criteria for once daily administration, targeting robust inhibition of 11β-HSD1 enzyme for the first 12 h period after dosing followed by an “inhibition holiday” so that the potential for hypothalamic–pituitary–adrenal (HPA) axis activation might be mitigated. 6n-2 was found to be well-tolerated in phase 1 clinical studies and represents a potential new treatment for type 2 diabetes, metabolic syndrome, and other human diseases modulated by glucocorticoid control.

Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

Science.gov (United States)

Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

2016-04-01

Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

Effects of whole body x-ray irradiation on induction by phenobarbital of rat liver glucose-6-phosphate dehydrogenase and glutathione reductase

Energy Technology Data Exchange (ETDEWEB)

Bitny-Szlachto, S.; Szyszko, A. (Wojskowy Inst. Higieny i Epidemiologii, Warsaw (Poland))

1979-01-01

In rats treated with phenobarbital (3x100 mg/kg, i.p.), liver G-6-P dehydrogenase activity increased by 70% in the cytosol and in the 9.000xg supernatant, and only by 20% in microsomes. Moreover, the phenobarbital treatment increased rat liver GSSG reductase activity by 30%. On the other hand, activity of the liver microsomal G-6-P dehydrogenase was found to increase by some 20% in whole body irradiated, both control and phenobarbital treated rats. In rats irradiated with 600 R prior to the first dose of the inducer there was not noted any increase in G-6-P dehydrogenase of the 9.000xg supernatant, and increase in the cytosol activity dropped to 38%. Thus, induction of the soluble liver G-6-P dehydrogenase by phenobarbital has turned out to be radiosensitive, whereas phenobarbital induction of GSSG reductase was unaffected by irradiation.

Influence of thorax irradiation on lactic dehydrogenase isoenzyme activity

International Nuclear Information System (INIS)

Valle, C.; Munnich, A.; Pasquier, C.

The right hemi-thorax of rats was irradiated with 1200 and 3000 rads ( 60 Co) and blood samples were taken sequentially. The five lactic dehydrogenase (LDH) isoenzymes which have proved to be useful as biochemical indicators of acute pulmonary injury in other experimental animals (dogs), were assayed, after irradiation, as a function of time and as a functon of dose. There was no significant change in LDH isoenzyme activities after lung irradiation in rats [fr

Inactivation of alcohol dehydrogenase (ADH) by ferryl derivatives of human hemoglobin.

Science.gov (United States)

Kowalczyk, Aleksandra; Puchała, Mieczysław; Wesołowska, Katarzyna; Serafin, Eligiusz

2007-01-01

In this paper, inactivation of alcohol dehydrogenase (ADH) by products of reactions of H2O2 with metHb has been studied. Inactivation of the enzyme was studied in two systems corresponding to two kinetic stages of the reaction. In the first system H2O2 was added to the mixture of metHb and ADH [the (metHb+ADH)+H2O2] system (ADH was present in the system since the moment of addition of H2O2 i. e. since the very beginning of the reaction of metHb with H2O2). In the second system ADH was added to the system 5 min after the initiation of the reaction of H2O2 with metHb [the (metHb+H2O2)5 min+ADH] system. In the first case all the products of reaction of H2O2 with metHb (non-peroxyl and peroxyl radicals and non-radical products, viz. hydroperoxides and *HbFe(IV)=O) could react with the enzyme causing its inactivation. In the second system, enzyme reacted almost exclusively with non-radical products (though a small contribution of reactions with peroxyl radicals cannot be excluded). ADH inactivation was observed in both system. Hydrogen peroxide alone did not inactivate ADH at the concentrations employed evidencing that enzyme inactivation was due exclusively to products of reaction of H2O2 with metHb. The rate and extent of ADH inactivation were much higher in the first than in the second system. The dependence of ADH activity on the time of incubation with ferryl derivatives of Hb can be described by a sum of three exponentials in the first system and two exponentials in the second system. Reactions of appropriate forms of the ferryl derivatives of hemoglobin have been tentatively ascribed to these exponentials. The extent of the enzyme inactivation in the second system was dependent on the proton concentration, being at the highest at pH 7.4 and negligible at pH 6.0. The reaction of H2O2 with metHb resulted in the formation of cross-links of Hb subunits (dimers and trimers). The amount of the dimers formed was much lower in the first system i. e. when the radical

Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis.

Science.gov (United States)

Huang, Emina H; Hynes, Mark J; Zhang, Tao; Ginestier, Christophe; Dontu, Gabriela; Appelman, Henry; Fields, Jeremy Z; Wicha, Max S; Boman, Bruce M

2009-04-15

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC), investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly, aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom, where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma, ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells, which are more numerous and broadly distributed in normal crypts, showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not), (b) generated them after implanting as few as 25 cells, and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus, ALDH1 seems to be a specific marker for identifying, isolating, and tracking human colonic SC during CRC development. These findings also support our original hypothesis, derived previously from mathematical modeling of crypt dynamics, that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development.

Cofactor specificity switch in Shikimate dehydrogenase by rational design and consensus engineering.

Science.gov (United States)

García-Guevara, Fernando; Bravo, Iris; Martínez-Anaya, Claudia; Segovia, Lorenzo

2017-08-01

Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Promysalin Elicits Species-Selective Inhibition of Pseudomonas aeruginosa by Targeting Succinate Dehydrogenase.

Science.gov (United States)

Keohane, Colleen E; Steele, Andrew D; Fetzer, Christian; Khowsathit, Jittasak; Van Tyne, Daria; Moynié, Lucile; Gilmore, Michael S; Karanicolas, John; Sieber, Stephan A; Wuest, William M

2018-02-07

Natural products have served as an inspiration to scientists both for their complex three-dimensional architecture and exquisite biological activity. Promysalin is one such Pseudomonad secondary metabolite that exhibits narrow-spectrum antibacterial activity, originally isolated from the rhizosphere. We herein utilize affinity-based protein profiling (AfBPP) to identify succinate dehydrogenase (Sdh) as the biological target of the natural product. The target was further validated in silico, in vitro, in vivo, and through the selection, and sequencing, of a resistant mutant. Succinate dehydrogenase plays an essential role in primary metabolism of Pseudomonas aeruginosa as the only enzyme that is involved both in the tricarboxylic acid cycle (TCA) and in respiration via the electron transport chain. These findings add credence to other studies that suggest that the TCA cycle is an understudied target in the development of novel therapeutics to combat P. aeruginosa, a significant pathogen in clinical settings.

DFT-based prediction of reactivity of short-chain alcohol dehydrogenase

Science.gov (United States)

Stawoska, I.; Dudzik, A.; Wasylewski, M.; Jemioła-Rzemińska, M.; Skoczowski, A.; Strzałka, K.; Szaleniec, M.

2017-06-01

The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, ( S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2' ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R2 = 0.93) and a good prediction of the reaction kinetics (R2 = 0.89). The observed relations were validated in prediction of log K eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.

The activity of dehydrogenases in the uterus of C57B mice after X-irradiation and serotonin treatment

International Nuclear Information System (INIS)

Mazur, L.

1978-01-01

In C57B female mice, irradiated with 500 R and/or treated with serotonin (5-hydroxytryptamine), the activity of dehydrogenases in the uterus was studied on the fourth day of pregnancy. The reduction of 2,3,5-triphenyltetrazolium chloride to formazane by the uterine tissue was taken as the measure of such activity. The activity of dehydrogenases in the uterus of irradiated mice was distinctly lower than in non-irradiated controls. This activity was also depressed after serotonin treatment, the level of enzyme activity being dose-dependent. In females injected with serotonin and then irradiated, the activity of dehydrogenases was higher than in those irradiated only. The radioprotective effect was more pronounced in mice injected with serotonin alone on the third day of pregnancy i.e. shortly before irradiation, than in those injected on the second and the third day. (author)

[Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

Science.gov (United States)

Khunderyakova, N V; Zakharchenko, A V; Zakharchenko, M V; Muller, H; Fedotcheva, I; Kondrashova, M N

2015-01-01

Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress.

Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells.

Science.gov (United States)

Lee, Su Jeong; Park, Jeen-Woo

2014-04-01

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells.

Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

OpenAIRE

Wisselink, H. Wouter; Mars, Astrid E.; van der Meer, Pieter; Eggink, Gerrit; Jeroen Hugenholtz

2004-01-01

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (

Structural and functional characterization of plant aminoaldehyde dehydrogenase from Pisum sativum with a broad specificity for natural and synthetic aminoaldehydes

Czech Academy of Sciences Publication Activity Database

Tylichová, M.; Kopečný, D.; Moréra, S.; Briozzo, P.; Lenobel, René; Snégaroff, J.; Šebela, M.

2010-01-01

Roč. 396, č. 4 (2010), s. 870-882 ISSN 0022-2836 R&D Projects: GA ČR GA522/08/0555; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : aminoaldehyde dehydrogenase * betaine aldehyde dehydrogenase * NAD+ complex Subject RIV: CE - Biochemistry Impact factor: 4.008, year: 2010

Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis

DEFF Research Database (Denmark)

Andersen, H.W.; Pedersen, M.B.; Hammer, Karin

2001-01-01

enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control (C- LDH(F1)J=-1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate...

Assessing the stereoselectivity of Serratia marcescens CECT 977 2,3-butanediol dehydrogenase

NARCIS (Netherlands)

Medici, R.; Stammes, J.K.; Otten, L.G.; Hanefeld, U.; Kwakernaak, Stender

2017-01-01

α-Hydroxy ketones and vicinal diols constitute well-known building blocks in organic synthesis. Here we describe one enzyme that enables the enantioselective synthesis of both building blocks starting from diketones. The enzyme 2,3-butanediol dehydrogenase (BudC) from S. marcescens CECT 977 belongs

Investigations regarding the anthropic impact on the Krebs cycle dehydrogenases system on certain wood-species in mining areas, Suceava county

Directory of Open Access Journals (Sweden)

Marius Viorel Oniciuc

2013-03-01

Full Text Available The Krebs cycle, a second stage of cellular respiration that occurs in the mitochondrion of the leafcell and consist in a multistep processes plays a central role in catabolism of organic fuel molecules. The miningextraction technologies for both underground and surface, the preparation of copper ore and barite applied in Tarnia,respectively to the sulphur in Calimani Mountain and the excess of these elements in natural environment may causemalfunction of ecosystem. The dehydrogenases of Krebs cycle can give information on the type and the duration of theeffects of pollutants on the metabolic activity in leaves, to subsequent area pollution, therefore, the aim of the presentstudy has been to determine these effects on this enzymatic system activity. For this reason, the isocitrate dehydrogenase,the -ketoglutate dehydrogenase, the succinate ehydrogenase and the malate dehydrogenase activity was determined using the spectrophotometric method with triphenyl-tetrazolium and the analysis of experimental results shows the differences from one sample to another sample of closely related species specificity, but also the effect of environmentalfactors.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa.

Science.gov (United States)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-02-01

The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.

Scanning mutagenesis of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

Directory of Open Access Journals (Sweden)

Nagib eAhsan

2012-07-01

Full Text Available The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform scanning mutagenesis of the residues flanking the site of phosphorylation. Consistent with the results from phylogenetic analysis of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp.

OpenAIRE

Xu, J; Johnson, R C

1995-01-01

Escherichia coli aldB was identified as a gene that is negatively regulated by Fis but positively regulated by RpoS. The complete DNA sequence determined in this study indicates that aldB encodes a 56.3-kDa protein which shares a high degree of homology with an acetaldehyde dehydrogenase encoded by acoD of Alcaligenes eutrophus and an aldehyde dehydrogenase encoded by aldA of Vibrio cholerae and significant homology with a group of other aldehyde dehydrogenases from prokaryotes and eukaryotes...

The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

Czech Academy of Sciences Publication Activity Database

Smolková, Katarína; Ježek, Petr

2012-01-01

Roč. 2012, č. 2012 (2012), ID273947 ISSN 1687-8876 R&D Projects: GA ČR GPP301/12/P381; GA ČR(CZ) GAP302/10/0346 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : isocitrate dehydrogenase 2 * Krebs cycle * cancer cells Subject RIV: ED - Physiology

An L-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of D-sorbose with enzymatic or electrochemical cofactor regeneration

DEFF Research Database (Denmark)

Gauer, Sabrina; Wang, Zhijie; Otten, Harm

2014-01-01

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an L-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consistin...

Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

Science.gov (United States)

Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw; Stridh, Malin H; Zaganas, Ioannis; Skytt, Dorte M; Schousboe, Arne; Bak, Lasse K; Enard, Wolfgang; Pääbo, Svante; Waagepetersen, Helle S

2017-03-01

A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO 2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [ 3 H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of 13 C and 14 C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO 2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488. © 2016 Wiley Periodicals, Inc.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model.

Directory of Open Access Journals (Sweden)

Sameer Udhane

Full Text Available Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs. Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute versus long-term (chronic stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

Science.gov (United States)

Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

2015-11-01

Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

5FU and oxaliplatin-containing chemotherapy in two dihydropyrimidine dehydrogenase-deficient patients

NARCIS (Netherlands)

Reerink, O; Mulder, NH; Szabo, BG; Hospers, GAP

2004-01-01

Patients with a germline mutation leading to a deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme are at risk from developing severe toxicity on the administration of 5FU-containing chemotherapy. We report on the implications of this inborn genetic error in two patients who received 5FU

NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

Directory of Open Access Journals (Sweden)

Ekaterina Yu. Bezsudnova

2016-01-01

Full Text Available We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution, three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å, and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å. The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

Sequence of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Nicotiana plumbaginifolia and phylogenetic origin of the gene family.

Science.gov (United States)

Habenicht, A; Quesada, A; Cerff, R

1997-10-01

A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.

15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis.

Science.gov (United States)

Myung, Seung-Jae; Rerko, Ronald M; Yan, Min; Platzer, Petra; Guda, Kishore; Dotson, Angela; Lawrence, Earl; Dannenberg, Andrew J; Lovgren, Alysia Kern; Luo, Guangbin; Pretlow, Theresa P; Newman, Robert A; Willis, Joseph; Dawson, Dawn; Markowitz, Sanford D

2006-08-08

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a prostaglandin-degrading enzyme that is highly expressed in normal colon mucosa but is ubiquitously lost in human colon cancers. Herein, we demonstrate that 15-PGDH is active in vivo as a highly potent suppressor of colon neoplasia development and acts in the colon as a required physiologic antagonist of the prostaglandin-synthesizing activity of the cyclooxygenase 2 (COX-2) oncogene. We first show that 15-PGDH gene knockout induces a marked 7.6-fold increase in colon tumors arising in the Min (multiple intestinal neoplasia) mouse model. Furthermore, 15-PGDH gene knockout abrogates the normal resistance of C57BL/6J mice to colon tumor induction by the carcinogen azoxymethane (AOM), conferring susceptibility to AOM-induced adenomas and carcinomas in situ. Susceptibility to AOM-induced tumorigenesis is mediated by a marked induction of dysplasia, proliferation, and cyclin D1 expression throughout microscopic aberrant crypt foci arising in 15-PGDH null colons and is concomitant with a doubling of prostaglandin E(2) in 15-PGDH null colonic mucosa. A parallel role for 15-PGDH loss in promoting the earliest steps of colon neoplasia in humans is supported by our finding of a universal loss of 15-PGDH expression in microscopic colon adenomas recovered from patients with familial adenomatous polyposis, including adenomas as small as a single crypt. These models thus delineate the in vivo significance of 15-PGDH-mediated negative regulation of the COX-2 pathway and moreover reveal the particular importance of 15-PGDH in opposing the neoplastic progression of colonic aberrant crypt foci.

Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

International Nuclear Information System (INIS)

Batzer, M.A.

1988-01-01

Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from

Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

Science.gov (United States)

Baggetto, L G; Lehninger, A L

1987-05-29

Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

Idiopathic intracranial hypertension, hormones, and 11ß-hydroxysteroid dehydrogenases

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Markey KA

2016-04-01

Full Text Available Keira A Markey,1 Maria Uldall,2 Hannah Botfield,1 Liam D Cato,1 Mohammed A L Miah,1 Ghaniah Hassan-Smith,1 Rigmor H Jensen,2 Ana M Gonzalez,1 Alexandra J Sinclair1 1Neurometabolism, Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK; 2Danish Headache Center, Clinic of Neurology, Rigshospitalet-Glostrup, University of Copenhagen, Glostrup, Denmark Abstract: Idiopathic intracranial hypertension (IIH results in raised intracranial pressure (ICP leading to papilledema, visual dysfunction, and headaches. Obese females of reproductive age are predominantly affected, but the underlying pathological mechanisms behind IIH remain unknown. This review provides an overview of pathogenic factors that could result in IIH with particular focus on hormones and the impact of obesity, including its role in neuroendocrine signaling and driving inflammation. Despite occurring almost exclusively in obese women, there have been a few studies evaluating the mechanisms by which hormones and adipokines exert their effects on ICP regulation in IIH. Research involving 11ß-hydroxysteroid dehydrogenase type 1, a modulator of glucocorticoids, suggests a potential role in IIH. Improved understanding of the complex interplay between adipose signaling factors such as adipokines, steroid hormones, and ICP regulation may be key to the understanding and future management of IIH. Keywords: 11beta-hydroxysteroid dehydrogenase type 1, steroid and adipokines, obesity, leptin

Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1.

LENUS (Irish Health Repository)

Semjonous, Nina M

2011-01-01

Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1\\/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)\\/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.

Enzymatic conversion of CO2 to CH3OH via reverse dehydrogenase cascade biocatalysis: Quantitative comparison of efficiencies of immobilized enzyme systems

DEFF Research Database (Denmark)

Marpani, Fauziah Binti; Pinelo, Manuel; Meyer, Anne S.

2017-01-01

A designed biocatalytic cascade system based on reverse enzymatic catalysis by formate dehydrogenase (EC 1.2.1.2), formaldehyde dehydrogenase (EC 1.2.1.46), and alcohol dehydrogenase (EC 1.1.1.1) can convert carbon dioxide (CO2) to methanol (CH3OH) via formation of formic acid (CHOOH......) and formaldehyde (CHOH) during equimolar cofactor oxidation of NADH to NAD+. This reaction is appealing because it represents a double gain: (1) reduction of CO2 and (2) an alternative to fossil fuel based production of CH3OH. The present review evaluates the efficiency of different immobilized enzyme systems...

Antiviral activity of A771726, the active metabolite of leflunomide, against Junín virus.

Science.gov (United States)

Sepúlveda, Claudia S; García, Cybele C; Damonte, Elsa B

2018-05-01

The aim of this study was to investigate the effect of A771726, the active metabolite of leflunomide, (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad against the infection with Junín virus (JUNV), agent of Argentine hemorrhagic fever (AHF). The treatment with non-cytotoxic concentrations of A771726 of Vero and A549 cells infected with JUNV inhibited virus replication in a dose-dependent manner, as determined by virus yield reduction assay. The antiviral effectiveness of A771726 was not importantly affected by the multiplicity of infection and the virus strain. Moreover, the combination of A771726 and ribavirin had a significantly more potent antiviral activity than each single drug treatment. Mechanistic studies showed that the main action of A771726 is exerted before 6 h of JUNV infection. Accordingly, inhibition of viral RNA synthesis was detected in treated infected cells by real time RT-PCR. The exogenous addition of uridine or orotic acid produced a partial reversal of the inhibitory effect of A771726 on infective virus production whereas a total reversion was detected on JUNV RNA synthesis, probably by restoration of the enzymatic activity of dihydroorotate dehydrogenase (DHODH) and the intracellular pyrimidine pools. In conclusion, these results suggest that the antiviral target would be viral RNA synthesis through pyrimidine depletion, but any other effect of the compound on JUNV infection cannot be excluded. This study opens the possibility of the therapeutic application of a wide spectrum host-targeted compound alone or in combination with ribavirin to combat AHF as well as other human pathogenic arenaviruses. © 2018 Wiley Periodicals, Inc.

Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

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Aceves-Ramos, A; de la Torre, P; Hinojosa, L; Ponce, A; García-Villegas, R; Laclette, J P; Bobes, R J; Romano, M C

2014-07-01

The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species. Copyright © 2014 Elsevier Inc. All rights reserved.

Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

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Gordon, G L; Doelle, H W

1976-08-16

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

Post-irradiation repairing processes of glucose-6-phosphate dehydrogenase and catalase from Hansenula Polymorpha yeast

International Nuclear Information System (INIS)

Postolache, Carmen; Postolache, Cristian; Dinu, Diana; Dinischiotu, Anca; Sahini, Victor Emanuel

2002-01-01

The post-irradiation repairing mechanisms of two Hansenula Polymorpha yeast enzymes, glucose-6-phosphate dehydrogenase and catalase, were studied. The kinetic parameters of the selected enzymes were investigated over one month since the moment of γ-irradiation with different doses in the presence of oxygen. Dose dependent decrease of initial reaction rates was noticed for both enzymes. Small variation of initial reaction rate was recorded for glucose-6-phosphate dehydrogenase over one month, with a decreasing tendency. No significant electrophoretic changes of molecular forms of this enzyme were observed after irradiation. Continuous strong decrease of catalase activity was evident for the first 20 days after irradiation. Partial recovery process of the catalytic activity was revealed by this study. (authors)

Hit discovery of Mycobacterium tuberculosis inosine 5'-monophosphate dehydrogenase, GuaB2, inhibitors.

Science.gov (United States)

Sahu, Niteshkumar U; Singh, Vinayak; Ferraris, Davide M; Rizzi, Menico; Kharkar, Prashant S

2018-04-18

Tuberculosis remains a global concern. There is an urgent need of newer antitubercular drugs due to the development of resistant forms of Mycobacterium tuberculosis (Mtb). Inosine 5'-monophosphate dehydrogenase (IMPDH), guaB2, of Mtb, required for guanine nucleotide biosynthesis, is an attractive target for drug development. In this study, we screened a focused library of 73 drug-like molecules with desirable calculated/predicted physicochemical properties, for growth inhibitory activity against drug-sensitive MtbH37Rv. The eight hits and mycophenolic acid, a prototype IMPDH inhibitor, were further evaluated for activity on purified Mtb-GuaB2 enzyme, target selectivity using a conditional knockdown mutant of guaB2 in Mtb, followed by cross-resistance to IMPDH inhibitor-resistant SRMV2.6 strain of Mtb, and activity on human IMPDH2 isoform. One of the hits, 13, a 5-amidophthalide derivative, has shown growth inhibitory potential and target specificity against the Mtb-GuaB2 enzyme. The hit, 13, is a promising molecule with potential for further development as an antitubercular agent. Copyright © 2018 Elsevier Ltd. All rights reserved.

cis-chlorobenzene dihydrodiol dehydrogenase (TcbB) from Pseudomonas sp. strain P51, expressed in Escherichia coli DH5alpha(pTCB149), catalyzes enantioselective dehydrogenase reactions.

Science.gov (United States)

Raschke, H; Fleischmann, T; Van Der Meer, J R; Kohler, H P

1999-12-01

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5alpha(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (-)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3, 4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (-)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1, 2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.

cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions

Science.gov (United States)

Raschke, Henning; Fleischmann, Thomas; Van Der Meer, Jan Roelof; Kohler, Hans-Peter E.

1999-01-01

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (−)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols. PMID:10583971

Cis-Chlorobenzene dihydrodiol dehydrogenase (TcbB) from Pseudomonas sp. strain P51, expressed in Escherichia coli DH5{alpha}(pTCB149), catalyzes enantioselective dehydrogenase reactions

Energy Technology Data Exchange (ETDEWEB)

Raschke, H.; Fleischmann, T.; Meer, J.R. van der; Kohler, H.P.E.

1999-12-01

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5{alpha}(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (-)-cis-(S,2R) enantiomer remained unchanged, CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (-)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enatiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis.

Science.gov (United States)

Nair, Ramesh B; Bastress, Kristen L; Ruegger, Max O; Denault, Jeff W; Chapple, Clint

2004-02-01

Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.

GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice1

Science.gov (United States)

Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

2006-01-01

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis. PMID:16443696

Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate.

Science.gov (United States)

Kormanik, Kaitlyn; Kang, Heejung; Cuebas, Dean; Vockley, Jerry; Mohsen, Al-Walid

2012-12-01

Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (K(D app)) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the K(m) for phenylbutyryl-CoA were 0.2 mM 34(-1)·sec(-1) and 5.3 μM compared to 4.0 mM(-1)·sec(-1) and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism. Copyright © 2012 Elsevier Inc. All rights reserved.

Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic bacterium isolated from a sterilization drying oven

Directory of Open Access Journals (Sweden)

Maximiliano J. Amenábar

2012-02-01

Full Text Available Glutamate dehydrogenase from axenic bacterial cultures of anew microorganism, called GWE1, isolated from the interior ofa sterilization drying oven, was purified by anion-exchange andmolecular-exclusion liquid chromatography. The apparent molecularmass of the native enzyme was 250.5 kDa and wasshown to be an hexamer with similar subunits of molecularmass 40.5 kDa. For glutamate oxidation, the enzyme showedan optimal pH and temperature of 8.0 and 70oC, respectively.In contrast to other glutamate dehydrogenases isolated frombacteria, the enzyme isolated in this study can use both NAD+and NADP+ as electron acceptors, displaying more affinity forNADP+ than for NAD+. No activity was detected with NADHor NADPH, 2-oxoglutarate and ammonia. The enzyme was exceptionallythermostable, maintaining more than 70% of activityafter incubating at 100oC for more than five hours suggestingbeing one of the most thermoestable enzymes reported inthe family of dehydrogenases. [BMB reports 2012; 45(2: 91-95

Modified expression of cytoplasmic isocitrate dehydrogenase electrophoretic isoforms in seminal plasma of men with sertoli-cell-only syndrome and seminoma.

Science.gov (United States)

Starita-Geribaldi, Mireille; Samson, Michel; Guigonis, Jean-Marie; Pointis, Georges; Fenichel, Patrick

2008-06-01

Two isoforms of human cytoplasmic isocitrate dehydrogenase (IDPc) of close molecular weights and different isoelectric points were identified in human seminal plasma (SP) by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). These two isoforms were detected in the normospermic men SP and their expressions were markedly altered in patients with testicular seminoma, the most frequent testicular germ cell cancer (TGCC): increase of the more acidic spot and decrease of the more basic one. Since oligospermia has been considered as a high risk pathological condition for developing a testicular cancer, the two IDPc isoforms were analyzed in SP of a group of secretory azoospermic patients. In this group the two spots displayed similar variations of expression to those observed in testicular seminoma. These results propose IDPc as a promising SP biomarker of testicular seminoma. Whether IDPc alteration in secretory azoospermia is predictive of testicular seminoma remains to be elucidated. (c) 2007 Wiley-Liss, Inc.

In vitro hydrogen production by glucose dehydrogenase and hydrogenase

Energy Technology Data Exchange (ETDEWEB)

Woodward, J. [Oak Ridge National Lab., TN (United States)

1996-10-01

A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

Expression and prognostic value of lactate dehydrogenase-A and -D subunits in human uterine myoma and uterine sarcoma.

Science.gov (United States)

Song, Ke-Juan; Yu, Xiao-Ni; Lv, Teng; Chen, Yu-Long; Diao, Yu-Chao; Liu, Su-Li; Wang, Yan-Kui; Yao, Qin

2018-04-01

This study aimed to determine the expression of lactate dehydrogenase (LDH)-A and LDH-D in patients with uterine myoma, cellular leiomyoma (CLM), and uterine sarcoma and to evaluate their prognostic significance. Protein expression levels of LDH-A and LDH-D were determined in tissue samples from 86 patients (26 uterine myoma, 10 CLM, 50 uterine sarcoma) by immunohistochemistry and their associations with clinicopathologic parameters and outcomes were analyzed in patients with uterine sarcoma. The positivity rates for LDH-A and LDH-D were significantly higher in patients with uterine sarcoma compared with those with uterine myoma or CLM (P sarcoma were classified as having uterine leiomyosarcoma (LMS), malignant endometrial stromal sarcoma, and malignant mixed Mullerian tumor, with 5-year overall survival rates of 59%, 71%, and 29%, respectively (P sarcoma. Furthermore, the overexpressions of LDH-A and LDH-D in uterine sarcoma patients may contribute to further understanding of the mechanism of LDH in tumor metabolism in uterine sarcoma. Positive expression of LDH-A in patients with LMS may act as a potential prognostic biomarker in these patients.

Identification of novel PfDHODH inhibitors as antimalarial agents via pharmacophore-based virtual screening followed by molecular docking and in vivo antimalarial activity.

Science.gov (United States)

Vyas, V K; Qureshi, G; Ghate, M; Patel, H; Dalai, S

2016-06-01

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) catalyses the fourth reaction of de novo pyrimidine biosynthesis in parasites, and represents an important target for the treatment of malaria. In this study, we describe pharmacophore-based virtual screening combined with docking study and biological evaluation as a rational strategy for identification of novel hits as antimalarial agents. Pharmacophore models were established from known PfDHODH inhibitors using the GALAHAD module with IC50 values ranging from 0.033 μM to 142 μM. The best pharmacophore model consisted of three hydrogen bond acceptor, one hydrogen bond donor and one hydrophobic features. The pharmacophore models were validated through receiver operating characteristic and Günere-Henry scoring methods. The best pharmacophore model as a 3D search query was searched against the IBS database. Several compounds with different structures (scaffolds) were retrieved as hit molecules. Among these compounds, those with a QFIT value of more than 81 were docked in the PfDHODH enzyme to further explore the binding modes of these compounds. In silico pharmacokinetic and toxicities were predicted for the best docked molecules. Finally, the identified hits were evaluated in vivo for their antimalarial activity in a parasite inhibition assay. The hits reported here showed good potential to become novel antimalarial agents.

Dimerization interface of 3-hydroxyacyl-CoA dehydrogenase tunes the formation of its catalytic intermediate.

Directory of Open Access Journals (Sweden)

Yingzhi Xu

Full Text Available 3-Hydroxyacyl-CoA dehydrogenase (HAD, EC 1.1.1.35 is a homodimeric enzyme localized in the mitochondrial matrix, which catalyzes the third step in fatty acid β-oxidation. The crystal structures of human HAD and subsequent complexes with cofactor/substrate enabled better understanding of HAD catalytic mechanism. However, numerous human diseases were found related to mutations at HAD dimerization interface that is away from the catalytic pocket. The role of HAD dimerization in its catalytic activity needs to be elucidated. Here, we solved the crystal structure of Caenorhabditis elegans HAD (cHAD that is highly conserved to human HAD. Even though the cHAD mutants (R204A, Y209A and R204A/Y209A with attenuated interactions on the dimerization interface still maintain a dimerization form, their enzymatic activities significantly decrease compared to that of the wild type. Such reduced activities are in consistency with the reduced ratios of the catalytic intermediate formation. Further molecular dynamics simulations results reveal that the alteration of the dimerization interface will increase the fluctuation of a distal region (a.a. 60-80 that plays an important role in the substrate binding. The increased fluctuation decreases the stability of the catalytic intermediate formation, and therefore the enzymatic activity is attenuated. Our study reveals the molecular mechanism about the essential role of the HAD dimerization interface in its catalytic activity via allosteric effects.

On-column ligand exchange for structure-based drug design: a case study with human 11β-hydroxysteroid dehydrogenase type 1

International Nuclear Information System (INIS)

Qin, Wenying; Judge, Russell A.; Longenecker, Kenton L.; Solomon, Larry R.; Harlan, John E.

2012-01-01

An on-column ligand- and detergent-exchange method was developed to obtain ligand–protein complexes for an adamantane series of compounds with 11β-HSD1 after a variety of other complexation methods had failed. An interesting byproduct of the method was the observation of artificial trimers in the crystal structures. Successfully forming ligand–protein complexes with specific compounds can be a significant challenge in supporting structure-based drug design for a given protein target. In this respect, an on-column ligand- and detergent-exchange method was developed to obtain ligand–protein complexes of an adamantane series of compounds with 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) after a variety of other complexation methods had failed. This report describes the on-column exchange method and an unexpected byproduct of the method in which artificial trimers were observed in the structures

15-Hydroxyprostaglandin dehydrogenase inactivation as a mechanism of resistance to celecoxib chemoprevention of colon tumors.

LENUS (Irish Health Repository)

Yan, Min

2009-06-09

Pharmacologic inhibitors of the prostaglandin-synthesizing COX-2 oncogene prevent the development of premalignant human colon adenomas. However, resistance to treatment is common. In this study, we show that the adenoma prevention activity of the COX-2 inhibitor celecoxib requires the concomitant presence of the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) tumor suppressor gene, and that loss of 15-PGDH expression imparts resistance to celecoxib\\'s anti-tumor effects. We first demonstrate that the adenoma-preventive activity of celecoxib is abrogated in mice genetically lacking 15-PGDH. In FVB mice, celecoxib prevents 85% of azoxymethane-induced tumors >1 mm in size, but is essentially inactive in preventing tumor induction in 15-PGDH-null animals. Indeed, celecoxib treated 15-PGDH null animals develop more tumors than do celecoxib naive WT mice. In parallel with the loss of tumor prevention activity, celecoxib-mediated suppression of colonic PGE(2) levels is also markedly attenuated in 15-PGDH-null versus WT mice. Finally, as predicted by the murine models, humans with low colonic 15-PGDH levels also exhibit celecoxib resistance. Specifically, in a colon adenoma prevention trial, in all cases tested, individuals who developed new adenomas while receiving celecoxib treatment were also found as having low colonic 15-PGDH levels.

Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations

DEFF Research Database (Denmark)

Rehn, Gustav; Pedersen, Asbjørn Toftgaard; Woodley, John

2016-01-01

alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables...

Alcohol dehydrogenase 3 genotype as a risk factor for upper aerodigestive tract cancers

DEFF Research Database (Denmark)

Nishimoto, Inês Nobuko; Pinheiro, Nidia A; Rogatto, Silvia R

2004-01-01

OBJECTIVE: To assess alcohol dehydrogenase 3 (ADH3) polymorphism at position Ile349Val as indicator of risk factor for upper aerodigestive tract (UADT) cancer to verify its association with UADT cancer in nonalcoholic or nonsmoking individuals. DESIGN: Cross-sectional study. SETTING: Primary care...

Tissue carnitine homeostasis in very-long-chain acyl-CoA dehydrogenase-deficient mice

NARCIS (Netherlands)

Spiekerkoetter, Ute; Tokunaga, Chonan; Wendel, Udo; Mayatepek, Ertan; Ijlst, Lodewijk; Vaz, Frederic M.; van Vlies, Naomi; Overmars, Henk; Duran, Marinus; Wijburg, Frits A.; Wanders, Ronald J.; Strauss, Arnold W.

2005-01-01

Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD) is the most common long-chain fatty acid oxidation defect and presents with heterogeneous clinical manifestations. Accumulation of long-chain acylcarnitines and deficiency of free carnitine have often been proposed to play an important

Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress

Czech Academy of Sciences Publication Activity Database

Pospíšilová, H.; Jiskrová, E.; Vojta, P.; Mrízová, K.; Kokáš, F.; Majeská Čudějková, M.; Bergougnoux, V.; Plíhal, O.; Klimešová, J.; Novák, Ondřej; Dzurová, L.; Frébort, I.; Galuszka, P.

2016-01-01

Roč. 33, č. 5 (2016), s. 692-705 ISSN 1871-6784 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : ROOT-GROWTH * OXIDASE/DEHYDROGENASE GENES * BETA-GLUCOSIDASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.813, year: 2016

Positive selection on D-lactate dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus.

Science.gov (United States)

Zhang, Jifeng; Gong, Guangyu; Wang, Xiao; Zhang, Hao; Tian, Weidong

2015-08-01

Lactobacillus delbrueckii has been widely used for yogurt fermentation. It has genes encoding both D- and L-type lactate dehydrogenases (LDHs) that catalyse the production of L(+) or D(-) stereoisomer of lactic acid. D-lactic acid is the primary lactate product by L. delbrueckii, yet it cannot be metabolised by human intestine. Since it has been domesticated for long time, an interesting question arises regarding to whether the selection pressure has affected the evolution of both L-LDH and D-LDH genes in the genome. To answer this question, in this study the authors first investigated the evolution of these two genes by constructing phylogenetic trees. They found that D-LDH-based phylogenetic tree could better represent the phylogenetic relationship in the acidophilus complex than L-LDH-based tree. They next investigated the evolutions of LDH genes of L. delbrueckii at amino acid level, and found that D-LDH gene in L. delbrueckii is positively selected, possibly a consequence of long-term domestication. They further identified four amino acids that are under positive selection. One of them, V261, is located at the centre of three catalytic active sites, indicating likely functional effects on the enzyme activity. The selection from the domestication process thus provides direction for future engineering of D-LDH.

11β-Hydroxysteroid Dehydrogenases and Hypertension in the Metabolic Syndrome.

Science.gov (United States)

Bailey, Matthew A

2017-11-14

The metabolic syndrome describes a clustering of risk factors-visceral obesity, dyslipidaemia, insulin resistance, and salt-sensitive hypertension-that increases mortality related to cardiovascular disease, type 2 diabetes, cancer, and non-alcoholic fatty liver disease. The prevalence of these concurrent comorbidities is ~ 25-30% worldwide, and metabolic syndrome therefore presents a significant global public health burden. Evidence from clinical and preclinical studies indicates that glucocorticoid excess is a key causal feature of metabolic syndrome. This is not increased systemic in circulating cortisol, rather increased bioavailability of active glucocorticoids within tissues. This review examines the role of covert glucocorticoid excess on the hypertension of the metabolic syndrome. Here, the role of the 11β-hydroxysteroid dehydrogenase enzymes, which exert intracrine and paracrine control over glucocorticoid signalling, is examined. 11βHSD1 amplifies glucocorticoid action in cells and contributes to hypertension through direct and indirect effects on the kidney and vasculature. The deactivation of glucocorticoid by 11βHSD2 controls ligand access to glucocorticoid and mineralocorticoid receptors: loss of function promotes salt retention and hypertension. As for hypertension in general, high blood pressure in the metabolic syndrome reflects a complex interaction between multiple systems. The clear association between high dietary salt, glucocorticoid production, and metabolic disorders has major relevance for human health and warrants systematic evaluation.

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans.

Science.gov (United States)

Puentes-Cala, Edinson; Liebeke, Manuel; Markert, Stephanie; Harder, Jens

2018-05-01

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprene and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a dithiothreitol:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism ( ctm ) in Castellaniella defragrans and were annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB and CtmAB in E. coli demonstrated that limonene dehydrogenase activity required both subunits carrying each a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest a placement in EC 1

Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics

Czech Academy of Sciences Publication Activity Database

Kádek, Alan; Kavan, Daniel; Marcoux, J.; Stojko, J.; Felice, A.K.G.; Cianférani, S.; Ludwig, R.; Halada, Petr; Man, Petr

2017-01-01

Roč. 1861, č. 2 (2017), s. 157-167 ISSN 0304-4165 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LQ1604 Institutional support: RVO:61388971 Keywords : Cellobiose dehydrogenase * Direct electron transfer * Electrostatic interaction Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

Science.gov (United States)

Dave, Khyati K.; Punekar, Narayan S.

2015-01-01

Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production. PMID:26683313

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis.

Science.gov (United States)

Stiti, Naim; Missihoun, Tagnon D; Kotchoni, Simeon O; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected ArabidopsisALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities.

Action of sulphite on plant malate dehydrogenase

Energy Technology Data Exchange (ETDEWEB)

Ziegler, I.

1974-01-01

SO/sub 3//sup 2 -/ acts on NAD- and NADP-dependent malate dehydrogenase in several ways. Firstly, SO/sub 3//sup 2 -/ favours the appearance of low MW species (65000 and 39000 daltons) in Sephadex gel chromatography. Secondly, the enzyme from which is obtained by gel chromatography with dithioerythritol plus nucleotide cofactor is changed in the presence of SO/sub 3//sup 2 -/. This is indicated by the appearance of a linear reaction (instead of curvilinear), and by the abolition of the biphasic sigmoidal kinetics on varying substrate and cofactor concentrations. Thus the inhibition of initial velocity at high substrate or cofactor concentrations is even more marked than at lower ones. Thirdly, SO/sub 3//sup 2 -/ strongly reduces the activity in substrate saturating conditions.

Metabolism of excised embryos of Lupinus luteus L. VI. An electrophoretic analysis of some dehydrogenases in cultured embryos as compared with the normal seedling axes

Directory of Open Access Journals (Sweden)

J. Czosnowski

2015-01-01

Full Text Available The electrophoretic patterns (disc electrophoresis of the studied dehydrogenases: glucose-6-phosphate - (A, malate - (B, glutamate - (C, alcohol - (D and lactate dehydrogenase (E, in the axial organs of isolated Lupinus luteus embryos and seedlings cultivated over 12 days are characterized by great similarities. With time, after the third day of cultivation the patterns begin to become less deyeloped. Analyses performed during the first 10 hours of imbibition of seed parts indicate that the maximal development of isozyme patterns occurs during the third hour after which the patterns become poorer. The most uniform type of pattern. and the lowest number of isozymes was shown by glutamate dehydrogenase, the richest pattern was shown by malate dehydrogenase. No band common for a 11 the 27 experimental elements was found.

Glucose-6-phosphate dehydrogenase activity decreases during storage of leukoreduced red blood cells

NARCIS (Netherlands)

Peters, Anna L.; van Bruggen, Robin; de Korte, Dirk; van Noorden, Cornelis J. F.; Vlaar, Alexander P. J.

2016-01-01

During storage, the activity of the red blood cell (RBC) antioxidant system decreases. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress by producing NADPH. G6PD function of RBC transfusion products is reported to remain stable during storage, but activity

Alcohol consumption and type 2 diabetes: Influence of genetic variation in alcohol dehydrogenase

NARCIS (Netherlands)

Beulens, J.W.J.; Rimm, E.B.; Hendriks, H.F.J.; Hu, F.B.; Manson, J.E.; Hunter, D.J.; Mukamal, K.J.

2007-01-01

OBJECTIVE - We sought to investigate whether a polymorphism in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. RESEARCH DESIGN AND METHODS - In nested case-control studies of 640 women with incident diabetes and 1,000 control

Bioelectrochemical fuel cell and sensor based on quinoprotein alcohol dehydrogenase

Energy Technology Data Exchange (ETDEWEB)

Davis, G; Hill, H A.O.; Aston, W J; Higgins, I J; Turner, A P.F.

1983-09-01

A biofuel cell, yielding a stable and continuous low-power output, based on the enzymatic oxidation of methanol to formic acid has been designed and investigated. The homogeneous kinetics of the electrochemically-coupled enzymatic oxidation reaction were investigated and optimized. The biofuel cell also functioned as a sensitive method for the detection of primary alcohols. A method for medium-scale preparation of the enzyme alcohol dehydrogenase (alcohol: (acceptor) oxidoreductase, EC 1.1.99.8) is described. (Refs. 14).

Calcium inhibition of the NAD+-linked isocitrate dehydrogenase from blowfly flight muscle mitochondria.

Science.gov (United States)

Bulos, B A; Thomas, B J; Sacktor, B

1984-08-25

Free Ca2+ was shown to inhibit the NAD+-isocitrate dehydrogenase from blowfly flight muscle mitochondria. Inhibition by free Ca2+ concentrations of 40 microM or greater was found in the absence or presence of ADP and citrate, two known activators of the enzyme. Calcium decreased the affinity of the enzyme for its substrate, the magnesium DL-isocitrate chelate; no change in the apparent V of the reaction was observed. Calcium was inhibitory when activity was measured in the presence of fixed concentrations of magnesium DL-isocitrate chelate in the presence of several fixed concentrations of either free isocitrate3-, an activator, or free Mg2+, an inhibitor of the enzyme. That NAD+-isocitrate dehydrogenase from blowfly flight muscle mitochondria was not activated by micromolar free Ca2+ is consistent with the view that calcium does not play a role in regulating the flux through the tricarboxylate cycle in this species.

Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

Science.gov (United States)

Tuck, Laura R; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D; Campopiano, Dominic J; Clarke, David J; Marles-Wright, Jon

2016-02-22

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

Prevalence of Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency in Estonia

DEFF Research Database (Denmark)

Joost, K; Ounap, K; Zordania, R

2012-01-01

The aim of our study was to evaluate the prevalence of long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) in the general Estonian population and among patients with symptoms suggestive of fatty acid oxidation (FAO) defects. We collected DNA from a cohort of 1,040 anonymous newborn blo...... prevalence of LCHADD in Estonia would be 1: 91,700....

Galactonolactone Dehydrogenase Requires a Redox-Sensitive Thiol for Optimal Production of Vitamin C1.

NARCIS (Netherlands)

Leferink, N.G.H.; Duijn, van E.; Barendregt, A.; Heck, A.J.R.; Berkel, van W.J.H.

2009-01-01

The mitochondrial flavoenzyme L-galactono--lactone dehydrogenase (GALDH) catalyzes the ultimate step of vitamin C biosynthesis in plants. We found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is inactivated by hydrogen peroxide due to selective oxidation of cysteine (Cys)-340,

Biochemical and cytochemical evaluation of heterozygote individuals with glucose-6-phosphate dehydrogenase deficiency

NARCIS (Netherlands)

Gurbuz, Nilgun; Aksu, Tevfik Aslan; van Noorden, Cornelis J. F.

2005-01-01

The aim of this study was to diagnose heterozygous glucose-6-phosphate dehydrogenase (G6PD) deficient females by an inexpensive cytochemical G6PD staining method that is easy to perform, allowing diagnosis of G6PD deficiency without cumbersome genetic analysis. Three subject groups were included in

The Alcohol Dehydrogenase Kinetics Laboratory: Enhanced Data Analysis and Student-Designed Mini-Projects

Science.gov (United States)

Silverstein, Todd P.

2016-01-01

A highly instructive, wide-ranging laboratory project in which students study the effects of various parameters on the enzymatic activity of alcohol dehydrogenase has been adapted for the upper-division biochemistry and physical biochemistry laboratory. Our two main goals were to provide enhanced data analysis, featuring nonlinear regression, and…

Alcohol consumption and type 2 diabetes - Influence of genetic variation in alcohol dehydrogenase

NARCIS (Netherlands)

Beulens, J.W.J.; Rimm, E.B.; Hendriks, H.F.J.; Hu, F.B.; Manson, J.E.; Hunter, D.J.; Mukamal, K.J.

2007-01-01

OBJECTIVE-We sought to investigate whether a polymorphism I in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. RESEARCH DESIGN AND METHODS-In nested case-control studies of 640 women with incident diabetes and 1,000 control subjects

Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

Science.gov (United States)

Sola-Carvajal, Agustín; García-García, María Inmaculada; Sánchez-Carrón, Guiomar; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

2012-11-01

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described

Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme.

Science.gov (United States)

Ogura, Ryutaro; Wakamatsu, Taisuke; Mutaguchi, Yuta; Doi, Katsumi; Ohshima, Toshihisa

2014-06-10

An NAD(+)-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. The enzyme required NAD(+) and NADH as cofactors for oxidative deamination and reductive amination, respectively, but not NADP(+) or NADPH. l-Trp was the preferred substrate for deamination, though l-Phe was deaminated at a much lower rate. The enzyme exclusively aminated 3-indolepyruvate; phenylpyruvate was inert. The pH optima for the deamination of l-Trp and amination of 3-indolpyruvate were 11.0 and 7.5, respectively. For deamination of l-Trp, maximum enzymatic activity was observed at 45°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°C. Unlike l-Trp dehydrogenases from higher plants, NpTrpDH activity was not activated by metal ions. Typical Michaelis-Menten kinetics were observed for NAD(+) and l-Trp for oxidative deamination, but with reductive amination there was marked substrate inhibition by 3-indolepyruvate. NMR analysis of the hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that NpTrpDH has a pro-S (B-type) stereospecificity similar to the Glu/Leu/Phe/Val dehydrogenase family. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Directory of Open Access Journals (Sweden)

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

Overexpression, crystallization and preliminary X-­ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

Science.gov (United States)

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

2006-01-01

The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-­phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M) of 3.64 Å3 Da−1 and a solvent content of 66%. PMID:16511285

The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease

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Andreas Plaitakis

2017-02-01

Full Text Available Glutamate dehydrogenase (GDH is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P+ to NAD(PH. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate, lipid biosynthesis (via oxidative generation of citrate, and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1 is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth

Stabilizing Effects of Deep Eutectic Solvents on Alcohol Dehydrogenase Mediated Systems

OpenAIRE

Fatima Zohra Ibn Majdoub Hassani; Ivan Lavandera; Joseph Kreit

2016-01-01

This study explored the effects of different organic solvents, temperature, and the amount of glycerol on the alcohol dehydrogenase (ADH)-catalysed stereoselective reduction of different ketones. These conversions were then analyzed by gas chromatography. It was found that when the amount of deep eutectic solvents (DES) increases, it can improve the stereoselectivity of the enzyme although reducing its ability to convert the substrate into the corresponding alcohol. Moreover, glycerol was fou...

Oxidation of aromatic alcohols by purified methanol dehydrogenase from Methylosinus trichosporium.

OpenAIRE

Mountfort, D O

1990-01-01

Methanol dehydrogenase was found to be present in subcellular preparations of methanol-grown Methylosinus trichosporium and occurred almost wholly in the soluble fraction of the cell. The enzyme, purified by DEAE-Sephadex and Sephadex G-100 chromatography, showed broad specificity toward different substrates and oxidized the aromatic alcohols benzyl, vanillyl, and veratryl alcohols in addition to a range of aliphatic primary alcohols. No enzyme activity was found toward the corresponding alde...

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

Energy Technology Data Exchange (ETDEWEB)

Plapp, Bryce V.; Savarimuthu, Baskar Raj; Ferraro, Daniel J.; Rubach, Jon K.; Brown, Eric N.; Ramaswamy, S. (Iowa)

2017-07-07

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ~1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production

DEFF Research Database (Denmark)

Thykær, Jette; Kildegaard, Kanchana Rueksomtawin; Noorman, H.

2008-01-01

was detected in either of the Delta gdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed......The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA...... continued beta-lactam production for the reference strains whereas the Delta gdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results...

Control of Glycolysis by Glyceraldehyde-3-Phosphate Dehydrogenase in Streptococcus cremoris and Streptococcus lactis

NARCIS (Netherlands)

POOLMAN, B; BOSMAN, B; KONINGS, WN

1987-01-01

The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity. The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and

Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish.

OpenAIRE

Biery, B. J.; Stein, D. E.; Morton, D. H.; Goodman, S. I.

1996-01-01

The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span approximately 7 kb. Fibroblast DNA from 64 unrelated glutaric acidemia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms fou...

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

Czech Academy of Sciences Publication Activity Database

Popelková, H.; Fraaije, M. W.; Novák, Ondřej; Frébortová, Jitka; Bilyeu, K. D.; Frébort, I.

2006-01-01

Roč. 398, č. 1 (2006), s. 113-124 ISSN 0264-6021 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin * cytokinin dehydrogenase (CKX) * flavoprotein Subject RIV: CE - Biochemistry Impact factor: 4.100, year: 2006

Proteomic comparison of Entamoeba histolytica and Entamoeba dispar and the role of E. histolytica alcohol dehydrogenase 3 in virulence.

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Paul H Davis

Full Text Available The protozoan intestinal parasite Entamoeba histolytica infects millions of people worldwide and is capable of causing amebic dysentery and amebic liver abscess. The closely related species Entamoeba dispar colonizes many more individuals, but this organism does not induce disease. To identify molecular differences between these two organisms that may account for their differential ability to cause disease in humans, we used two-dimensional gel-based (DIGE proteomic analysis to compare whole cell lysates of E. histolytica and E. dispar. We observed 141 spots expressed at a substantially (>5-fold higher level in E. histolytica HM-1:IMSS than E. dispar and 189 spots showing the opposite pattern. Strikingly, 3 of 4 proteins consistently identified as different at a greater than 5-fold level between E. histolytica HM-1:IMSS and E. dispar were identical to proteins recently identified as differentially expressed between E. histolytica HM-1:IMSS and the reduced virulence strain E. histolytica Rahman. One of these was E. histolytica alcohol dehydrogenase 3 (EhADH3. We found that E. histolytica possesses a higher level of NADP-dependent alcohol dehydrogenase activity than E. dispar and that some EhADH3 can be localized to the surface of E. histolytica. Episomal overexpression of EhADH3 in E. histolytica trophozoites resulted in only subtle phenotypic differences in E. histolytica virulence in animal models of amebic colitis and amebic liver abscess, making it difficult to directly link EhADH3 levels to virulence differences between E. histolytica and less-pathogenic Entamoeba.

Heat-stable, FE-dependent alcohol dehydrogenase for aldehyde detoxification

Science.gov (United States)

Elkins, James G.; Clarkson, Sonya

2018-04-24

The present invention relates to microorganisms and polypeptides for detoxifying aldehydes associated with industrial fermentations. In particular, a heat-stable, NADPH- and iron-dependent alcohol dehydrogenase was cloned from Thermoanaerobacter pseudethanolicus 39E and displayed activity against a number of aldehydes including inhibitory compounds that are produced during the dilute-acid pretreatment process of lignocellulosic biomass before fermentation to biofuels. Methods to use the microorganisms and polypeptides of the invention for improved conversion of bio mass to biofuel are provided as well as use of the enzyme in metabolic engineering strategies for producing longer-chain alcohols from sugars using thermophilic, fermentative microorganisms.

[Genetic variations in alcohol dehydrogenase, drinking habits and alcoholism

DEFF Research Database (Denmark)

Tolstrup, J.S.; Rasmussen, S.; Tybjaerg-Hansen, A.

2008-01-01

Alcohol is degraded primarily by alcohol dehydrogenase (ADH), and genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. By genotyping 9,080 white men and women from the general population, we found that men and women with ADH1B slow versus fast alcohol...... degradation drank approximately 30% more alcohol per week and had a higher risk of everyday and heavy drinking, and of alcoholism. Individuals with ADH1C slow versus fast alcohol degradation had a higher risk of heavy drinking Udgivelsesdato: 2008/8/25...

Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha

NARCIS (Netherlands)

Klei, I.J. van der; Faber, K.N.; Keizer-Gunnink, I.; Gietl, C.; Harder, W.; Veenhuis, M.

1993-01-01

We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector

FUNCTIONAL-ANALYSIS OF THE N-TERMINAL PREPEPTIDES OF WATERMELON MITOCHONDRIAL AND GLYOXYSOMAL MALATE-DEHYDROGENASES

NARCIS (Netherlands)

LEHNERER, M; KEIZERGUNNIK, [No Value; VEENHUIS, M; GIETL, C

1994-01-01

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate : NAD(+) oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in

cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions

OpenAIRE

Raschke, Henning; Fleischmann, Thomas; Van Der Meer, Jan Roelof; Kohler, Hans-Peter E.

1999-01-01

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of ...

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

Science.gov (United States)

Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

2010-03-01

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

Immobilization of malate dehydrogenase on carbon nanotubes for development of malate biosensor.

Science.gov (United States)

Ruhal, A; Rana, J S; Kumar, S; Kumar, A

2012-12-22

An amperometric malic acid biosensor was developed by immobilizing malate dehydrogenase on multi-walled carbon nanotubes (MWCNT) coated on screen printed carbon electrode. The screen printed carbon electrode is made up of three electrodes viz., carbon as working, platinum as counter and silver as reference electrode. Detection of L-malic acid concentration provides important information about the ripening and shelf life of the fruits. The NADP specific malate dehydrogenase was immobilized on carboxylated multiwalled carbon nanotubes using cross linker EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide] on screen printed carbon electrode. An amperometric current was measured by differential pulse voltammetry (DPV) which increases with increasing concentrations of malic acid at fixed concentration of NADP. Enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The detection limit of malic acid by the sensor was 60 - 120 μM and sensitivity of the sensor was 60 μM with a response time of 60s. The usual detection methods of malic acid are nonspecific, time consuming and less sensitive. However, an amperometric malic acid nanosensor is quick, specific and more sensitive for detection of malic acid in test samples.

Immobilisation and characterisation of glucose dehydrogenase immobilised on ReSyn: a proprietary polyethylenimine support matrix

CSIR Research Space (South Africa)

Twala, BV

2010-01-01

Full Text Available Immobilisation of enzymes is of considerable interest due to the advantages over soluble enzymes, including improved stability and recovery. Glucose Dehydrogenase (GDH) is an important biocatalytic enzyme due to is ability to recycle the biological...

Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans

International Nuclear Information System (INIS)

Li, Zhijie; Zhai, Yujia; Fang, Junnan; Zhou, Qiangjun; Geng, Yunqi; Sun, Fei

2010-01-01

Very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been crystallized in space group C2 and its X-ray diffraction data set has been collected to 1.6 Å resolution. Unlike other VLCADs that were reported to form dimers, the purified cVLCAD was found as a homotetrameric protein according to static light-scattering measurements. Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid β-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12 mg ml −1 ) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100 mM Tris–HCl pH 8.0, 150 mM sodium chloride, 200 mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3 Å, α = γ = 90.0, β = 124.0°. A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient V M of 2.76 Å 3 Da −1 and a solvent content of 55%

Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

Science.gov (United States)

Duka, Tetyana; Collins, Zachary; Anderson, Sarah M; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

2017-07-01

The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Dual enzymatic dynamic kinetic resolution by Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase and Candida antarctica lipase B

KAUST Repository

Karume, Ibrahim; Musa, Musa M.; Bsharat, Odey; Takahashi, Masateru; Hamdan, Samir; El Ali, Bassam

2016-01-01

The immobilization of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) using sol–gel method enables its use to racemize enantiopure alcohols in organic media. Here, we report the racemization of enantiopure phenyl

Exercise-induced pyruvate dehydrogenase activation is not affected by 7 days of bed rest

DEFF Research Database (Denmark)

Kiilerich, Kristian; Jørgensen, Stine Ringholm; Biensø, Rasmus Sjørup

2011-01-01

To test the hypothesis that physical inactivity impairs the exercise-induced modulation of pyruvate dehydrogenase (PDH), 6 healthy normally physically active male subjects completed 7 days of bed rest. Before and immediately after the bed rest, the subjects completed an OGTT and a one-legged knee...

Direct evidence for the inactivation of branched-chain oxo-acid dehydrogenase by enzyme phosphorylation

International Nuclear Information System (INIS)

Odessey, R.

1980-01-01

The branched-chain 2-oxo-acid dehydrogenase (BCOAD) from mitochondria of several different rat tissues is inactivated by ATP and can be reactivated by incubation in Mg 2+ -containing buffers. Work carried out on the system from skeletal muscle mitochondria has shown that inactivation requires the cleavage of the γ-phosphate group of ATP and that modification is covalent. The non-metabolized ATP analog, p[NH]ppA, can block the inhibitory effect of ATP when added prior to ATP addition, but cannot reverse the inhibition of the inactivated dehydrogenase. These and other data raise the possibility that BCOAD may be regulated by enzyme phosphorylation. This hypothesis is supported by the finding that various procedures which separate the enzyme from its mitochondrial environment (e.g. detergent treatment, ammonium sulfate precipitation and freeze-thawing) do not alter the degree of inhibition induced by ATP in the mitochondrial preincubation. These experiments suggested the feasibility of labelling the enzyme with 32 P and purifying it. (Auth.)

Structure of halophilic malate dehydrogenase in multimolar KCl solutions from neutron scattering and ultracentrifugation

International Nuclear Information System (INIS)

Calmettes, P.

1987-01-01

The structure and solvent interactions of malate dehydrogenase from Halobacterium marismortui in multimolar KCl solvents are found to be similar to those in multimolar NaCl solvents reported previously (G. Zaccai, E. Wachtel and H. Eisenberg, J. Mol. Biol. 190 (1986) 97). KCl rather than NaCl is predominant in physiological medium. At salt concentrations up to about 3.0 M, the protein (a dimer of M 87000 g/mol) can be considered to occupy an invariant volume in which it is associated with about 4100 molecules of water and about 520 molecules of salt. At very low resolution, the enzyme particle appears to have a compact protein core and protruding protein parts in interaction with the water and salt components, structural features that are not observed in non-halophilic mitochondrial malate dehydrogenase. The above conclusions were drawn from the analysis of neutron scattering and ultracentrifugation data, and the complementarity of these approaches is discussed extensively. 24 refs.; 7 figs.; 4 tabs

The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

Directory of Open Access Journals (Sweden)

Katarína Smolková

2012-01-01

Full Text Available Isocitrate dehydrogenase 2 (IDH2 is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP+-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD+ accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction, which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall “anoxic” glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2—frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia—catalyze reductive carboxylation of 2-oxoglutarate and reduction to D-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations. D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factor α.

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Czech Academy of Sciences Publication Activity Database

Mráček, Tomáš; Holzerová, Eliška; Drahota, Zdeněk; Kovářová, Nikola; Vrbacký, Marek; Ješina, Pavel; Houštěk, Josef

2014-01-01

Roč. 1837, č. 1 (2014), s. 98-111 ISSN 0005-2728 R&D Projects: GA ČR(CZ) GPP303/10/P227; GA MŠk(CZ) LL1204 Grant - others:Univerzita Karlova(CZ) 750213 Institutional support: RVO:67985823 Keywords : mitochondrial glycerol-3-phosphate dehydrogenase * ROS production * supercomplex * in-gel ROS detection Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.353, year: 2014

Aldehyde dehydrogenase polymorphism in North American, South American, and Mexican Indian populations.

Science.gov (United States)

Goedde, H W; Agarwal, D P; Harada, S; Rothhammer, F; Whittaker, J O; Lisker, R

1986-01-01

While about 40% of the South American Indian populations (Atacameños, Mapuche, Shuara) were found to be deficient in aldehyde dehydrogenase isozyme I (ALDH2 or E2), preliminary investigations showed very low incidence of isozyme deficiency among North American natives (Sioux, Navajo) and Mexican Indians (mestizo). Possible implications of such trait differences on cross-cultural behavioral response to alcohol drinking are discussed. PMID:3953578

Riboflavin-Responsive Multiple Acyl-CoA Dehydrogenase Deficiency Associated with Hepatoencephalomyopathy and White Matter Signal Abnormalities on Brain MRI.

Science.gov (United States)

Vieira, Päivi; Myllynen, Päivi; Perhomaa, Marja; Tuominen, Hannu; Keski-Filppula, Riikka; Rytky, Seppo; Risteli, Leila; Uusimaa, Johanna

2017-06-01

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting both fatty acid and amino acid oxidation. It can manifest at any age, but riboflavin-responsiveness has mainly been described in less severely affected patients. We describe an infant with severe MADD presenting with profound hypotonia and hepatomegaly. Treatment with riboflavin improved his muscle strength, liver size, and biochemical markers. A homozygous mutation of electron transfer flavoprotein dehydrogenase ( ETFDH ) was found. His motor skills continued to progress until a fatal infection-triggered deterioration at the age of 34 months. We show changes in brain magnetic resonance imaging over the course of the disease, with profound white matter abnormalities during the deterioration phase. Aggregates of mitochondria with abnormal cristae in muscle electron microscopy were noticed already in infancy. An unusual lactate dehydrogenase (LDH) isoenzyme pattern with LDH-1 predominance was additionally observed. This case demonstrates riboflavin-responsiveness in a severely affected infant with both muscular and extramuscular involvement and further underlines the variable nature of this disease. Georg Thieme Verlag KG Stuttgart · New York.

Depleted aldehyde dehydrogenase 1A1 (ALDH1A1) reverses cisplatin resistance of human lung adenocarcinoma cell A549/DDP.

Science.gov (United States)

Wei, Yunyan; Wu, Shuangshuang; Xu, Wei; Liang, Yan; Li, Yue; Zhao, Weihong; Wu, Jianqing

2017-01-01

Cisplatin is the standard first-line chemotherapeutic agent for the treatment of non-small cell lung cancer (NSCLC). However, resistance to chemotherapy has been a major obstacle in the management of NSCLC. Aldehyde dehydrogenase 1A1 (ALDH1A1) overexpression has been observed in a variety of cancers, including lung cancer. The purpose of this study was to investigate the effect of ALDH1A1 expression on cisplatin resistance and explore the mechanism responsible. Reverse transcriptase-PCR was applied to measure the messenger RNA expression of ALDH1A1, while Western blot assay was employed to evaluate the protein expression of ALDH1A1, B-cell lymphoma 2, Bcl-2-like protein 4, phospho-protein kinase B (p-AKT) and AKT. A short hairpin RNA was used to knockdown ALDH1A1 expression. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effect of ALDH1A1 decrease on cell viability. The cell apoptotic rate was tested using flow cytometry assay. ALDH1A1 is overexpressed in cisplatin resistant cell line A549/DDP, compared with A549. ALDH1A1 depletion significantly decreased A549/DDP proliferation, increased apoptosis, and reduced cisplatin resistance. In addition, the phosphoinositide 3-kinase (PI3K) / AKT pathway is activated in A549/DDP, and ALDH1A1 knockdown reduced the phosphorylation level of AKT. Moreover, the combination of ALDH1A1-short hairpin RNA and PI3K/AKT pathway inhibitor LY294002 markedly inhibited cell viability, enhanced apoptotic cell death, and increased cisplatin sensitivity. These results suggest that ALDH1A1 depletion could reverse cisplatin resistance in human lung cancer cell line A549/DDP, and may act as a potential target for the treatment of lung cancers resistant to cisplatin. © 2016 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina.

Science.gov (United States)

Tangthirasunun, Narumon; Navarro, David; Garajova, Sona; Chevret, Didier; Tong, Laetitia Chan Ho; Gautier, Valérie; Hyde, Kevin D; Silar, Philippe; Berrin, Jean-Guy

2017-01-15

Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose

Electron transfer between a quinohemoprotein alcohol dehydrogenase and an electrode via a redox polymer network

NARCIS (Netherlands)

Stigter, E.C.A.; Jong, G.A.H. de; Jongejan, J.A.; Duine, J.A.; Lugt, J.P. van der; Somers, W.A.C.

1996-01-01

A quinohemoprotein alcohol dehydrogenase (QH-EDH) from Comamonas testosteroni was immobilized on an electrode in a redox polymer network consisting of a polyvinylpyridine partially N-complexed with osmiumbis-(bipyridine)chloride. The enzyme effectively transfers electrons to the electrode via the

Sulfoacetate released during the assimilation of taurine-nitrogen by Neptuniibacter caesariensis: purification of sulfoacetaldehyde dehydrogenase

Czech Academy of Sciences Publication Activity Database

Krejčík, Zdeněk; Denger, K.; Weinitschke, S.; Hollemeyer, K.; Pačes, Václav; Cook, A.M.; Smits, T.H.M.

2008-01-01

Roč. 190, č. 2 (2008), s. 159-168 ISSN 0302-8933 Institutional research plan: CEZ:AV0Z50520514 Keywords : assimilation of taurine-nitrogen * sulfoacetaldehyde dehydrogenase * sulfoacetate exporter Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.975, year: 2008

Struktuur en interaktie analyse van NAD+ en NAD+ analoga in horse liver alcohol dehydrogenase

NARCIS (Netherlands)

Beijer, N.A.

1988-01-01

Dit verslag beschrijft een studie naar de relatie tussen struktuur en funktie voor het co-enzym NAn+ en zijn analoga in de aktieve holte van het enzym Horse Liver Alcohol Dehydrogenase (LADH). De rol van NAD+ in enzymgekatalyseerde oxidatie-reduktie reakties is die van het bewerkstelligen van een

Engineering of Class II Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

DEFF Research Database (Denmark)

Ortiz, Roberto; Rahman, Mahbubur; Zangrilli, Beatrice

2017-01-01

The front cover artwork is provided by Prof. Lo Gorton from Lund University (Sweden) and his co-workers. The image shows mutated cellobiose dehydrogenase (CDH) immobilized on a graphite electrode and how preferentially glucose is oxidized by this enzyme. Read the full text of the Article at 10.1002...

Analysis of alcohol dehydrogenase inhibitors from Desmodium styracifolium using centrifugal ultrafiltration coupled with HPLC-MS

Directory of Open Access Journals (Sweden)

Liu Liangliang

2015-01-01

Full Text Available Alcohol dehydrogenase (ADH inhibitors play an important role in the treatment of human methanol or ethylene glycol poisoning and the suppression of acetaldehyde accumulation in alcoholics. In this study, centrifugal ultrafiltration coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS was utilized to screen and identify ADH inhibitors from ethyl acetate extract of Desmosium styracifolium (Osb. Merr. The experiment conditions of centrifugal ultrafiltration were optimized. At the optimum conditions (ADH concentration: 37.5 μg mL-1, incubation time: 90 min, pH: 7.0 and temperature: 15°C, formononetin and aromadendrin were successfully screened and identified from ethyl acetate extract of Desmodium styracifolium. The screening result was verified by ADH inhibition assays. The IC50 values of formononetin and aromadendrin were 70.8 and 84.7 μg mL-1, which were accorded with the binding degrees of them. Aromadendrin was first reported to have inhibitory activity on ADH. This method provided an effective way to screen active compounds from natural products.

Determination of estradiol, estrone and progesterone in serum and human endometrium in correlation to the content of steroid receptors and 17β-hydroxysteroid dehydrogenase activity during menstrual cycle

International Nuclear Information System (INIS)

Schmidt-Gollwitzer, M.; Eiletz, J.; Pachaly, J.

1977-01-01

A study has been carried out to compare the influence of estradiol estrone and progesterone on the estradiol and progesterone receptor levels and 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in human endometrium. The steroid hormone concentrations were measured simultaneously in both serum and endometrial tissue. The estradiol receptor levels were highest during the early proliferative phase and were inversely correlated to the endometrial tissue and serum concentrations of estradiol and progesterone. The highest progesterone binding capacity was found in endometrical cytosol during the late proliferative phase (midcycle) of the menstrual cycle. The midcycle peak of the progesterone receptor level correlated well with the first peak of the serum and tissue concentrations of estradiol. During,the luteal phase, in contrast to the proliferative phase, the progesterone receptor level decreased whereas serum progesterone concentrations were high. Estrone concentrations were higher in secretory than proliferative endometrium and were correlated to the increase of progesterone receptor content and 17β-HSD activity during early secretory phase. The 17β-HSD activity was approximately 10-fold higher during the early secretory than during the proliferative phase. The progesterone receptor level was highly correlated to the specific 17β-HSD activity of the microsomal fraction whereas a significant inverse correlation between the enzyme activity and the estradiol receptor level was observed. (orig.) [de

Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures

International Nuclear Information System (INIS)

Saw, Yu-Ting; Thompson, David; Vasiliou, Vasilis; Berkowitz, Ross S; Ng, Shu-Wing; Yang, Junzheng; Ng, Shu-Kay; Liu, Shubai; Singh, Surendra; Singh, Margit; Welch, William R; Tsuda, Hiroshi; Fong, Wing-Ping

2012-01-01

Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. The results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to

The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes

International Nuclear Information System (INIS)

Chiu, Hsiu-Ju; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

2010-01-01

The crystal structure of the prephenate dehydrogenase component of the bifunctional H. influenzae TyrA reveals unique structural differences between bifunctional and monofunctional TyrA enzymes. Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P) + -dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD + has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP + and NAD + . The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from

SIGNIFICANCE OF LACTATE DEHYDROGENASE AND ASPARTATE TRANSAMINASE AS BIOCHEMICAL MARKERS AND AS PREDICTORS OF SEVERITY OF PREGNANCY-INDUCED HYPERTENSION AND ITS COMPLICATIONS

Directory of Open Access Journals (Sweden)

Ramesh Sonowal

2017-03-01

Full Text Available BACKGROUND To compare serum Lactate Dehydrogenase (LDH and serum Aspartate Transaminase (AST of normotensive pregnant women with those of preeclamptic and eclamptic women. To determine the relationship of levels of serum lactate dehydrogenase and serum aspartate transaminase with severity of pregnancy-induced hypertension and its complications. MATERIALSAND METHODS The study was carried out on pregnant hypertensive patients attending outpatient department of Obstetrics and Gynaecology department, AMCH, Dibrugarh, Assam from 1 st July 2013 to 30 th June 2014. Normotensive pregnant women were taken as controls. Each serum sample from both the control group as well as study group was estimated for lactate dehydrogenase and aspartate transaminase using standard methods and a comparison is drawn and analysed using t-test and Chi-square test. RESULTS Serum lactate dehydrogenase and serum aspartate transaminase levels were higher in the study group in comparison to the study groups. The mean serum LDH was 198±30.03U/L in control group, whereas in preeclampsia and eclampsia, mean serum levels of LDH were 817±114U/L and 927±108U/L, respectively. The levels of the serum AST were found to be less than 600U/L in normotensive and preeclampsia patients and more than 600 U/L in eclampsia and other complications of PIH. CONCLUSION Serum lactate dehydrogenase and serum aspartate transaminase levels in patients suffering from preeclampsia and its complications are consistently higher compared to the normotensive pregnant patients. To determine the usefulness of inclusion of these enzymes along with other cardiac enzymes in the panel of investigations of pregnant women universally needs further large scale comparative studies.

Potentiation of insulin release in response to amino acid methyl esters correlates to activation of islet glutamate dehydrogenase activity

DEFF Research Database (Denmark)

Kofod, Hans; Lernmark, A; Hedeskov, C J

1986-01-01

Column perifusion of mouse pancreatic islets was used to study the ability of amino acids and their methyl esters to influence insulin release and activate islet glutamate dehydrogenase activity. In the absence of L-glutamine, L-serine and the methyl ester of L-phenylalanine, but neither L...... glutamate dehydrogenase activity showed that only the two methyl esters of L-phenylalanine and L-serine activated the enzyme. It is concluded that the mechanism by which methyl esters of amino acids potentiate insulin release is most likely to be mediated by the activation of pancreatic beta-cell glutamate...

Coupled reactions by coupled enzymes : alcohol to lactone cascade with alcohol dehydrogenase-cyclohexanone monooxygenase fusions

NARCIS (Netherlands)

Aalbers, Friso S; Fraaije, Marco W

2017-01-01

The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP(+) to oxidize cyclohexanol to form cyclohexanone and NADPH. Both

The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

Science.gov (United States)

Zaganas, Ioannis V; Kanavouras, Konstantinos; Borompokas, Nikolas; Arianoglou, Giovanna; Dimovasili, Christina; Latsoudis, Helen; Vlassi, Metaxia; Mastorodemos, Vasileios

2014-01-01

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

DEFF Research Database (Denmark)

Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen

2014-01-01

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been...... prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered...... hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense...

Lactate dehydrogenase (LDH isoenzymes patterns in ocular tumours

Directory of Open Access Journals (Sweden)

Singh Rajendra

1991-01-01

Full Text Available Estimation of lactate dehydrogenase (LDH isoenzymes in the serum and aqueous humor was carried out in 15 cases of benign ocular tumour, 15 cases of malignant tumor and 15 normal cases. Cases of both sexes aged between 1 year and 75 years were included. LDH, isoenzymes specially LDH4 and LDH5 are higher and LDH1 and LDH2 lower in sera of patients with malignant tumor specially retinoblastoma as compared to benign tumor cases and control cases. LDH isoenzymes in aqueous humor are significantly higher and show a characteristic pattern in retinoblastoma cases, the concentration was presumably too low in the control, malignant tumor other than retinoblastoma and benign tumor cases as its fractionation was not possible.

Immobilization of alcohol dehydrogenase on ceramic silicon carbide membranes for enzymatic CH3 OH production

DEFF Research Database (Denmark)

Zeuner, Birgitte; Ma, Nicolaj; Berendt, Kasper

2018-01-01

BACKGROUND Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyzes oxidation of CH3OH to CHOH during NAD+ reduction to NADH. ADH can also accelerate the reverse reaction, which is studied as part of cascadic enzymatic conversion of CO2 to CH3OH. In the present study, immobilization of ADH onto macropor......BACKGROUND Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyzes oxidation of CH3OH to CHOH during NAD+ reduction to NADH. ADH can also accelerate the reverse reaction, which is studied as part of cascadic enzymatic conversion of CO2 to CH3OH. In the present study, immobilization of ADH onto......‐of‐concept for the use of NaOH‐treated SiC membranes for covalent enzyme immobilization and biocatalytic efficiency improvement of ADH during multiple reaction cycles. These data have implications for the development of robust extended enzymatic reactions....

Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance

Czech Academy of Sciences Publication Activity Database

Šindelář, Luděk; Šindelářová, Milada

2002-01-01

Roč. 215, - (2002), s. 862-869 ISSN 0032-0935 R&D Projects: GA ČR GA522/99/1264 Institutional research plan: CEZ:AV0Z5038910 Keywords : Glucose 6 phosphate dehydrogenase * Nicotiana (viral infection) * Plant viruses Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.960, year: 2002

Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles.

Science.gov (United States)

Nicolau, Eduardo; Méndez, Jessica; Fonseca, José J; Griebenow, Kai; Cabrera, Carlos R

2012-06-01

Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and remarkable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the enzyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectrochemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanoparticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future. Copyright © 2011 Elsevier B.V. All rights reserved.

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Science.gov (United States)

Augustin, Peter; Toplak, Marina; Fuchs, Katharina; Gerstmann, Eva Christine; Prassl, Ruth; Winkler, Andreas; Macheroux, Peter

2018-01-01

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria. PMID:29301933

Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

Science.gov (United States)

Meng, Fantao; Xu, Yan

2010-04-01

An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco

OpenAIRE

Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N.; Marshall, David; Hancock, Robert D.; Lapierre, Catherine; Morreele, Kris; Boerjane, Wout

2011-01-01

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was cha...

Changes of α-glycerophosphate dehydrogenase activity in fatty liver of rats by amino acid imbalance

International Nuclear Information System (INIS)

Ogura, Masaji; Katsunuma, Eiichi; Akabane, Tomoko; Ogawa, Seiichi

1976-01-01

The previous study on the lipogenesis in the fatty livers of rats, which was induced by feeding the diet with imbalanced amino acid, revealed that the induction of this type of fatty livers was due mainly to the acceleration of triglyceride synthesis by the increase in both synthesis and esterification of fatty acid in the livers. Although many studies have been carried out on the dietary control of α-glycerophosphate dehydrogenase activity in rat livers, the enzyme change in amino acid imbalance has not been reported. In the present study, in order to elucidate the difference in the supply of glycerol moiety of triglyceride due to the imbalance, the change of the α-glycerophosphate dehydrogenase activity in livers was investigated. The experimental diets were 8% casein basal diet and basal + 0.3% DL-methionine imbalanced diet. 5 rats of each group were killed after 0.5 and 10 days on the diet, and the analysis of the lipid content in the livers and the determination of the α-glycerophosphate dehydrogenase activity were carried out. The linear response of the enzyme activity to time and protein concentration was obtained. The development of fatty livers was observed in the imbalanced diet group in the feeding period of 10 days. It was found that the specific activity of the imbalanced diet group increased significantly in 5 and 10 days as compared with that of the basal diet group. The elevation in the enzyme activity may suggest that the supply of α-glycerophosphate for triglyceride synthesis is also increased in this type of fatty livers. (Kako, I.)

Heterologous Expression of Aldehyde Dehydrogenase in Lactococcus lactis for Acetaldehyde Detoxification at Low pH.

Science.gov (United States)

Lyu, Yunbin; LaPointe, Gisèle; Zhong, Lei; Lu, Jing; Zhang, Chong; Lu, Zhaoxin

2018-02-01

Aldehyde dehydrogenase (E.C. 1.2.1.x) can catalyze detoxification of acetaldehydes. A novel acetaldehyde dehydrogenase (istALDH) from the non-Saccharomyces yeast Issatchenkia terricola strain XJ-2 has been previously characterized. In this work, Lactococcus lactis with the NIsin Controlled Expression (NICE) System was applied to express the aldehyde dehydrogenase gene (istALDH) in order to catalyze oxidation of acetaldehyde at low pH. A recombinant L. lactis NZ3900 was obtained and applied for the detoxification of acetaldehyde as whole-cell biocatalysts. The activity of IstALDH in L. lactis NZ3900 (pNZ8148-istALDH) reached 36.4 U mL -1 when the recombinant cells were induced with 50 ng mL -1 nisin at 20 °C for 2 h. The IstALDH activity of recombinant L. lactis cells showed higher stability at 37 °C and pH 4.0 compared with the crude enzyme. L. lactis NZ3900 (pNZ8148-istALDH) could convert acetaldehyde at pH 2.0 while the crude enzyme could not. Moreover, the resting cells of L. lactis NZ3900 (pNZ8148-istALDH) showed a 2.5-fold higher activity and better stability in catalyzing oxidation of acetaldehyde at pH 2.0 compared with that of Escherichia coli expressing the IstALDH. Taken together, the L. lactis cells expressing recombinant IstALDH are potential whole-cell biocatalysts that can be applied in the detoxification of aldehydes.

Developmental changes in rat liver branched-chain 2-oxo acid dehydrogenase.

OpenAIRE

May, E E; May, M E; Aftring, R P; Buse, M G

1982-01-01

Branched-chain 2-oxo acid dehydrogenase catalyses the first irreversible step in the degradation of the branched-chain amino acids leucine, isoleucine and valine. With specifically labelled 4-methyl-2-oxo[1-14C]pentanoate as substrate, the enzyme's activity was measured in rat liver homogenates. Activity (per g wet wL of liver or per mg of protein) increased most rapidly during the perinatal period (2 days before to 1 day after birth), reaching approximately adult values by the time of weanin...

Role of pyruvate dehydrogenase inhibition in the development of hypertrophy in the hyperthyroid rat heart: a combined magnetic resonance imaging and hyperpolarized magnetic resonance spectroscopy study.

Science.gov (United States)

Atherton, Helen J; Dodd, Michael S; Heather, Lisa C; Schroeder, Marie A; Griffin, Julian L; Radda, George K; Clarke, Kieran; Tyler, Damian J

2011-06-07

Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg x kg(-1) x d(-1)). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022 ± 0.0002 versus 0.0055 ± 0.0005 second(-1); P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-(13)C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100 ± 20 versus 200 ± 30 mg; P=0.04) despite no change in the increase observed in cardiac output. This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic

Genetic Polymorphisms of the Mitochondrial Aldehyde Dehydrogenase ALDH2 Gene in a Large Ethnic Hakka Population in Southern China.

Science.gov (United States)

Zhong, Zhixiong; Hou, Jingyuan; Li, Bin; Zhang, Qifeng; Li, Cunren; Liu, Zhidong; Yang, Min; Zhong, Wei; Zhao, Pingsen

2018-04-06

BACKGROUND Human mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a critical role in the detoxification of the ethanol metabolite acetaldehyde. The ALDH2*2 (rs671) gene variant is mainly absent among Europeans but is prevalent in populations in East Asia. The aim of this study was to investigate ALDH2*2 mutant alleles and genotype frequencies in the Hakka population of China. MATERIAL AND METHODS Between January 2016 and June 2017, 7,966 unrelated individuals were recruited into the study from the Hakka ethnic population residing in the Meizhou area of Guangdong Province, China, who provided venous blood samples. Genotyping of ALDH2 genotypes were determined using a gene chip platform and confirmed by DNA sequencing. RESULTS In the 7,966 individuals from the Hakka population of China in this study, the frequencies of the ALDH2 genotypes *1/*1, *1/*2 and *2/*2 were 52.03%, 39.67%, and 8.30%, respectively; 47.97% of the individuals were found to carry the ALDH2*2 genotype, which was associated with a deficiency in the aldehyde dehydrogenase (ALDH2) enzyme activity. The frequency of the ALDH2*2 allele was lower than that previously reported in the Japanese population but higher than that reported in other Oriental populations. CONCLUSIONS The findings of this study have provided new information on the ALDH2 gene polymorphisms in the Hakka ethnic population residing in the Meizhou area of Guangdong Province, China, including an understanding of the origin of the atypical ALDH2*2 allele. Also, the study findings may be relevant to the primary care of patients in China.

Cellular defense against UVB-induced phototoxicity by cytosolic NADP+-dependent isocitrate dehydrogenase

International Nuclear Information System (INIS)

Jo, Seung-Hee; Lee, So-Hyun; Suk Chun, Hang; Min Lee, Su; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-01-01

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

Science.gov (United States)

Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-03-29

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco.

Science.gov (United States)

Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N; Marshall, David; Hancock, Robert D; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

2011-12-01

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.

Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling

Directory of Open Access Journals (Sweden)

María Francisca eAguayo

2015-02-01

Full Text Available Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. NAD+-dependent SORBITOL DEHYDROGENASE (SDH, E.C. 1.1.1.14 from Arabidopsis thaliana L. (AtSDH is capable of oxidizing several polyols including sorbitol, ribitol and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S and C4 (R polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid (EDTA, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn. (Hemiptera: Aleyrodidae SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S and C4 (R.

EFFECTS OF AMARANTHS’ SEEDS ON DEHYDROGENASE ACTIVITY AND GASES EMISSION IN METHANOGENIC BIOREACTORS

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Victor COVALIOV

2015-12-01

Full Text Available The influence of amaranths‘ seeds as the source of squalene on the dehydrogenase activity and efficiency of methane production were investigated in methanogenic bench-scale (5000 ml bioreactors used to treat the mixture of distillery wastes and farmyard manure. The adding of amaranth seeds to the methanogenic bioreactor has an inhibitory effect on the dehydrogenase activity and stimulates the process of methanogenesis. Dehydrogenase activity decreased with the increase of doses of squalene and its trend had a close connection with doses (R2=0.77-0.78. The methane content in the total amount of gases is 65.3-71.3% in a bioreactor with the additive of amaranth seeds in a dose of 50 mg l-1, which is 22.1% higher than in the the control bioreactor without additives. The increase in squalene concentration higher than 0.0005% is not rational because its stimulating effect on the methanogenic process decreases. Anaerobic digestion of alcohol distillery industry wastes with manure is a complex nonlinear time-varying microbiological process. Dehydrogenase activity trends in the experiment are described by the power function for 5 hours observations and by the logarithmic function for 120 hours of observations. Trends of CH4 are described by the polynomial function in all periods of testing. Correlation coefficients are 0.37 and 0.70 for CH4 after 5 and 120 hours of the anaerobic digestion. Dehydrogenase activity is in the close negative connection with the amount of gases, including methane. Correlation analysis between dehydrogenase activity and the release of gases has revealed the moderate and strongly negative link during 24 hours after the start of the experiment.EFECTUL SEMINŢELOR DE AMARANT ASUPRA ACTIVITĂŢII DEHIDROGENAZEI ŞI EMISIEI GAZELOR ÎN BIOREACTOARELE METANOGENEÎn bioreactoare metanogene unite consecutiv, cu volum de 5000 ml, utilizate pentru tratarea amestecului de borhot de la distilarea alcoolului cu gunoi de grajd, a fost

Evaluation of Serum Lactate Dehydrogenase Activity in a Virtual Environment

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V.M.T. Trindade

2013-05-01

Full Text Available Introduction: Lactate dehydrogenase is a citosolic enzyme involved in reversible transformation of pyruvate to lactate. It participates in anaerobic glycolysis of skeletal muscle and red blood cells, in liver gluconeogenesis and in aerobic metabolism of heart muscle. The determination of its activity helps in the diagnosis of various diseases, because it is increased in serum of patients suffering from myocardial infarction, acute hepatitis, muscular dystrophy and cancer. This paper presents a learning object, mediated by computer, which contains the simulation of the laboratory determination serum lactate dehydrogenase activity measured by the spectrophotometric method, based in the decrease of absorbance at 340 nm. Materials and Methods: Initially, pictures and videos were obtained recording the procedure of the methodology. The most representative images were selected, edited and inserted into an animation developed with the aid of the tool Adobe ® Flash ® CS3. The validation of the object was performed by the students of Biochemistry I (Pharmacy-UFRGS from the second semester of 2009 and both of 2010. Results and Discussion: The analysis of students' answers revealed that 80% attributed the excellence of the navigation program, the display format and to aid in learning. Conclusion: Therefore, this software can be considered an adequate teaching resource as well as an innovative support in the construction of theoretical and practical knowledge of Biochemistry. Available at: http://www6.ufrgs.br/gcoeb/LDH

Investigation of the Amycolatopsis sp. strain ATCC 39116 vanillin dehydrogenase and its impact on the biotechnical production of vanillin.

Science.gov (United States)

Fleige, Christian; Hansen, Gunda; Kroll, Jens; Steinbüchel, Alexander

2013-01-01

The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDH(ATCC 39116)). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDH(ATCC 39116) was purified to apparent electrophoretic homogeneity and exhibited NAD(+)-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin.

Escherichia coli pyruvate dehydrogenase complex: particle masses of the complex and component enzymes measured by scanning transmission electron microscopy

International Nuclear Information System (INIS)

CaJacob, C.A.; Frey, P.A.; Hainfeld, J.F.; Wall, J.S.; Yang, H.

1985-01-01

Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM). The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6). The masses of the component enzymes are 2.06 X 10(5) for the dimeric pyruvate dehydrogenase (E1), 1.15 X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and 2.20 X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme. STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions. The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex. The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2)

Magnetic resonance and fluorescence studies on pyruvate dehydrogenase complexes and their small molecular weight constituents

NARCIS (Netherlands)

Grande, H.J.

1976-01-01

The articles presented in this thesis do not describe at first glance one well-defined subject. They are, however, in fact connected by one central theme: the study of large enzyme aggregates by molecular physical methods. Chosen was the pyruvate dehydrogenase complex (PDC) because of its

Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic ¤Nicotiana sylvestris¤

DEFF Research Database (Denmark)

Michalecka, A.M.; Agius, S.C.; Møller, I.M.

2004-01-01

The plant respiratory chain contains a complex setup of non-energy conserving NAD(P)H dehydrogenases, the physiological consequences of which are highly unclear. An expression construct for the potato (Solanum tuberosum L., cv. Desiree) ndb1 gene, a homologue of bacterial and fungal type II NAD...

l-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.

NARCIS (Netherlands)

Leferink, N.G.H.; Berg, van den W.A.M.; Berkel, van W.J.H.

2008-01-01

l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant

Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase

International Nuclear Information System (INIS)

Szymańska-Majchrzak, Jolanta; Pałka, Katarzyna; Kańska, Marianna

2017-01-01

Synthesis of 3-fluoro-[2- 2 H]-L-alanine (3-F-[ 2 H]-L-Ala) in reductive amination of 3-fluoropyruvic acid catalysed by L-alanine dehydrogenase (AlaDH) was described. Fluorine derivative was used to study oxidative deamination catalysed by AlaDH applied kinetic (for 3-F-L-Ala in H 2 O - KIE’s on V max : 1.1; on V max /K M : 1.2; for 3-F-L-Ala in 2 H 2 O – on V max : 1.4; on V max /K M : 2.1) and solvent isotope effect methods (for 3-F-L-Ala - SIE’s on V max : 1.0; on V max /K M : 0.87; for 3-F-[2- 2 H]-L-Ala – on V max : 1.4; on V max /K M : 1.5). Studies explain some details of reaction mechanism. - Highlights: • Synthesis of 3-fluoro-[2- 2 H]-L-alanine was performed. • The reactions were catalysed using the enzyme L-alanine dehydrogenase. • Performed reactions involved fluorinated analogues of L-alanine. • Solvent isotope effects of deuterium were determined. • Kinetic isotope effects were determined for obtained 3-fluoro-L-alanine. • The mechanism of reaction catalysed by L-alanine dehydrogenase was proposed.

PURIFICATION AND CHARACTERIZATION OF AN OXYGEN-LABILE, NAD-DEPENDENT ALCOHOL-DEHYDROGENASE FROM DESULFOVIBRIO-GIGAS

NARCIS (Netherlands)

HENSGENS, CMH; VONCK, J; VANBEEUMEN, J; VANBRUGGEN, EFJ; HANSEN, TA

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (K(m), 0.15 mM) and 1-propanol (K(m), 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the

Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study

Czech Academy of Sciences Publication Activity Database

Kopečný, D.; Briozzo, P.; Popelková, H.; Šebela, M.; Končitíková, R.; Spíchal, Lukáš; Nisler, Jaroslav; Madzak, C.; Frébort, Ivo; Laloue, M.; Houba-Herin, N.

2010-01-01

Roč. 92, č. 8 (2010), s. 1052-1062 ISSN 0300-9084 R&D Projects: GA ČR GA522/08/0555; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : benzylurea * crystal structure * cytokinin oxidase/dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 3.787, year: 2010

Deracemization of Secondary Alcohols by using a Single Alcohol Dehydrogenase

KAUST Repository

Karume, Ibrahim

2016-03-01

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. We developed a single-enzyme-mediated two-step approach for deracemization of secondary alcohols. A single mutant of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase enables the nonstereoselective oxidation of racemic alcohols to ketones, followed by a stereoselective reduction process. Varying the amounts of acetone and 2-propanol cosubstrates controls the stereoselectivities of the consecutive oxidation and reduction reactions, respectively. We used one enzyme to accomplish the deracemization of secondary alcohols with up to >99% ee and >99.5% recovery in one pot and without the need to isolate the prochiral ketone intermediate.

Crystal structures of type IIIH NAD-dependent D-3-phosphoglycerate dehydrogenase from two thermophiles

International Nuclear Information System (INIS)

Kumar, S.M.; Pampa, K.J.; Manjula, M.; Hemantha Kumar, G.; Kunishima, Naoki; Lokanath, N.K.

2014-01-01

Highlights: • Determined the crystal structures of PGDH from two thermophiles. • Monomer is composed of nucleotide binding domain and substrate binding domain. • Crystal structures of type III H PGDH. - Abstract: In the L-Serine biosynthesis, D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the inter-conversion of D-3-phosphoglycerate to phosphohydroxypyruvate. PGDH belongs to 2-hydroxyacid dehydrogenases family. We have determined the crystal structures of PGDH from Sulfolobus tokodaii (StPGDH) and Pyrococcus horikoshii (PhPGDH) using X-ray diffraction to resolution of 1.77 Å and 1.95 Å, respectively. The PGDH protomer from both species exhibits identical structures, consisting of substrate binding domain and nucleotide binding domain. The residues and water molecules interacting with the NAD are identified. The catalytic triad residues Glu-His-Arg are highly conserved. The residues involved in the dimer interface and the structural features responsible for thermostability are evaluated. Overall, structures of PGDHs with two domains and histidine at the active site are categorized as type III H and such PGDHs structures having this type are reported for the first time

15-hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes.

Science.gov (United States)

Kim, Hak-Jae; Lee, Sora; Lee, Haw-Yong; Won, Hansol; Chang, Sung-Hae; Nah, Seong-Su

2015-09-01

15-Hydroxyprostaglandin dehydrogenase (HPGD) is the key enzyme responsible for the metabolic inactivation of prostaglandin E2 (PGE2) catabolism. PGE2 is one of the predominant catabolic factors involved in rheumatoid arthritis (RA). However, the expression and regulation of HPGD in RA fibroblast‑like synoviocyte (FLS) remain to be elucidated. Disease‑modifying anti‑rheumatic drugs (DMARDs) are the most important anti‑arthritic drugs, which reduce the effect of joint injury. The aim of the present study was to assess the expression of HPGD in RA tissues and cells, and normal synovial tissues and cells. The effect of the most popular DMARDs, hydroxychloroquine, on the expression of HPGD in RA‑FLS was also investigated. Western blotting and immunohistochemical analysis demonstrated that the expression levels of HPGD in human synovium were lower in RA synovium compared with the normal and OA synovium. In RA‑FLS, the expression of HPGD was increased following treatment with several DMARDs, including sulfasalazine, methotrexate, and hydroxychloroquine. Hydroxychloroquine (10 µM) treatment induced the phosphorylation of ERK, SAPK/JNK and p38. Hydroxychloroquine induced a decrease in the release of PGE2, which was restored by mitogen‑activated protein (MAP) kinase pathway inhibitors. Hydroxychloroquine may therefore, affect the pathogenesis of RA through the MAP kinase pathway by regulating the expression of HPGD.

Glucose-6-Phosphate Dehydrogenase: Update and Analysis of New Mutations around the World

Science.gov (United States)

Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; Ortega-Cuellar, Daniel; González-Valdez, Abigail; Castillo-Rodríguez, Rosa Angélica; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

2016-01-01

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme in the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain an adequate reducing environment in the cells and is especially important in red blood cells (RBC). Given its central role in the regulation of redox state, it is understandable that mutations in the gene encoding G6PD can cause deficiency of the protein activity leading to clinical manifestations such as neonatal jaundice and acute hemolytic anemia. Recently, an extensive review has been published about variants in the g6pd gene; recognizing 186 mutations. In this work, we review the state of the art in G6PD deficiency, describing 217 mutations in the g6pd gene; we also compile information about 31 new mutations, 16 that were not recognized and 15 more that have recently been reported. In order to get a better picture of the effects of new described mutations in g6pd gene, we locate the point mutations in the solved three-dimensional structure of the human G6PD protein. We found that class I mutations have the most deleterious effects on the structure and stability of the protein. PMID:27941691

Redox Balance in Lactobacillus reuteri DSM20016: Roles of Iron-Dependent Alcohol Dehydrogenases in Glucose/ Glycerol Metabolism.

Directory of Open Access Journals (Sweden)

Lu Chen

Full Text Available Lactobacillus reuteri, a heterofermentative bacterium, metabolizes glycerol via a Pdu (propanediol-utilization pathway involving dehydration to 3-hydroxypropionaldehyde (3-HPA followed by reduction to 1,3-propandiol (1,3-PDO with concomitant generation of an oxidized cofactor, NAD+ that is utilized to maintain cofactor balance required for glucose metabolism and even for oxidation of 3-HPA by a Pdu oxidative branch to 3-hydroxypropionic acid (3-HP. The Pdu pathway is operative inside Pdu microcompartment that encapsulates different enzymes and cofactors involved in metabolizing glycerol or 1,2-propanediol, and protects the cells from the toxic effect of the aldehyde intermediate. Since L. reuteri excretes high amounts of 3-HPA outside the microcompartment, the organism is likely to have alternative alcohol dehydrogenase(s in the cytoplasm for transformation of the aldehyde. In this study, diversity of alcohol dehydrogenases in Lactobacillus species was investigated with a focus on L. reuteri. Nine ADH enzymes were found in L. reuteri DSM20016, out of which 3 (PduQ, ADH6 and ADH7 belong to the group of iron-dependent enzymes that are known to transform aldehydes/ketones to alcohols. L. reuteri mutants were generated in which the three ADHs were deleted individually. The lagging growth phenotype of these deletion mutants revealed that limited NAD+/NADH recycling could be restricting their growth in the absence of ADHs. Notably, it was demonstrated that PduQ is more active in generating NAD+ during glycerol metabolism within the microcompartment by resting cells, while ADH7 functions to balance NAD+/NADH by converting 3-HPA to 1,3-PDO outside the microcompartment in the growing cells. Moreover, evaluation of ADH6 deletion mutant showed strong decrease in ethanol level, supporting the role of this bifuctional alcohol/aldehyde dehydrogenase in ethanol production. To the best of our knowledge, this is the first report revealing both internal and

17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

International Nuclear Information System (INIS)

Mendoza-Hernandez, G.; Lopez-Solache, I.; Rendon, J.L.; Diaz-Sanchez, V.; Diaz-Zagoya, J.C.

1988-01-01

The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively

Metabolism of Oxo-Bile Acids and Characterization of Recombinant 12α-Hydroxysteroid Dehydrogenases from Bile Acid 7α-Dehydroxylating Human Gut Bacteria.

Science.gov (United States)

Doden, Heidi; Sallam, Lina A; Devendran, Saravanan; Ly, Lindsey; Doden, Greta; Daniel, Steven L; Alves, João M P; Ridlon, Jason M

2018-05-15

Bile acids are important cholesterol-derived nutrient signaling hormones, synthesized in the liver, that act as detergents to solubilize dietary lipids. Bile acid 7α-dehydroxylating gut bacteria generate the toxic bile acids deoxycholic acid and lithocholic acid from host bile acids. The ability of these bacteria to remove the 7-hydroxyl group is partially dependent on 7α-hydroxysteroid dehydrogenase (HSDH) activity, which reduces 7-oxo-bile acids generated by other gut bacteria. 3α-HSDH has an important enzymatic activity in the bile acid 7α-dehydroxylation pathway. 12α-HSDH activity has been reported for the low-activity bile acid 7α-dehydroxylating bacterium Clostridium leptum ; however, this activity has not been reported for high-activity bile acid 7α-dehydroxylating bacteria, such as Clostridium scindens , Clostridium hylemonae , and Clostridium hiranonis Here, we demonstrate that these strains express bile acid 12α-HSDH. The recombinant enzymes were characterized from each species and shown to preferentially reduce 12-oxolithocholic acid to deoxycholic acid, with low activity against 12-oxochenodeoxycholic acid and reduced activity when bile acids were conjugated to taurine or glycine. Phylogenetic analysis suggests that 12α-HSDH is widespread among Firmicutes , Actinobacteria in the Coriobacteriaceae family, and human gut Archaea IMPORTANCE 12α-HSDH activity has been established in the medically important bile acid 7α-dehydroxylating bacteria C. scindens , C. hiranonis , and C. hylemonae Experiments with recombinant 12α-HSDHs from these strains are consistent with culture-based experiments that show a robust preference for 12-oxolithocholic acid over 12-oxochenodeoxycholic acid. Phylogenetic analysis identified novel members of the gut microbiome encoding 12α-HSDH. Future reengineering of 12α-HSDH enzymes to preferentially oxidize cholic acid may provide a means to industrially produce the therapeutic bile acid ursodeoxycholic acid. In

Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

Science.gov (United States)

Sukpipat, Wiphat; Komeda, Hidenobu; Prasertsan, Poonsuk; Asano, Yasuhisa

2017-01-01

Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with K m =16.1 mM and k cat /K m =67.0 min -1 mM -1 , while l-arabitol was also a substrate for the enzyme with K m =31.1 mM and k cat /K m =6.5 min -1  mM -1 . Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Electron-transfer mediator for a NAD-glucose dehydrogenase-based glucose sensor.

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Kim, Dong-Min; Kim, Min-yeong; Reddy, Sanapalli S; Cho, Jaegeol; Cho, Chul-ho; Jung, Suntae; Shim, Yoon-Bo

2013-12-03

A new electron-transfer mediator, 5-[2,5-di (thiophen-2-yl)-1H-pyrrol-1-yl]-1,10-phenanthroline iron(III) chloride (FePhenTPy) oriented to the nicotinamide adenine dinucleotide-dependent-glucose dehydrogenase (NAD-GDH) system was synthesized through a Paal-Knorr condensation reaction. The structure of the mediator was confirmed by Fourier-transform infrared spectroscopy, proton and carbon nucler magnetic resonance spectroscopy, and mass spectroscopy, and its electron-transfer characteristic for a glucose sensor was investigated using voltammetry and impedance spectroscopy. A disposable amperometric glucose sensor with NAD-GDH was constructed with FePhenTPy as an electron-transfer mediator on a screen printed carbon electrode (SPCE) and its performance was evaluated, where the addition of reduces graphene oxide (RGO) to the mediator showed the enhanced sensor performance. The experimental parameters to affect the analytical performance and the stability of the proposed glucose sensor were optimized, and the sensor exhibited a dynamic range between 30 mg/dL and 600 mg/dL with the detection limit of 12.02 ± 0.6 mg/dL. In the real sample experiments, the interference effects by acetaminophen, ascorbic acid, dopamine, uric acid, caffeine, and other monosaccharides (fructose, lactose, mannose, and xylose) were completely avoided through coating the sensor surface with the Nafion film containing lead(IV) acetate. The reliability of proposed glucose sensor was evaluated by the determination of glucose in artificial blood and human whole blood samples.

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

DEFF Research Database (Denmark)

Jensen, T G; Andresen, B S; Bross, P

1992-01-01

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter...... and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild......-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild...

Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae

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Yang Wang

2016-01-01

Full Text Available Testis-specific lactate dehydrogenase (LDH-C4 is one of the lactate dehydrogenase (LDH isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4, Lactate Dehydrogenase B4 (LDH-B4, and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE and native polyacrylamide gel electrophoresis (PAGE. The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L, LDH-B4 (Ki = 23.800 mmol/L, and LDH-C4 (Ki = 65.500 mmol/L. These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii.

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Datta, Suprama; Annapure, Uday S; Timson, David J

2017-04-30

Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met 177 , compared with the equivalent residue Leu 196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met 177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity. © 2017 The Author(s).

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and early-onset liver cirrhosis in two siblings

NARCIS (Netherlands)

van Maldergem, L.; Tuerlinckx, D.; Wanders, R. J.; Vianey-Saban, C.; van Hoof, F.; Martin, J. J.; Fourneau, C.; Gillerot, Y.; Bachy, A.

2000-01-01

We present the clinical, pathological, biochemical, and molecular results on an infant girl with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency and data on her deceased elder brother for whom this condition was retrospectively diagnosed. Clinical signs were liver enlargement and

Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily

NARCIS (Netherlands)

Machielsen, M.P.; Uria, A.R.; Kengen, S.W.M.; Oost, van der J.

2006-01-01

The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The

Sorbitol dehydrogenase of Aspergillus niger, SdhA, is part of the oxido-reductive D-galactose pathway and essential for D-sorbitol catabolism.

Science.gov (United States)

Koivistoinen, Outi M; Richard, Peter; Penttilä, Merja; Ruohonen, Laura; Mojzita, Dominik

2012-02-17

In filamentous fungi D-galactose can be catabolised through the oxido-reductive and/or the Leloir pathway. In the oxido-reductive pathway D-galactose is converted to d-fructose in a series of steps where the last step is the oxidation of d-sorbitol by an NAD-dependent dehydrogenase. We identified a sorbitol dehydrogenase gene, sdhA (JGI53356), in Aspergillus niger encoding a medium chain dehydrogenase which is involved in D-galactose and D-sorbitol catabolism. The gene is upregulated in the presence of D-galactose, galactitol and D-sorbitol. An sdhA deletion strain showed reduced growth on galactitol and growth on D-sorbitol was completely abolished. The purified enzyme converted D-sorbitol to D-fructose with K(m) of 50±5 mM and v(max) of 80±10 U/mg. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa

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Saúl Gómez-Manzo

2015-01-01

Full Text Available Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH and the aldehyde dehydrogenase (ALDH. We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6 and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

Increased expression of mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type 2 in human atria during atrial fibrillation.

Science.gov (United States)

De-An, Pei; Li, Li; Zhi-Yun, Xu; Jin-Yu, Huang; Zheng-Ming, Xu; Min, Wang; Qiang, Yao; Shi-Eng, Huang

2010-01-01

Atrialfibrillation (AF) is associated with the activation of the renin-angiotensin-aldosterone system in the atria. It is not clear whether the expression of a mineralocorticoid receptor (MR), or 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), conferring aldosterone specificity to the MR, in patients with AF is altered. Patients with AF may be associated with increased expression of MR and 11betaHSD2 in the atria. Atrial tissue samples of 25 patients with rheumatic heart valve disease undergoing a valve replacement operation were examined. A total of 13 patients had chronic persistent AF (>6 mo) and 12 patients had no history of AF. The MR and 11betaHSD2 expression were analyzed at the mRNA and protein level. The localization of MR and 11betaHSD2 in atrial tissue was performed using specific immunohistochemistry staining. The results of real-time quantitative polymerase chain reaction (PCR) showed that AF groups, in comparison with sinus rhythm, had a higher mRNA expression level of MR or 11betaHSD2 (all P atrial tissue were also significantly increased in patients with AF compared with patients with sinus rhythm (P atrial interstitial fibrosis in patients with AF. These findings may have an important impact on the treatment of AF with aldosterone antagonists. Copyright 2010 Wiley Periodicals, Inc.

Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors.

Science.gov (United States)

Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Jayaprakasan, Vetrichelvan; Jayaram, Hiremagalur N; Boyd, Donald B; Jain, Manojkumar D; Grifantini, Mario

2005-03-15

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2'-MeAD (1) and T-3'-MeAD (2) containing, respectively, a methyl group at the ribose 2'-C-, and 3'-C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2'-C-methyl- or 3'-C-methyl-adenosine 5'-monophosphate with 2',3'-O-isopropylidene-tiazofurin 5'-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2'-MeAD and T-3'-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was comparable to that of parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2'-MeAD and T-3'-MeAD were found to inhibit the growth of K562 cells (IC(50) 30.7 and 65.0muM, respectively).

Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum.

Science.gov (United States)

Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

2016-06-20

Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production.

A survey of cyclic replacements for the central diamide moiety of inhibitors of inosine monophosphate dehydrogenase.

Science.gov (United States)

Dhar, T G Murali; Liu, Chunjian; Pitts, William J; Guo, Junquing; Watterson, Scott H; Gu, Henry; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Barrish, Joel C; Hollenbaugh, Diane; Iwanowicz, Edwin J

2002-11-04

A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

Science.gov (United States)

Gerszon, Joanna; Serafin, Eligiusz; Buczkowski, Adam; Michlewska, Sylwia; Bielnicki, Jakub Antoni; Rodacka, Aleksandra

2018-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

Characterization of a dehydrogenase activity responsible for oxidation of 11-cis-retinol in the retinal pigment epithelium of mice with a disrupted RDH5 gene. A model for the human hereditary disease fundus albipunctatus.

NARCIS (Netherlands)

Jang, G.F.; Hooser, J.P. van; Kuksa, V.; McBee, J.K.; He, Y.G.; Janssen, J.J.M.; Driessen, C.A.G.G.; Palczewski, K.

2001-01-01

In the vertebrate retina, the final step of visual chromophore production is the oxidation of 11-cis-retinol to 11-cis-retinal. This reaction is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs), prior to the chromophore rejoining with the visual pigment apo-proteins. The RDH5 gene encodes a

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

Science.gov (United States)

Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

2014-01-01

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

Retinaldehyde dehydrogenase 1 deficiency inhibits PPARγ-mediated bone loss and marrow adiposity.

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Nallamshetty, Shriram; Le, Phuong T; Wang, Hong; Issacsohn, Maya J; Reeder, David J; Rhee, Eun-Jung; Kiefer, Florian W; Brown, Jonathan D; Rosen, Clifford J; Plutzky, Jorge

2014-10-01

PPARγ, a ligand-activated nuclear receptor, regulates fundamental aspects of bone homeostasis and skeletal remodeling. PPARγ-activating anti-diabetic thiazolidinediones in clinical use promote marrow adiposity, bone loss, and skeletal fractures. As such, delineating novel regulatory pathways that modulate the action of PPARγ, and its obligate heterodimeric partner RXR, may have important implications for our understanding and treatment of disorders of low bone mineral density. We present data here establishing retinaldehyde dehydrogenase 1 (Aldh1a1) and its substrate retinaldehyde (Rald) as novel determinants of PPARγ-RXR actions in the skeleton. When compared to wild type (WT) controls, retinaldehyde dehydrogenase-deficient (Aldh1a1(-/-)) mice were protected against bone loss and marrow adiposity induced by either the thiazolidinedione rosiglitazone or a high fat diet, both of which potently activate the PPARγ-RXR complex. Consistent with these results, Rald, which accumulates in vivo in Aldh1a1(-/-) mice, protects against rosiglitazone-mediated inhibition of osteoblastogenesis in vitro. In addition, Rald potently inhibits in vitro adipogenesis and osteoclastogenesis in WT mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) respectively. Primary Aldh1a1(-/-) HSCs also demonstrate impaired osteoclastogenesis in vitro compared to WT controls. Collectively, these findings identify Rald and retinoid metabolism through Aldh1a1 as important novel modulators of PPARγ-RXR transactivation in the marrow niche. Copyright © 2014 Elsevier Inc. All rights reserved.

Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies.

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Julia Penna-Coutinho

Full Text Available The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2. The IC(50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.

Cardiac-specific overexpression of aldehyde dehydrogenase 2 exacerbates cardiac remodeling in response to pressure overload

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Sujith Dassanayaka

2018-07-01

Full Text Available Pathological cardiac remodeling during heart failure is associated with higher levels of lipid peroxidation products and lower abundance of several aldehyde detoxification enzymes, including aldehyde dehydrogenase 2 (ALDH2. An emerging idea that could explain these findings concerns the role of electrophilic species in redox signaling, which may be important for adaptive responses to stress or injury. The purpose of this study was to determine whether genetically increasing ALDH2 activity affects pressure overload-induced cardiac dysfunction. Mice subjected to transverse aortic constriction (TAC for 12 weeks developed myocardial hypertrophy and cardiac dysfunction, which were associated with diminished ALDH2 expression and activity. Cardiac-specific expression of the human ALDH2 gene in mice augmented myocardial ALDH2 activity but did not improve cardiac function in response to pressure overload. After 12 weeks of TAC, ALDH2 transgenic mice had larger hearts than their wild-type littermates and lower capillary density. These findings show that overexpression of ALDH2 augments the hypertrophic response to pressure overload and imply that downregulation of ALDH2 may be an adaptive response to certain forms of cardiac pathology. Keywords: Heart failure, Hypertrophy, Oxidative stress, Aldehydes, Cardiac remodeling, Hormesis