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Sample records for nucleotide dehydrogenases

  1. Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans.

    PubMed Central

    Harms, N; de Vries, G E; Maurer, K; Hoogendijk, J; Stouthamer, A H

    1987-01-01

    A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3. Screening of this clone bank with antibodies raised against P. denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins. The nucleotide sequence of the P. denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene. DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp. strain AM1 and Methylobacterium organophilum. Images PMID:3114231

  2. A nucleotide-controlled conformational switch modulates the activity of eukaryotic IMP dehydrogenases.

    PubMed

    Buey, Rubén M; Fernández-Justel, David; Marcos-Alcalde, Íñigo; Winter, Graeme; Gómez-Puertas, Paulino; de Pereda, José María; Luis Revuelta, José

    2017-06-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities. Thereby, the comparison of their high-resolution structures defines the mechanistic and structural details of a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity of eukaryotic IMPDHs. Remarkably, retinopathy-associated mutations lie within the mechanical hinges of the conformational change, highlighting its physiological relevance. Our results expand the mechanistic repertoire of Bateman domains and pave the road to new approaches targeting IMPDHs.

  3. Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene.

    PubMed Central

    Kim, S F; Baek, S J; Pack, M Y

    1991-01-01

    An allosteric L-(+)-lactate dehydrogenase gene of Lactobacillus casei ATCC 393 was cloned in Escherichia coli, and the nucleotide sequence of the gene was determined. The gene was composed of an open reading frame of 981 bp, starting with a GTG codon and ending with a TAA codon. The sequences for the promoter and ribosome binding site were identified, and a sequence for a structure resembling a rho-independent transcription terminator was also found. Images PMID:1768113

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

    PubMed Central

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

    2015-01-01

    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. PMID:26558346

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase.

    PubMed

    Moye, W S; Amuro, N; Rao, J K; Zalkin, H

    1985-07-15

    The yeast GDH1 gene encodes NADP-dependent glutamate dehydrogenase. This gene was isolated by complementation of an Escherichia coli glutamate auxotroph. NADP-dependent glutamate dehydrogenase was overproduced 6-10-fold in Saccharomyces cerevisiae bearing GDH1 on a multicopy plasmid. The nucleotide sequence of the 1362-base pair coding region and 5' and 3' flanking sequences were determined. Transcription start sites were located by S1 nuclease mapping. Regulation of GDH1 was not maintained when the gene was present on a multicopy plasmid. Protein secondary structure predictions identified a region with potential to form the dinucleotide-binding domain. The amino acid sequences of the yeast and Neurospora crassa enzymes are 63% conserved. Unlike the N. crassa gene, yeast GDH1 has no introns.

  7. Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase.

    PubMed Central

    Grant, P M; Tellam, J; May, V L; Strauss, A W

    1986-01-01

    We have determined the complete sequence of the rat mitochondrial malate dehydrogenase (mMDH) precursor derived from nucleotide sequence of the cDNA. A single synthetic oligodeoxynucleotide probe was used to screen a rat atrial cDNA library constructed in lambda gt10. A 1.2 kb full-length cDNA clone provided the first complete amino acid sequence of pre-mMDH. The 1014 nucleotide-long open reading frame encodes the 314 residue long mature mMDH protein and a 24 amino acid NH2-terminal extension which directs mitochondrial import and is cleaved from the precursor after import to generate mature mMDH. The amino acid composition of the transit peptide is polar and basic. The pre-mMDH transit peptide shows marked homology with those of two other enzymes targeted to the rat mitochondrial matrix. Images PMID:3755817

  8. Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

    PubMed Central

    Thomas, Elaine C.; Gunter, Jennifer H.; Webster, Julie A.; Schieber, Nicole L.; Oorschot, Viola; Parton, Robert G.; Whitehead, Jonathan P.

    2012-01-01

    We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease. PMID:23236438

  9. Kinetic Characterization of Reduced Pyridine Nucleotide Dehydrogenases (Duroquinone-Dependent) in Cucurbita Microsomes 1

    PubMed Central

    Pupillo, Paolo; Valenti, Vincenzo; De Luca, Letizia; Hertel, Rainer

    1986-01-01

    Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting Km of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting Km of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S0.5 of about 30 micromolar and production of H2O and H2O2, without superoxide involvement. The ratio NAD(P)H:O2 averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to O2 without release of intermediates. PMID:16664630

  10. Investigation of single nucleotide polymorphisms based on the intronic sequences of the propylene alcohol dehydrogenase gene in Chinese tobacco genotypes

    PubMed Central

    Wei, Ji-Cheng; Qiu, En-Jian; Guo, Hui-Yan; Hao, Ai-Ping; Chen, Rong-Ping

    2014-01-01

    A pair of primers was designed to amplify the propylene alcohol dehydrogenase gene sequence based on the cDNA sequence of the tobacco allyl-alcohol dehydrogenase gene. All introns were sequenced using traditional polymerase chain reaction (PCR) methods and T-A cloning. The sequences from common tobacco (Nicotiana tabaccum L.) and rustica tobacco (Nicotiana rustica L.) were analysed between the third intron and the fourth intron of the propylene alcohol dehydrogenase gene. The results showed that the alcohol dehydrogenase gene is a low-copy nuclear gene. The intron sequences have a combination of single nucleotide polymorphisms and length polymorphisms between common tobacco and rustica tobacco, which are suitable to identify the different germplasms. Furthermore, there are some single nucleotide polymorphism sites in the target sequence within common tobacco that can be used to distinguish intraspecific varieties. PMID:26740754

  11. Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

    PubMed Central

    Glesne, D A; Collart, F R; Huberman, E

    1991-01-01

    To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides. Images PMID:1717828

  12. Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

    PubMed Central

    Rule, G S; Pratt, E A; Chin, C C; Wold, F; Ho, C

    1985-01-01

    Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH. Images PMID:3882663

  13. Derivatives of cinnamic acid interact with the nucleotide binding site of mitochondrial aldehyde dehydrogenase. Effects on the dehydrogenase reaction and stimulation of esterase activity by nucleotides.

    PubMed

    Poole, R C; Bowden, N J; Halestrap, A P

    1993-04-22

    A wide variety of cinnamic acid derivatives are inhibitors of the low Km mitochondrial aldehyde dehydrogenase. Two of the most potent inhibitors are alpha-cyano-3,4-dihydroxythiocinnamamide (Ki0.6 microM) and alpha-cyano-3,4,5-trihydroxycinnamonitrile (Ki2.6 microM). With propionaldehyde as substrate the inhibition by these compounds was competitive with respect to NAD+. alpha-Fluorocinnamate was a much less effective inhibitor of the enzyme, with mixed behaviour towards NAD+, but with a major competitive component. These cinnamic acid derivatives were ineffective as inhibitors of the aldehyde dehydrogenase-catalysed hydrolysis of p-nitrophenyl acetate, but inhibited the ability of NAD+ and NADH to activate this activity. Inhibition of the stimulation of esterase activity was competitive with respect to NAD+ and NADH, and the derived Ki values were the same as for inhibition of dehydrogenase activity. NAD+, but not acetaldehyde, could elute the low Km aldehyde dehydrogenase from alpha-cyanocinnamate-Sepharose, to which the enzyme binds specifically (Poole RC and Halestrap AP, Biochem J 259: 105-110, 1989). The cinnamic acid derivatives have little effect on lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase or a high Km aldehyde dehydrogenase present in rat liver mitochondria. It is concluded that some cinnamic acid derivatives are potent inhibitors of the low Km aldehyde dehydrogenase, by competing with NAD+/NADH for binding to the enzyme. They are much less effective as inhibitors of other NAD(+)-dependent dehydrogenases.

  14. Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit.

    PubMed

    Fisher-Wellman, Kelsey H; Lin, Chien-Te; Ryan, Terence E; Reese, Lauren R; Gilliam, Laura A A; Cathey, Brook L; Lark, Daniel S; Smith, Cody D; Muoio, Deborah M; Neufer, P Darrell

    2015-04-15

    Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+/NADH) and anabolic (NADP+/NADPH) processes integrate during metabolism to maintain cellular redox homoeostasis, however, is unknown. The present work identifies a continuously cycling mitochondrial membrane potential (ΔΨm)-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced, however, is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyses the regeneration of NADPH from NADH at the expense of ΔΨm. The net effect is an automatic fine-tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy-expenditure rates, consistent with their well-known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homoeostasis is maintained and body weight is defended during periods of positive and negative energy balance.

  15. Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy consuming redox circuit

    PubMed Central

    Fisher-Wellman, Kelsey H.; Lin, Chien-Te; Ryan, Terence E.; Reese, Lauren R.; Gilliam, Laura A. A.; Cathey, Brook L.; Lark, Daniel S.; Smith, Cody D.; Muoio, Deborah M.; Neufer, P. Darrell

    2015-01-01

    SUMMARY Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+:NADH) and anabolic (NADP+:NADPH) processes integrate during metabolism to maintain cellular redox homeostasis however is unknown. The present work identifies a continuously cycling, mitochondrial membrane potential-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced however is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyzes the regeneration of NADPH from NADH at the expense of the mitochondrial membrane potential. The net effect is an automatic fine tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy expenditure rates, consistent with their well known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homeostasis is maintained and body weight is defended during periods of positive and negative energy balance. PMID:25643703

  16. Association of ferredoxin-NADP oxidoreductase with the chloroplastic pyridine nucleotide dehydrogenase complex in barley leaves

    PubMed

    Jose Quiles M; Cuello

    1998-05-01

    Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2'-monophosphoadenosine-5'-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2'-monophosphoadenosine-5'-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex.

  17. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-)

    SciTech Connect

    Hirono, A.; Beutler, E. )

    1988-06-01

    Glucose-6-phosphate dehydrogenase A(-) is a common variant in Blacks that causes sensitivity to drug- and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3{prime} end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C{sup 33} {yields} G, G{sup 202} {yields} A, and A{sup 376} {yields} G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The findings of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.

  18. [Comparative analysis of nucleotide sequences of lactate dehydrogenase (LDH) gene and LDH epitopes of Plasmodium vivax and Plasmodium falciparum].

    PubMed

    Jiang, Li; Wang, Zhen-yu; Ma, Xiao-jiang; Zhang, Xiao-ping; Cai, Li

    2010-04-01

    To analyze the difference of nucleotide sequences of lactate dehydrogenase (LDH) gene and LDH epitopes of Plasmodium vivax and P. falciparum. Specific primers were designed to amplify the full-length LDH gene sequence of P. vivax and P. falciparum (GenBank accession number: DQ198262 and DQ060151 respectively). The PCR products were sequenced and compared. The epitopes of objective LDH antigens were predicted by SYFPEITHI software. Pv-LDH and Pf-LDH genes were cloned into prokaryotic plasmid pET28a, then expressed in E. coli BL21(DE3) with isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction. The immunogenicity of the recombinants Pv-LDH and Pf-LDH was analyzed by Western blotting and neutralization ELISA assays. Pf-LDH gene was same to reference sequences(DQ198262), while there is a single nucleotide difference at the position 666 between Pv-LDH gene and reference sequences (DQ060151). The coding region of the two genes contained 951 bp encoding a 316-amino-acid residue. Compared with Pf-LDH, Pv-LDH showed a nucleotide sequence identity of 75.1%, and an amino acid sequence identity of 90.2%. T cell epitope prediction indicated that there were 28 human leukocyte antigen (HLA) types which could recognize pLDH antigen epitopes. The common or similar epitopes accounted for about 75% of the predicted 180 epitopes. The number of specific epitopes of Pv-LDH and Pf-LDH proteins was 38 and 45, respectively. Western blotting analysis showed that the Pv-LDH recombinant antigen reacted with the sera of malaria patients, and the reactivity was much lower than that of sera of immunized rabbit. Neutralization ELISA showed that about 70.3% reactivity of Pv-LDH polyclonal antibodies could be suppressed by Pv-LDH, while only 30.5% by Pf-LDH. There are differences in DNA sequences of LDH gene and LDH epitopes between P. vivax and P. falciparum. The antibodies induced by the specific epitopes account for a small proportion in the antibody repertoire.

  19. Regulation of adenine nucleotide translocase and glycerol 3-phosphate dehydrogenase expression by thyroid hormones in different rat tissues.

    PubMed Central

    Dümmler, K; Müller, S; Seitz, H J

    1996-01-01

    Thyroid hormone (T3)-dependent gene expression of the adenine nucleotide translocase (ANT) and the FAD-linked glycerol 3-phosphate dehydrogenase (mGPDH) was investigated in several rat tissues. Both proteins provide an important link between cytosolic and mitochondrial metabolic pathways and seem to be involved in the stimulation of mitochondrial oxygen consumption in response to T3. Here we show that two ANT isoforms are expressed in rat, the muscle-specific ANT1 form and the ubiquitous ANT2 form. The expression of ANT1 mRNA is not sensitive to T3 whereas the amount of ANT2 mRNA is increased 7-9-fold in liver and heart within 12-48 h after T3 application. Little or no effect of T3 on ANT2 mRNA was observed in kidney and brain. The mRNA changes are paralleled by an increase in ANT protein, thus explaining the accelerated ADP/ATP exchange observed in mitochondria isolated from hyperthyroid rats. The key role of ANT2 in the control of hyperthyroid metabolism is evident because the expression of the mersalyl-sensitive phosphate carrier and the mitochondrial creatine kinase mRNA, which are functionally linked to ANT, did not respond to T3. Similarly to the ADP/ATP exchange, the transfer of cytosolic NADH to the respiratory chain via the glycerophosphate shuttle is very sensitive to T3. Recently we demonstrated the 10-15-fold induction of mGPDH mRNA in rat liver after administration of T3 [Müller and Seitz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10581-10585]. Here we show that, in contrast with ANT2, the time course of induction is fast (4-6 h). Furthermore, mGPDH mRNA is induced 6-fold by T3 in heart and 4-fold in kidney. From these results we conclude that the T3-mediated transcriptional induction leading to increased activity of ANT2 and mGPDH contributes considerably to the increase in mitochondrial oxygen consumption in rat tissues. PMID:8760382

  20. Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

    NASA Technical Reports Server (NTRS)

    White, H. B., III; Kaplan, N. O.

    1972-01-01

    The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

  1. Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

    NASA Technical Reports Server (NTRS)

    White, H. B., III; Kaplan, N. O.

    1972-01-01

    The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

  2. Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae.

    PubMed

    Nagasu, T; Hall, B D

    1985-01-01

    The isolation of the Saccharomyces cerevisiae gene for NADP-dependent glutamate dehydrogenase (NADP-GDH) by cross hybridization to the Neurospora crassa am gene, known to encode for NADP-GDH is described. Two DNA fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence. A yeast shuttle vector (CV13) carrying either to the cloned fragments complements the gdh- strain of S. cerevisiae and directs substantial overproduction of NADP-GDH. One of the cloned fragments was sequenced, and the deduced amino acid (aa) sequence of the yeast NADP-GDH is 64% homologous to N. crassa, 51% to Escherichia coli and 24% to bovine NADP-GDHs.

  3. Association of Ferredoxin-NADP Oxidoreductase with the Chloroplastic Pyridine Nucleotide Dehydrogenase Complex in Barley Leaves1

    PubMed Central

    José Quiles, María; Cuello, Juan

    1998-01-01

    Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2′-monophosphoadenosine-5′-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2′-monophosphoadenosine-5′-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex. PMID:9576793

  4. Engineering the nucleotide coenzyme specificity and sulfhydryl redox sensitivity of two stress-responsive aldehyde dehydrogenase isoenzymes of Arabidopsis thaliana.

    PubMed

    Stiti, Naim; Adewale, Isaac O; Petersen, Jan; Bartels, Dorothea; Kirch, Hans-Hubert

    2011-03-15

    Lipid peroxidation is one of the consequences of environmental stress in plants and leads to the accumulation of highly toxic, reactive aldehydes. One of the processes to detoxify these aldehydes is their oxidation into carboxylic acids catalyzed by NAD(P)+-dependent ALDHs (aldehyde dehydrogenases). We investigated kinetic parameters of two Arabidopsis thaliana family 3 ALDHs, the cytosolic ALDH3H1 and the chloroplastic isoform ALDH3I1. Both enzymes had similar substrate specificity and oxidized saturated aliphatic aldehydes. Catalytic efficiencies improved with the increase of carbon chain length. Both enzymes were also able to oxidize α,β-unsaturated aldehydes, but not aromatic aldehydes. Activity of ALDH3H1 was NAD+-dependent, whereas ALDH3I1 was able to use NAD+ and NADP+. An unusual isoleucine residue within the coenzyme-binding cleft was responsible for the NAD+-dependence of ALDH3H1. Engineering the coenzyme-binding environment of ALDH3I1 elucidated the influence of the surrounding amino acids. Enzyme activities of both ALDHs were redox-sensitive. Inhibition was correlated with oxidation of both catalytic and non-catalytic cysteine residues in addition to homodimer formation. Dimerization and inactivation could be reversed by reducing agents. Mutant analysis showed that cysteine residues mediating homodimerization are located in the N-terminal region. Modelling of the protein structures revealed that the redox-sensitive cysteine residues are located at the surfaces of the subunits.

  5. Nucleotide sequence of medium-chain acyl-CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue

    SciTech Connect

    Kelly, D.P.; Kim, J.J.; Billadello, J.J.; Hainline, B.E.; Chu, T.W.; Strauss, A.W.

    1987-06-01

    Medium-chain acyl-CoA dehydrogenase is one of three similar enzymes that catalyze the initial step of fatty acid ..beta..-oxidation. Definition of the primary structure of MCAD and the tissue distribution of its mRNA is of biochemical and clinical importance because of the recent recognition of inherited MCAD deficiency in humans. The MCAD mRNA nucleotide sequence was determined from two overlapping cDNA clones isolated from human liver and placental cDNA libraries, respectively. The MCAD mRNA includes a 1263-base-pair coding region and a 738-base-pair 3'-nontranslated region. A partial amino acid sequence (137 residues) determined on peptides derived from MCAD purified from porcine liver confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human MCAD cDNA with the partial protein sequence of the porcine MCAD revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis shows that MCAD mRNA is expressed in a variety of rat (2.2 kilobases) and human (2.4 kilobases) tissues. Blot hybridization of RNA prepared from cultured skin fibroblasts from a patient with MCAD deficiency disclosed that mRNA was present and of similar size of MCAD mRNA derived from control fibroblasts. The isolation and characterization of MCAD cDNA is an important step in the definition of the defect underlying its metabolic consequences.

  6. Evaluation of clinical value of single nucleotide polymorphisms of dihydropyrimidine dehydrogenase gene to predict 5-fluorouracil toxicity in 60 colorectal cancer patients in China.

    PubMed

    Zhang, Xin; Sun, Butong; Lu, Zhenxia

    2013-01-01

    Dihydropyrimidine dehydrogenase (DPD) activity could be affected by single nucleotide polymorphisms (SNPs), resulting in either no effect, partial or complete loss of DPD activity. To evaluate if SNPs of DPD can be used to predict 5-FU toxicity, we evaluated five SNPs of DPD (14G1A, G1156T, G2194A, T85C and T464A) by TaqMan real time PCR in 60 colorectal cancer patients. Clinical data demonstrated that there was higher correlation between DPD activity and toxic effects of 5-FU (p<0.05). Six patients were positive for G2194A detection, which were all heterozygous. Two patients had lower DPD activities (< 3) with higher toxic effects (≥ stage III) while one patient was also positive for T85C detection. Ten patients were positive for T85C detection. Two patients were homozygous with lower DPD activities and higher toxic effects. Two patients were positive for the T464A detection, which were heterozygous with lower DPD activity and higher toxic effects and also positive for T85C detection. These data clearly indicated that the T464A and homozygous of the T85C are stronger biomarkers to predict the 5-FU toxicity. Our study significantly indicated that the detection for G2194A, T85C and T464A could predict ~13% of 5-FU severe toxic side effects.

  7. Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

    PubMed Central

    Nobukuni, Y; Mitsubuchi, H; Endo, F; Akaboshi, I; Asaka, J; Matsuda, I

    1990-01-01

    A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5'-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3'-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp. Images PMID:2365818

  8. Molecular cloning, nucleotide sequencing, and expression of genes encoding alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii and the mesophile Clostridium beijerinckii.

    PubMed

    Peretz, M; Bogin, O; Tel-Or, S; Cohen, A; Li, G; Chen, J S; Burstein, Y

    1997-08-01

    Proteins play a pivotal role in thermophily. Comparing the molecular properties of homologous proteins from thermophilic and mesophilic bacteria is important for understanding the mechanisms of microbial adaptation to extreme environments. The thermophile Thermoanaerobacter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinckii contain an NADP(H)-linked, zinc-containing secondary alcohol dehydrogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, sequenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 nucleotides long. The TB adh gene encoded an amino acid sequence identical to that of the purified TBADH. Alignment of the deduced amino acid sequences of the TB and CB adh genes showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons with A or T in the third position. Multiple sequence alignment of ADHs from different sources revealed that two (Cys-46 and His-67) of the three ligands for the catalytic Zn atom of the horse-liver ADH are preserved in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. The substrate specificities and thermostabilities of the TBADH and CBADH expressed inE. coli were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T. brockii enzyme.

  9. E. coli histidine triad nucleotide binding protein 1 (ecHinT) is a catalytic regulator of D-alanine dehydrogenase (DadA) activity in vivo.

    PubMed

    Bardaweel, Sanaa; Ghosh, Brahma; Chou, Tsui-Fen; Sadowsky, Michael J; Wagner, Carston R

    2011-01-01

    Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.

  10. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

    An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

  11. Nucleotide sequence of Phaseolus vulgaris L. alcohol dehydrogenase encoding cDNA and three-dimensional structure prediction of the deduced protein

    PubMed Central

    Amelia, Kassim; Khor, Chin Yin; Shah, Farida Habib; Bhore, Subhash J.

    2015-01-01

    Background: Common beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken. Objective: The objective of this study was to elucidate P. vulgaris L. AD (PvAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein. Materials and Methods: positive and negative strands of the PvAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvAD protein were predicted by using the PHYRE automatic fold recognition server. Results: The sequencing results analysis showed that PvAD cDNA is 1294 bp in length. It's open reading frame encodes for a protein that contains 371 amino acids. Deduced protein sequence analysis showed the presence of putative substrate binding, catalytic Zn binding, and NAD binding sites. Results indicate that the predicted 3D structure of PvAD protein is analogous to the experimentally determined crystal structure of s-nitrosoglutathione reductase from an Arabidopsis species. Conclusions: The 1294 bp long PvAD cDNA encodes for 371 amino acid long protein that contains conserved domains required for biological functions of AD. The predicted deduced PvAD protein's 3D structure reflects the analogy with the crystal structure of

  12. Regulation of p-nitroanisole O-demethylation in perfused rat liver. Adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase.

    PubMed Central

    Kauffman, F C; Evans, R K; Reinke, L A; Thurman, R G

    1979-01-01

    Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and "malic" enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for "malic" enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes. PMID:44195

  13. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  14. Lactate dehydrogenase test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003471.htm Lactate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Lactate dehydrogenase (LDH) is a protein that helps produce energy ...

  15. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a protein that ...

  16. Evidence that adrenal hexose-6-phosphate dehydrogenase can effect microsomal P450 cytochrome steroidogenic enzymes.

    PubMed

    Foster, Christy A; Mick, Gail J; Wang, Xudong; McCormick, Kenneth

    2013-09-01

    The role of adrenal hexose-6-phosphate dehydrogenase in providing reducing equivalents to P450 cytochrome steroidogenic enzymes in the endoplasmic reticulum is uncertain. Hexose-6-phosphate dehydrogenase resides in the endoplasmic reticulum lumen and co-localizes with the bidirectional enzyme 11β-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11β-hydroxysteroid dehydrogenase 1 with NADPH electrons via channeling. Intracellularly, two compartmentalized reactions generate NADPH upon oxidation of glucose-6-phosphate: cytosolic glucose-6-phosphate dehydrogenase and microsomal hexose-6-phosphate dehydrogenase. Because some endoplasmic reticulum enzymes require an electron donor (NADPH), it is conceivable that hexose-6-phosphate dehydrogenase serves in this capacity for these pathways. Besides 11β-hydroxysteroid dehydrogenase 1, we examined whether hexose-6-phosphate dehydrogenase generates reduced pyridine nucleotide for pivotal adrenal microsomal P450 enzymes. 21-hydroxylase activity was increased with glucose-6-phosphate and, also, glucose and glucosamine-6-phosphate. The latter two substrates are only metabolized by hexose-6-phosphate dehydrogenase, indicating that requisite NADPH for 21-hydroxylase activity was not via glucose-6-phosphate dehydrogenase. Moreover, dihydroepiandrostenedione, a non-competitive inhibitor of glucose-6-phosphate dehydrogenase, but not hexose-6-phosphate dehydrogenase, did not curtail activation by glucose-6-phosphate. Finally, the most compelling observation was that the microsomal glucose-6-phosphate transport inhibitor, chlorogenic acid, blunted the activation by glucose-6-phosphate of both 21-hydroxylase and 17-hydroxylase indicating that luminal hexose-6-phosphate dehydrogenase can supply NADPH for these enzymes. Analogous kinetic observations were found with microsomal 17-hydroxylase. These findings indicate that hexose-6-phosphate dehydrogenase can be a source, but not exclusively so, of NADPH

  17. Regulation of heart muscle pyruvate dehydrogenase kinase

    PubMed Central

    Cooper, Ronald H.; Randle, Philip J.; Denton, Richard M.

    1974-01-01

    1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [32P]phosphate from [γ-32P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100μm) and cyclic 3′:5′-nucleotides (at 10μm) had no significant effect on kinase activity. 3. The Km for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76μm. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The Km for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9–25.4μm. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The Km for pyruvate in the pyruvate dehydrogenase reaction was 35.5μm. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25–500μm. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate

  18. Plant Formate Dehydrogenase

    SciTech Connect

    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.

  19. Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

    PubMed Central

    Lampel, K A; Uratani, B; Chaudhry, G R; Ramaley, R F; Rudikoff, S

    1986-01-01

    The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase. Images PMID:3082854

  20. Plant Cyclic Nucleotide Signalling

    PubMed Central

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc

    2007-01-01

    The presence of the cyclic nucleotides 3′,5′-cyclic adenyl monophosphate (cAMP) and 3′,5′-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed. PMID:19704553

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

    PubMed Central

    Ueshima, Sakuko; Muramatsu, Hisashi; Nakajima, Takanori; Yamamoto, Hiroaki; Kato, Shin-ichiro; Misono, Haruo; Nagata, Shinji

    2010-01-01

    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-thienyl)serine 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. PMID:21048868

  2. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    SciTech Connect

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.; Huberman, E.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  3. POLYOL DEHYDROGENASES OF AZOTOBACTER AGILIS

    PubMed Central

    Marcus, Leon; Marr, Allen G.

    1961-01-01

    Marcus, Leon (University of California, Davis), and Allen G. Marr. Polyol dehydrogenases of Azotobacter agilis. J. Bacteriol. 82:224–232. 1961.—Two soluble diphosphopyridine-linked polyol dehydrogenases are formed by Azotobacter agilis (A. vinelandii). The first, d-mannitol dehydrogenase is induced by d-mannitol and all of the pentitols except l-arabitol. Ribitol is an excellent inducer of mannitol dehydrogenase although it is not metabolized, nor does the enzyme act upon it. This allows study of the gratuitous induction of mannitol dehydrogenase. Of the polyols tested, mannitol dehydrogenase oxidizes d-mannitol, d-arabitol, d-rhamnitol, and perseitol, demonstrating its requirement for substrates bearing the d-manno configuration. The corresponding 2-ketoses, d-fructose, d-xylulose, and presumably d-rhamnulose, and perseulose are reduced. The second enzyme, l-iditol dehydrogenase is induced only by polyols containing the d-xylo configuration, i.e., sorbitol and xylitol. l-Iditol dehydrogenase oxidizes d-xylo polyols seven times faster than it does d-ribo polyols. Substrates oxidized include l-iditol, sorbitol, xylitol, and ribitol. The corresponding 2-ketoses, l-sorbose, d-fructose, d-xylulose, and d-ribulose, are reduced. The two polyol dehydrogenases have been separated and purified by chromatography on a modified cellulose ion exchanger. PMID:13766585

  4. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  5. Prokaryotic Nucleotide Excision Repair

    PubMed Central

    Kisker, Caroline; Kuper, Jochen; Van Houten, Bennett

    2013-01-01

    Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation. PMID:23457260

  6. Prokaryotic nucleotide excision repair.

    PubMed

    Kisker, Caroline; Kuper, Jochen; Van Houten, Bennett

    2013-03-01

    Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation.

  7. LACTIC DEHYDROGENASES OF PSEUDOMONAS NATRIEGENS.

    PubMed

    WALKER, H; EAGON, R G

    1964-07-01

    Walker, Hazel (University of Georgia, Athens), and R. G. Eagon. Lactic dehydrogenases of Pseudomonas natriegens. J. Bacteriol. 88:25-30. 1964.-Lactic dehydrogenases specific for d- and l-lactate were demonstrated in Pseudomonas natriegens. The l-lactic dehydrogenase showed considerable heat stability, and 40% of the activity remained in extracts after heating at 60 C for 10 min. An essential thiol group for enzyme activity was noted. The results of these experiments were consistent with the view that lactate was dehydrogenated initially by a flavin cofactor and that electrons were transported through a complete terminal oxidase system to oxygen. The intracellular site of these lactic dehydrogenases was shown to be the cell membrane. It was suggested that the main physiological role of these lactic dehydrogenases is that of lactate utilization.

  8. LACTIC DEHYDROGENASES OF PSEUDOMONAS NATRIEGENS

    PubMed Central

    Walker, Hazel; Eagon, R. G.

    1964-01-01

    Walker, Hazel (University of Georgia, Athens), and R. G. Eagon. Lactic dehydrogenases of Pseudomonas natriegens. J. Bacteriol. 88:25–30. 1964.—Lactic dehydrogenases specific for d- and l-lactate were demonstrated in Pseudomonas natriegens. The l-lactic dehydrogenase showed considerable heat stability, and 40% of the activity remained in extracts after heating at 60 C for 10 min. An essential thiol group for enzyme activity was noted. The results of these experiments were consistent with the view that lactate was dehydrogenated initially by a flavin cofactor and that electrons were transported through a complete terminal oxidase system to oxygen. The intracellular site of these lactic dehydrogenases was shown to be the cell membrane. It was suggested that the main physiological role of these lactic dehydrogenases is that of lactate utilization. Images PMID:14197895

  9. EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

    EPA Science Inventory

    The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

  10. Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

    ERIC Educational Resources Information Center

    Strumilo, Slawomir

    2005-01-01

    Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

  11. Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

    ERIC Educational Resources Information Center

    Strumilo, Slawomir

    2005-01-01

    Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

  12. EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

    EPA Science Inventory

    The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

  13. Pyridine nucleotide redox abnormalities in diabetes.

    PubMed

    Ido, Yasuo

    2007-07-01

    In addition to hyperglycemia, diabetes is associated with increased levels of circulating free fatty acids, lactate, and branched chain amino acids, all of which produce an excessive reduced form of pyridine nucleotides NADH (reductive stress) in the cytosol and mitochondria. Our studies suggest that cytosolic NADH reductive stress under high glucose is largely caused by increased flux of glucose through polyol (sorbitol) pathway consisting of aldose reductase and sorbitol dehydrogenase. Inhibition of aldose reductase that blocks the polyol pathway has been shown to ameliorate diabetic neuropathy in humans. Cytosolic NADH reductive stress is predicted to increase production of diglycerides, reactive oxygen species, and methylglyoxal. Recent studies indicate that increasing NADH affects gene expression through the NADH activating transcriptional co-repressor, C-terminal binding protein (CtBP). In addition, it has been shown that the NADH utilizing enzyme, glyceraldehyde-3-phosphate dehydrogenase, participates as transcriptional regulator. These findings testify to the importance of NADH redox balance in cell biology and pathogenesis of diabetes and its complications. For example, through CtBP, the high NADH to NAD(+) ratio decreases an expression of SirT1, the protein inducing longevity and anti-apoptosis. This review covers metabolic cascades causing reductive stress and oxidative stress in diabetes after a brief introduction of the redox concept.

  14. Nucleotide signalling during inflammation

    PubMed Central

    Idzko, Marco; Ferrari, Davide; Eltzschig, Holger K.

    2014-01-01

    Inflammatory conditions are associated with the extracellular release of nucleotides, particularly ATP. In the extracellular compartment, ATP predominantly functions as a signalling molecule through the activation of purinergic P2 receptors. Metabotropic P2Y receptors are G-protein-coupled, whereas ionotropic P2X receptors are ATP-gated ion channels. Here we discuss how signalling events through P2 receptors alter the outcomes of inflammatory or infectious diseases. Recent studies implicate a role for P2X/P2Ysignalling in mounting appropriate inflammatory responses critical for host defence against invading pathogens or tumours. Conversely, P2X/P2Y signalling can promote chronic inflammation during ischaemia and reperfusion injury, inflammatory bowel disease or acute and chronic diseases of the lungs. Although nucleotide signalling has been used clinically in patients before, research indicates an expanding field of opportunities for specifically targeting individual P2 receptors for the treatment of inflammatory or infectious diseases. PMID:24828189

  15. Lactate dehydrogenase-elevating virus

    USDA-ARS?s Scientific Manuscript database

    This book chapter describes the taxonomic classification of Lactate dehydrogenase-elevating virus (LDV). Included are: host, genome, classification, morphology, physicochemical and physical properties, nucleic acid, proteins, lipids, carbohydrates, geographic range, phylogenetic properties, biologic...

  16. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  17. Nucleotide cleaving agents and method

    DOEpatents

    Que, Jr., Lawrence; Hanson, Richard S.; Schnaith, Leah M. T.

    2000-01-01

    The present invention provides a unique series of nucleotide cleaving agents and a method for cleaving a nucleotide sequence, whether single-stranded or double-stranded DNA or RNA, using and a cationic metal complex having at least one polydentate ligand to cleave the nucleotide sequence phosphate backbone to yield a hydroxyl end and a phosphate end.

  18. Wiring of PQQ-dehydrogenases.

    PubMed

    Laurinavicius, Valdas; Razumiene, Julija; Ramanavicius, Arunas; Ryabov, Alexander D

    2004-12-15

    The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.

  19. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  20. Template polymerization of nucleotide analogues

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.

    1991-01-01

    Recent work on the template-directed reactions of the natural D-nucleotides has made it clear that l-nucleotides and nucleotide-like derivatives of other sugars would strongly inhibit the formation of long oligonucleotides. Consequently, attention is focusing on molecules simpler than nucleotides that might have acted as monomers of an information transfer system. We have begun a general exploration of the template directed reactions of diverse peptide analogues. I will present work by Dr. Taifeng Wu on oxidative oligomerization of phosphorothioates and of Dr. Mary Tohidi on the cyclic polymerization of nucleoside and related cyclic pyrophosphates.

  1. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  2. Glucose-6-Phosphate Dehydrogenase Revisited

    PubMed Central

    O'Connell, Jerome T.; Henderson, Alfred R.

    1984-01-01

    Hemolytic diseases associated with drugs have been recognized since antiquity. Many of these anemias have been associated with oxidizing agents and deficiencies in the intraerythrocytic enzyme glucose-6-phosphate dehydrogenase. This paper outlines the discovery, prevalence, and variants of this enzyme. Methods of diagnosis of associated anemias are offered. PMID:6502728

  3. Increased riboflavin production by manipulation of inosine 5'-monophosphate dehydrogenase in Ashbya gossypii.

    PubMed

    Buey, Rubén M; Ledesma-Amaro, Rodrigo; Balsera, Mónica; de Pereda, José María; Revuelta, José Luis

    2015-11-01

    Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii.

  4. [Isolation PQQ biosynthesis gene cluster from Gluconobacter oxydans based on sorbose-dehydrogenase activity].

    PubMed

    Gao, Shuying; Xiong, Xionghua; Wang, Jianhua; Zhang, Weicai

    2010-08-01

    To isolate PQQ biosynthesis gene cluster from Gluconobacter oxydans H24 based on sorbose-dehydrogenase activity. A library of Gluconobacter oxydans H24 genomic DNA was constructed with host strains Escherichia coli JM109s, which was integrated of sdh gene at the ptsG site on the chromosome of JM109. By detecting sorbose-dehydrogenase activity, clone of PQQ biosynthesis was isolated and subcloned. A positive clone was isolated from Gluconobacter oxydans H24 genomic DNA library. Within the 5,400-base-pair DNA fragment five reading frames are presented, corresponding to five of the pqq genes (pqqABCDE). The nucleotide and amino acid sequence showed highly homology to pqq genes of other bacteria. The pqqABCDE gene cluster was successfully isolated from Gluconobacter oxydans H24 by sorbose dehydrogenase activity.

  5. Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase from Alcaligenes faecalis and evolution of amine dehydrogenases.

    PubMed

    Chistoserdov, A Y

    2001-08-01

    The nucleotide sequence of the aromatic amine utilization (aau) gene region from Alcaligenes faecalis contained nine genes (orf-1, aauBEDA, orf-2, orf-3, orf-4 and hemE) transcribed in the same direction. The aauB and aauA genes encode the periplasmic aromatic amine dehydrogenase (AADH) large and small subunit polypeptides, respectively, and were homologous to mauB and mauA, the genes for the large and small subunits of methylamine dehydrogenase (MADH). aauE and aauD are homologous to mauE and mauD and apparently carry out the same function of transport and folding of the small subunit polypeptide in the periplasm. No analogues of the mauF, mauG, mauL, mauM and mauN genes responsible for biosynthesis of tryptophan tryptophylquinone (the prosthetic group of amine dehydrogenases) were found in the aau cluster. orf-2 was predicted to encode a small periplasmic monohaem c-type cytochrome. No biological function can be assigned to polypeptides encoded by orf-1, orf-3 and orf-4 and mutations in these genes appeared to be lethal. Mutants generated by insertions into mauD were not able to use phenylethylamine, tyramine and tryptamine as a source of carbon and phenylethylamine, 3'-hydroxytyramine (dopamine) and tyramine as a source of nitrogen, indicating that AADH is the only enzyme involved in utilization of primary amines in A. faecalis. AADH genes are present in Alcaligenes xylosoxydans subsp. xylosoxydans, but not in other beta- and gamma-proteobacteria. Phylogenetic analysis of amine dehydrogenases (MADH and AADH) indicated that AADH and MADH evolutionarily diverged before separation of proteobacteria into existing subclasses.

  6. Labeled nucleotide phosphate (NP) probes

    DOEpatents

    Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  7. [Nucleotide receptors and renal function].

    PubMed

    Jankowski, Maciej

    2014-01-01

    Kidney plays a key role in homeostasis of human body. It has heterogenic structure and is characterized by complicated vascular beds and numbers of sympathetic nerves endings. Nucleotides receptors are involved in the regulation of blood flow, a fundamental process for renal function. Plasma is filtrated in renal glomerulus and activity of nucleotides receptors located on cells of glomerular filter modifies the physi- cochemical properties of filter and affects the filtration process. Electrolytes, water and low molecular weight molecules are reabsorbed from tubular fluid or secreted into fluid in proximal and distal tubules. Glomerular filtration rate and activity of tubular processes are regulated via nucleotides receptors by glomerulotubularbalance and tubuloglomerular feedback. Nucleotides receptors are involved in systemic regulation of blood pressure and carbohydrate metabolism.

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

    PubMed Central

    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

  9. Characterization of retinaldehyde dehydrogenase 3

    PubMed Central

    Graham, Caroline E.; Brocklehurst, Keith; Pickersgill, Richard W.; Warren, Martin J.

    2005-01-01

    RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the β-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488. PMID:16241904

  10. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  11. [Sorbitol-6-Phosphate Dehydrogenase Gene Polymorhism in Malus Mill. (Rosaceae)].

    PubMed

    Boris, K V; Kudryavtsev, A M; Kochieva, E Z

    2015-11-01

    The sorbitol-6-phosphate dehydrogenase gene (S6PDH) sequences of six representatives of the genus Malus, which belong to five different taxonomic sections, were examined for the first time. The exon-intron structure and polymorphism of the nucleotide and amino acid sequences of these genes was characterized. The intraspecific polymorphism of the S6PDH gene was assessed for the first time in 40 Russian and foreign apple (Malus domestica) cultivars. It was demonstrated that the interspecific polymorphism level of the S6PDH coding sequences in the studied. representatives of the genus Malus was 4%, and the intraspecific polymorphism level of M. domestica cultivars was very low, constituting 0.96%.

  12. Heterogeneity of liver alcohol dehydrogenase on starch-gel electrophoresis

    PubMed Central

    McKinley-McKee, J. S.; Moss, D. W.

    1965-01-01

    1. Purified horse-liver alcohol dehydrogenase is heterogeneous on starch-gel electrophoresis in several buffer systems. 2. The electrophoretic pattern is altered by the addition to the buffers of oxidized or reduced coenzymes, isobutyramide, metal ions or metal-chelating agents. 3. The effect of coenzymes on the pattern suggests that the major cause of the observed heterogeneity is not the existence of isoenzymes, but the presence in the enzyme preparations of coenzyme–enzyme complexes or complexes with other nucleotides similar to, but less reactive than, the coenzymes. 4. Metal ions and chelating agents influence the electrophoretic separation by partial denaturation and inactivation of the enzyme. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:4285890

  13. Method To Identify Specific Inhibiutors Of Imp Dehydrogenase

    DOEpatents

    Collart, Frank R.; Huberman, Eliezer

    2000-11-28

    This invention relates to methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, IMP dehydrogenase (IMPDH). IMPDH is an essential enzyme found in all free-living organisms from humans to bacteria and is an important therapeutic target. The invention allows the identification of specific inhibitors of any IMPDH enzyme which can be expressed in a functional form in a recombinant host cell. A variety of eukaryotic or prokaryotic host systems commonly used for the expression of recombinant proteins are suitable for the practice of the invention. The methods are amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods such as antisense molecule production. Utilization of exogenous guanosine as a control component of the methods allows for the identification of inhibitors specific for IMPDH rather than other causes of decreased cell proliferation.

  14. Metalated nucleotide chemisorption on hydroxyapatite.

    PubMed

    Benedetti, Michele; Antonucci, Daniela; De Castro, Federica; Girelli, Chiara R; Lelli, Marco; Roveri, Norberto; Fanizzi, Francesco P

    2015-12-01

    The experiments here reported evidence on the importance of the residual charge of a nucleotide derivative, for the adsorption on nHAP (hydroxyapatite nanocrystals), in water solution. We found that the simple presence of phosphates on the nucleotide derivative does not guarantee adsorption on nHAP. On the other hand, we demonstrated that a cationic or neutral charge on a nucleotide derivative produces a strongly reduced chemical adsorption (chemisorption) whereas, in the presence of a net negative charge, relevant adsorption on nHAP is observed. The number of phosphates can only modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. Previous considerations are important to model the binding ability of phosphate bearing nucleotide derivatives or molecules on hydroxyapatite. The findings reported in the present paper could be relevant in bone tissue targeting or nHAP mediated drug delivery.

  15. Structural organization of the human sorbitol dehydrogenase gene (SORD)

    SciTech Connect

    Iwata, T.; Carper, D.; Popescu, N.C.

    1995-03-01

    The primary structure of human sorbitol dehydrogenase (SORD) was determined by cDNA and genomic cloning. The nucleotide sequence of the mRNA covers 2471 bp including an open reading frame that yields a protein of 356 amino acid residues. The gene structure of SORD spans approximatley 30 kb divided into 9 exons and 8 introns. The gene was localized to chromosome 15q21.1 by in situ hybridization. Two transcription initiation sites were detected. Three Sp1 sites and a repetitive sequence (CAAA){sub 5} were observed in the 5{prime} noncoding region; no classical TATAA or CCAAT elements were found. The related alcohol dehydrogenases and {zeta}-crystallin have the same gene organization split by 8 introns, but no splice points coincide between SORD and these gene types. The deduced amino acid sequence of the SORD structure differs at a few positions from the directly determined protein sequence, suggesting allelic forms of the enzyme. High levels of SORD transcripts were observed in lens and kidney, as judged from Northern blot analysis. 42 refs., 7 figs., 1 tab.

  16. Crystal structure of a chimaeric bacterial glutamate dehydrogenase

    SciTech Connect

    Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.; Khan, Amir R.

    2016-05-23

    Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

  17. Single nucleotide polymorphisms in alcohol dehydrogenase genes among some Indian populations.

    PubMed

    Rao, V R; Bhaskar, L V K S; Annapurna, C; Reddy, A G; Thangaraj, K; Rao, A Papa; Singh, Lalji

    2007-01-01

    Seven ADH genes, identified until now, located in the long arm of human chromosome 4, produce seven different isozymes involved in the metabolism of ethanol to acetaldehyde. Of the more than 500 SNPs reported in the coding and non-coding regions of these genes in the world databases, 11 are more extensively studied. Three SNPs, ADH1B Arg47His (Exon3), ADH1B Arg369Cys (Exon9) and ADH1C Val349Ile (Exon8), are functionally validated in terms of phenotype-genotype correlations and are in specific linkage disequilibrium (LD) with non-coding SNPs. However, the frequency of each SNP and configuration of LD varies among populations. The Indian populations studied were conspicuous by the complete absence of African specific allele ADH1B*369Cys, the negligible frequency of East Asian specific ADH1B*47His allele and the presence of a novel SNP ADH1B A3529G (Intron3). The ADH1C*349Ile was the only functional allele polymorphic with a strong LD block in all the populations studied and the high F(st) value observed for the non-coding ADH1B Rsa1 variant was in conformity with world populations. Copyright 2007 Wiley-Liss, Inc.

  18. Structure of NADP+-dependent glutamate dehydrogenase from Escherichia coli: Reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

    PubMed Central

    Sharkey, Michael A.; Oliveira, Tânia F.; Engel, Paul C.; Khan, Amir R.

    2013-01-01

    Summary Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD+ and/or NADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding Domain I followed by a nucleotide binding Domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the glutamate dehydrogenase (GDH) family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5Å resolution. Modelling of NADP+ in Domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine residue that follows the P7 aspartate is presumed to form a hydrogen bond to the 2’-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP+ recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD+ dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution toward the co-ordination of the 2’-ribose phosphate of NADP+. These studies clarify the sequence/structure relationships in bacterial glutamate dehydrogenases, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual specificity GDHs of animals. PMID:23879525

  19. Cephalopod alcohol dehydrogenase: purification and enzymatic characterization.

    PubMed

    Rosario Fernández, M; Jörnvall, H; Moreno, A; Kaiser, R; Parés, X

    1993-08-16

    Octopus, squid and cuttle-fish organs were examined for alcohol dehydrogenase activity. Only one form was detectable, with properties typical of mammalian class III alcohol dehydrogenase. The corresponding protein was purified from octopus and enzymatically characterized. Ion-exchange and affinity chromatography produced a pure protein in excellent yield (73%) after 1600-fold purification. Enzymatic parameters with several substrates were similar to those for the human class III alcohol dehydrogenase, demonstrating a largely conserved function of the enzyme through wide lines of divergence covering vertebrates, cephalopods and bacteria. The results establish the universal occurrence of class III alcohol dehydrogenase and its strictly conserved functional properties in separate living forms. The absence of other alcohol dehydrogenases in cephalopods is compatible with the emergence of the ethanol-active class I type at a later stage, in lineages leading to vertebrates.

  20. [The PQQ-dehydrogenases. A novel example of bacterial quinoproteins].

    PubMed

    Flores-Encarnación, Marcos; Sánchez-Cuevas, Mariano; Ortiz-Gutiérrez, Felipe

    2004-01-01

    The word "quinoprotein" describes four groups of different enzymes which have cofactors containing o-quinones. Pyrrolo-quinoline quinone (PQQ) is not covalently attached. PQQ is the cofactor of several quinoprotein bacterial dehydrogenases including glucose dehydrogenase (G-DH), alcohol dehydrogenase (A-DH) and aldehyde dehydrogenase (AL-DH). These dehydrogenases are located in the periplasm of Gram-negative bacteria. This report summarises the structural properties of quinoprotein dehydrogenases, such as the biological functions and biotechnological aspects more important.

  1. The EMBL Nucleotide Sequence Database.

    PubMed

    Stoesser, Guenter; Baker, Wendy; van den Broek, Alexandra; Camon, Evelyn; Garcia-Pastor, Maria; Kanz, Carola; Kulikova, Tamara; Leinonen, Rasko; Lin, Quan; Lombard, Vincent; Lopez, Rodrigo; Redaschi, Nicole; Stoehr, Peter; Tuli, Mary Ann; Tzouvara, Katerina; Vaughan, Robert

    2002-01-01

    The EMBL Nucleotide Sequence Database (aka EMBL-Bank; http://www.ebi.ac.uk/embl/) incorporates, organises and distributes nucleotide sequences from all available public sources. EMBL-Bank is located and maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK. In an international collaboration with DDBJ (Japan) and GenBank (USA), data are exchanged amongst the collaborating databases on a daily basis. Major contributors to the EMBL database are individual scientists and genome project groups. Webin is the preferred web-based submission system for individual submitters, whilst automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via FTP, email and World Wide Web interfaces. EBI's Sequence Retrieval System (SRS), a network browser for databanks in molecular biology, integrates and links the main nucleotide and protein databases plus many other specialized databases. For sequence similarity searching, a variety of tools (e.g. Blitz, Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. All resources can be accessed via the EBI home page at http://www.ebi.ac.uk.

  2. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies.

    PubMed Central

    MacKintosh, R W; Fewson, C A

    1988-01-01

    The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydes and the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was the most effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second most effective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcohol dehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being better substrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehyde was much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single small substituent group in the meta or para position were better substrates than any other benzaldehyde derivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan-1-al and octan-1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde when the assay concentration exceeded approx. 10 microM. Benzaldehyde dehydrogenase II, but not benzyl alcohol dehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagents iodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzaldehyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzyl alcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agents EDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausible metabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP. Benzaldehyde dehydrogenase II was

  3. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    PubMed

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  4. Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

    2016-04-01

    G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue.

  5. Opine dehydrogenases in marine invertebrates.

    PubMed

    Harcet, Matija; Perina, Drago; Pleše, Bruna

    2013-10-01

    It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD(+) ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.

  6. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  7. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  8. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  9. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  10. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  11. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

    PubMed Central

    Goldberg, D A

    1980-01-01

    The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments. Images PMID:6777776

  12. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication

    PubMed Central

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  13. The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

    PubMed Central

    Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

    2016-01-01

    Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

  14. Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria. Physical, chemical, and enzymatic properties.

    PubMed

    Yanagawa, H; Egami, F

    1976-06-25

    Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have been obtained in homogeneous state from asparagus mitochondria. They are flavin enzymes with 1 mol of FAD/mol of protein. Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have s20,w of 6.22 S, 6.39 S, and 5.91 S, respectively, and molecular weights of 111,000, 110,000, and 95,000 (sedimentation equilibrium) or 112,000, 112,000, and 92,000 (gel filtration). They are slightly acidic proteins with isoelectric points of 6.75, 5.75, and 6.80. Both asparagusate dehydrogenases catalyzed the reaction Asg(SH)2 + NAD+ equilibrium AsgS2 + NADH + H+ and exhibit lipoyl dehydrogenase and diaphorase activities. Lipoyl dehydrogenase is specific for lipoate and has no asparagusate dehydrogenase activity. NADP cannot replace NAD in any case. Optimum pH for substrate reduction of the three enzymes are near 5.9. Asparagusate dehydrogenases I and II have Km values of 21.5 mM and 20.0 mM for asparagusate and 3.0 mM and 3.3 mM for lipoate, respectively. Lipoyl dehydrogenase activity of asparagusate dehydrogenases is enhanced by NAD and surfactants such as lecithin and Tween 80, but asparagusate dehydrogenase activity is not enhanced. Asparagusate dehydrogenases are strongly inhibited by mercuric ion, p-chloromercuribenzoic acid, and N-ethylmaleimide. Amino acid composition of the three enzymes is presented and discussed.

  15. The Single Nucleotide Polymorphism Consortium

    NASA Technical Reports Server (NTRS)

    Morgan, Michael

    2003-01-01

    I want to discuss both the Single Nucleotide Polymorphism (SNP) Consortium and the Human Genome Project. I am afraid most of my presentation will be thin on law and possibly too high on rhetoric. Having been engaged in a personal and direct way with these issues as a trained scientist, I find it quite difficult to be always as objective as I ought to be.

  16. The Single Nucleotide Polymorphism Consortium

    NASA Technical Reports Server (NTRS)

    Morgan, Michael

    2003-01-01

    I want to discuss both the Single Nucleotide Polymorphism (SNP) Consortium and the Human Genome Project. I am afraid most of my presentation will be thin on law and possibly too high on rhetoric. Having been engaged in a personal and direct way with these issues as a trained scientist, I find it quite difficult to be always as objective as I ought to be.

  17. Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases.

    PubMed

    Britton, K L; Baker, P J; Rice, D W; Stillman, T J

    1992-11-01

    The family of glutamate dehydrogenases include a group of hexameric oligomers with a subunit M(r) of around 50,000, which are closely related in amino acid sequence and a smaller group of tetrameric oligomers based on a much larger subunit with M(r) 115,000. Sequence comparisons have indicated a low level of similarity between the C-terminal portion of the tetrameric enzymes and a substantial region of the polypeptide chain for the more widespread hexameric glutamate dehydrogenases. In the light of the solution of the three-dimensional structure of the hexameric NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequence for the C-terminal domain of the tetrameric Neurospora crassa glutamate dehydrogenase against the sequence and the molecular structure of that from C. symbiosum. This analysis reveals that the residues conserved between these two families are clustered in the three-dimensional structure and points to a remarkably similar layout of the glutamate-binding site and the active-site pocket, though with some differences in the mode of recognition of the nucleotide cofactor.

  18. Complementary DNA cloning and molecular evolution of opine dehydrogenases in some marine invertebrates.

    PubMed

    Kimura, Tomohiro; Nakano, Toshiki; Yamaguchi, Toshiyasu; Sato, Minoru; Ogawa, Tomohisa; Muramoto, Koji; Yokoyama, Takehiko; Kan-No, Nobuhiro; Nagahisa, Eizou; Janssen, Frank; Grieshaber, Manfred K

    2004-01-01

    The complete complementary DNA sequences of genes presumably coding for opine dehydrogenases from Arabella iricolor (sandworm), Haliotis discus hannai (abalone), and Patinopecten yessoensis (scallop) were determined, and partial cDNA sequences were derived for Meretrix lusoria (Japanese hard clam) and Spisula sachalinensis (Sakhalin surf clam). The primers ODH-9F and ODH-11R proved useful for amplifying the sequences for opine dehydrogenases from the 4 mollusk species investigated in this study. The sequence of the sandworm was obtained using primers constructed from the amino acid sequence of tauropine dehydrogenase, the main opine dehydrogenase in A. iricolor. The complete cDNA sequence of A. iricolor, H. discus hannai, and P. yessoensis encode 397, 400, and 405 amino acids, respectively. All sequences were aligned and compared with published databank sequences of Loligo opalescens, Loligo vulgaris (squid), Sepia officinalis (cuttlefish), and Pecten maximus (scallop). As expected, a high level of homology was observed for the cDNA from closely related species, such as for cephalopods or scallops, whereas cDNA from the other species showed lower-level homologies. A similar trend was observed when the deduced amino acid sequences were compared. Furthermore, alignment of these sequences revealed some structural motifs that are possibly related to the binding sites of the substrates. The phylogenetic trees derived from the nucleotide and amino acid sequences were consistent with the classification of species resulting from classical taxonomic analyses.

  19. Heteropolymorphism of mitochondrial NADH dehydrogenase subunit 3 gene for the population analysis of chum salmon, Oncorhynchus keta.

    PubMed

    Yoon, M; Choi, Y S; Jin, H J; Sohn, Y C; Lee, S K; Jin, D H

    2008-07-01

    Mitochondrial DNAs (mtDNAs) has been frequently used as genetic markers for the population genetic studies. In this study we used chum salmon (Oncorhynchus keta) from Korea, Japan andAmerica, and compared their mitochondrial NADH dehydrogenase subunit 3 (ND3) genes by DNA sequence analysis. Sequence variation was studied in the ND3 among total 11 individuals from three populations. The ND3 gene was amplified by PCR targeting parts of cytochrome oxidase III gene (COIII) and NADH dehydrogenase subunit 4L gene (ND4L). ND3 gene sequence, encoded 752 bps, presented some genetic variation in the chum salmon populations. The observed nucleotide variations inferred the distinct genetic differentiation of American salmons from Korean and Japanese chum salmons. Six sites of single nucleotide polymorphism (SNP) were explored in the ND3 locus. Denaturing gradient gel electrophoresis analysis also showed a clear heterogenous band in American salmons compared to Asian salmons.

  20. Applications of adenine nucleotide measurements in oceanography

    NASA Technical Reports Server (NTRS)

    Holm-Hansen, O.; Hodson, R.; Azam, F.

    1975-01-01

    The methodology involved in nucleotide measurements is outlined, along with data to support the premise that ATP concentrations in microbial cells can be extrapolated to biomass parameters. ATP concentrations in microorganisms and nucleotide analyses are studied.

  1. SDR-type human hydroxysteroid dehydrogenases involved in steroid hormone activation.

    PubMed

    Wu, Xiaoqiu; Lukacik, Petra; Kavanagh, Kathryn L; Oppermann, Udo

    2007-02-01

    Hydroxysteroid dehydrogenases catalyze the NAD(P)(H)-dependent oxidoreduction of hydroxyl and oxo-functions at distinct positions of steroid hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands of the androgen, estrogen and glucocorticoid classes, since the conversion "switches" between receptor ligands and their inactive metabolites. The major reversible activities found in mammals acting on steroid hormones comprise 3alpha-, 11beta- and 17beta-hydroxysteroid dehydrogenases, and for each group several distinct isozymes have been described. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of steroid hormone levels. Several isoforms constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and immunity.

  2. Shikimate dehydrogenase from Pinu sylvestris L. needles

    SciTech Connect

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

    1986-07-10

    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/sup +/, but also with NAD/sup +/. The values of K/sub m/ for shikimate, when NADP/sup +/ and NAD/sup +/ 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.

  3. Genetics Home Reference: dihydropyrimidine dehydrogenase deficiency

    MedlinePlus

    ... of the skin on the palms and soles (hand-foot syndrome); shortness of breath; and hair loss may also ... dehydrogenase deficiency , with its early-onset neurological symptoms, is a rare disorder. Its prevalence is ...

  4. Isocitrate dehydrogenase mutations in gliomas

    PubMed Central

    Waitkus, Matthew S.; Diplas, Bill H.; Yan, Hai

    2016-01-01

    Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate α-ketoglutarate (αKG), but recurrent mutations at Arg132 of IDH1 and Arg172 of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of αKG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits αKG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy. PMID:26188014

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

    PubMed

    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.

  6. European Nucleotide Archive in 2016

    PubMed Central

    Toribio, Ana Luisa; Alako, Blaise; Amid, Clara; Cerdeño-Tarrága, Ana; Clarke, Laura; Cleland, Iain; Fairley, Susan; Gibson, Richard; Goodgame, Neil; ten Hoopen, Petra; Jayathilaka, Suran; Kay, Simon; Leinonen, Rasko; Liu, Xin; Martínez-Villacorta, Josué; Pakseresht, Nima; Rajan, Jeena; Reddy, Kethi; Rosello, Marc; Silvester, Nicole; Smirnov, Dmitriy; Vaughan, Daniel; Zalunin, Vadim; Cochrane, Guy

    2017-01-01

    The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) offers a rich platform for data sharing, publishing and archiving and a globally comprehensive data set for onward use by the scientific community. With a broad scope spanning raw sequencing reads, genome assemblies and functional annotation, the resource provides extensive data submission, search and download facilities across web and programmatic interfaces. Here, we outline ENA content and major access modalities, highlight major developments in 2016 and outline a number of examples of data reuse from ENA. PMID:27899630

  7. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

    SciTech Connect

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

    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.

  8. Recent advances on the enantioselective synthesis of C-nucleosides inhibitors of inosine monophosphate dehydrogenase (IMPDH).

    PubMed

    Merino, Pedro; Ghirardello, Mattia; Tejero, Tomas; Delso, Ignacio; Matute, Rosa

    2014-01-01

    This review will describe the recent advances in the synthesis of C-nucleosides with inhibitory activity of inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the biosynthesis of guanine nucleotides. The review will cover synthetic approaches of structural analogues showing modifications in the furanose ring as well as in the heterocyclic base. Heterocyclic sugar nucleoside analogues in which the furanose ring has been replaced by a different heterocyclic ring including aza analogues, thioanalogues as well as dioxolanyl and isoxazolidinyl analogues are also considered.

  9. New nucleotide analogues with enhanced signal properties.

    PubMed

    Cherkasov, Dmitry; Biet, Thorsten; Bäuml, Englbert; Traut, Walther; Lohoff, Michael

    2010-01-01

    We describe synthesis and testing of a novel type of dye-modified nucleotides which we call macromolecular nucleotides (m-Nucs). Macromolecular nucleotides comprise a nucleotide moiety, a macromolecular linear linker, and a large macromolecular ligand carrying multiple fluorescent dyes. With incorporation of the nucleotide moiety into the growing nucleic acid strand during enzymatic synthesis, the macromolecular ligand together with the coupled dyes is bound to the nucleic acid. By the use of this new class of modified nucleotides, signals from multiple dye molecules can be obtained after a single enzymatic incorporation event. The modified nucleotides are considered especially useful in the fields of nanobiotechnology, where signal stability and intensity is a limiting factor.

  10. Nucleotide Metabolism and DNA Replication.

    PubMed

    Warner, Digby F; Evans, Joanna C; Mizrahi, Valerie

    2014-10-01

    The development and application of a highly versatile suite of tools for mycobacterial genetics, coupled with widespread use of "omics" approaches to elucidate the structure, function, and regulation of mycobacterial proteins, has led to spectacular advances in our understanding of the metabolism and physiology of mycobacteria. In this article, we provide an update on nucleotide metabolism and DNA replication in mycobacteria, highlighting key findings from the past 10 to 15 years. In the first section, we focus on nucleotide metabolism, ranging from the biosynthesis, salvage, and interconversion of purine and pyrimidine ribonucleotides to the formation of deoxyribonucleotides. The second part of the article is devoted to DNA replication, with a focus on replication initiation and elongation, as well as DNA unwinding. We provide an overview of replication fidelity and mutation rates in mycobacteria and summarize evidence suggesting that DNA replication occurs during states of low metabolic activity, and conclude by suggesting directions for future research to address key outstanding questions. Although this article focuses primarily on observations from Mycobacterium tuberculosis, it is interspersed, where appropriate, with insights from, and comparisons with, other mycobacterial species as well as better characterized bacterial models such as Escherichia coli. Finally, a common theme underlying almost all studies of mycobacterial metabolism is the potential to identify and validate functions or pathways that can be exploited for tuberculosis drug discovery. In this context, we have specifically highlighted those processes in mycobacterial DNA replication that might satisfy this critical requirement.

  11. Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets.

    PubMed

    Lenzen, S; Panten, U

    1983-12-01

    Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.

  12. Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14. A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily.

    PubMed

    van der Werf, M J; van der Ven, C; Barbirato, F; Eppink, M H; de Bont, J A; van Berkel, W J

    1999-09-10

    A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 degrees C and displays a broad substrate specificity with a preference for substituted cyclohexanols. When incubated with a diastereomeric mixture of (4R)- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the (6S)-carveol stereoisomers only. Kinetic studies with pure stereoisomers showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea suggests that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexible C-terminal part involved in monoterpene binding.

  13. Digitalis metabolism and human liver alcohol dehydrogenase.

    PubMed Central

    Frey, W A; Vallee, B L

    1980-01-01

    Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired. PMID:6987673

  14. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  15. Sorbitol dehydrogenase: structure, function and ligand design.

    PubMed

    El-Kabbani, O; Darmanin, C; Chung, R P-T

    2004-02-01

    Sorbitol dehydrogenase (SDH), a member of the medium-chain dehydrogenase/reductase protein family and the second enzyme of the polyol pathway of glucose metabolism, converts sorbitol to fructose strictly using NAD(+) as coenzyme. SDH is expressed almost ubiquitously in all mammalian tissues. The enzyme has attracted considerable interest due to its implication in the development of diabetic complications and thus its tertiary structure may facilitate the development of drugs for the treatment of diabetes sufferers. Modelling studies suggest that SDH is structurally homologous to mammalian alcohol dehydrogenase with respect to conserved zinc binding motif and a hydrophobic substrate-binding pocket. Recently, the three-dimensional (3-D) structure of a mammalian SDH was solved, and it was found that while the overall 3-D structures of SDH and alcohol dehydrogenase are similar, the zinc coordination in the active sites of the two enzymes is different. The available structural and biochemical information of SDH are currently being utilized in a structure-based approach to develop drugs for the treatment or prevention of the complications of diabetes. This review provides an overview of the recent advances in the structure, function and drug development fields of sorbitol dehydrogenase.

  16. Mosaic organization of DNA nucleotides

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Havlin, S.; Simons, M.; Stanley, H. E.; Goldberger, A. L.

    1994-01-01

    Long-range power-law correlations have been reported recently for DNA sequences containing noncoding regions. We address the question of whether such correlations may be a trivial consequence of the known mosaic structure ("patchiness") of DNA. We analyze two classes of controls consisting of patchy nucleotide sequences generated by different algorithms--one without and one with long-range power-law correlations. Although both types of sequences are highly heterogenous, they are quantitatively distinguishable by an alternative fluctuation analysis method that differentiates local patchiness from long-range correlations. Application of this analysis to selected DNA sequences demonstrates that patchiness is not sufficient to account for long-range correlation properties.

  17. Mosaic organization of DNA nucleotides

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Havlin, S.; Simons, M.; Stanley, H. E.; Goldberger, A. L.

    1994-01-01

    Long-range power-law correlations have been reported recently for DNA sequences containing noncoding regions. We address the question of whether such correlations may be a trivial consequence of the known mosaic structure ("patchiness") of DNA. We analyze two classes of controls consisting of patchy nucleotide sequences generated by different algorithms--one without and one with long-range power-law correlations. Although both types of sequences are highly heterogenous, they are quantitatively distinguishable by an alternative fluctuation analysis method that differentiates local patchiness from long-range correlations. Application of this analysis to selected DNA sequences demonstrates that patchiness is not sufficient to account for long-range correlation properties.

  18. Nucleotide Excision Repair in Eukaryotes

    PubMed Central

    Schärer, Orlando D.

    2013-01-01

    Nucleotide excision repair (NER) is the main pathway used by mammals to remove bulky DNA lesions such as those formed by UV light, environmental mutagens, and some cancer chemotherapeutic adducts from DNA. Deficiencies in NER are associated with the extremely skin cancer-prone inherited disorder xeroderma pigmentosum. Although the core NER reaction and the factors that execute it have been known for some years, recent studies have led to a much more detailed understanding of the NER mechanism, how NER operates in the context of chromatin, and how it is connected to other cellular processes such as DNA damage signaling and transcription. This review emphasizes biochemical, structural, cell biological, and genetic studies since 2005 that have shed light on many aspects of the NER pathway. PMID:24086042

  19. Nucleotide excision repair in humans

    PubMed Central

    Spivak, Graciela

    2015-01-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429

  20. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  1. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  2. Fundamental molecular differences between alcohol dehydrogenase classes.

    PubMed Central

    Danielsson, O; Atrian, S; Luque, T; Hjelmqvist, L; Gonzàlez-Duarte, R; Jörnvall, H

    1994-01-01

    Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism. Images PMID:8197167

  3. Identifying single nucleotides by tunnelling current

    NASA Astrophysics Data System (ADS)

    Tsutsui, Makusu; Taniguchi, Masateru; Yokota, Kazumichi; Kawai, Tomoji

    2010-04-01

    A major goal in medical research is to develop a DNA sequencing technique that is capable of reading an entire human genome at low cost. Recently, it was proposed that DNA sequencing could be performed by measuring the electron transport properties of the individual nucleotides in a DNA molecule. Here, we report electrical detection of single nucleotides using two configurable nanoelectrodes and show that electron transport through single nucleotides occurs by tunnelling. We also demonstrate statistical identification of the nucleotides based on their electrical conductivity, thereby providing an experimental basis for a DNA sequencing technology based on measurements of electron transport.

  4. Detailed kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase

    PubMed Central

    2011-01-01

    Background Mitochondrial 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex (OGDHC), a key regulatory point of tricarboxylic acid (TCA) cycle, plays vital roles in multiple pathways of energy metabolism and biosynthesis. The catalytic mechanism and allosteric regulation of this large enzyme complex are not fully understood. Here computer simulation is used to test possible catalytic mechanisms and mechanisms of allosteric regulation of the enzyme by nucleotides (ATP, ADP), pH, and metal ion cofactors (Ca2+ and Mg2+). Results A model was developed based on an ordered ter-ter enzyme kinetic mechanism combined with con-formational changes that involve rotation of one lipoic acid between three catalytic sites inside the enzyme complex. The model was parameterized using a large number of kinetic data sets on the activity of OGDHC, and validated by comparison of model predictions to independent data. Conclusions The developed model suggests a hybrid rapid-equilibrium ping-pong random mechanism for the kinetics of OGDHC, consistent with previously reported mechanisms, and accurately describes the experimentally observed regulatory effects of cofactors on the OGDHC activity. This analysis provides a single consistent theoretical explanation for a number of apparently contradictory results on the roles of phosphorylation potential, NAD (H) oxidation-reduction state ratio, as well as the regulatory effects of metal ions on ODGHC function. PMID:21943256

  5. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

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

    DOE PAGES

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; ...

    2015-01-09

    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 withmore » 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. As a result, these findings offer a potential strategy for further ligand optimization.« less

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

    PubMed

    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.

  8. Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

    PubMed

    Gharib, Ghazaleh; Rashid, Naeem; Bashir, Qamar; Gardner, Qura-Tul Ann Afza; Akhtar, Muhammad; Imanaka, Tadayuki

    2016-01-01

    Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.

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

    PubMed Central

    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

  10. PARALLEL INDUCTION OF d-ARABITOL AND d-SORBITOL DEHYDROGENASES1

    PubMed Central

    Scolnick, Edward M.; Lin, Edmund C. C.

    1962-01-01

    Scolnick, Edward M. (Harvard Medical School, Boston, Mass.) and Edmund C. C. Lin. Parallel induction of d-arabitol and d-sorbitol dehydrogenases. J. Bacteriol. 84:631–637. 1962.—Two inducible diphosphopyridine nucleotide-linked dehydrogenases are described in a bacterium isolated from the soil, Cellvibrio polyoltrophicus ATCC 14774. The first enzyme catalyzes the dehydrogenation of d-arabitol to d-xylulose and d-mannitol to d-fructose. The data suggest that in vivo this enzyme has the dual function of the utilization of both of these polyhydric alcohols. The second enzyme was found to act only on d-sorbitol, converting it to d-fructose. Evidence for its physiological function as a d-sorbitol dehydrogenase is also given. Both of these enzymes were found to be induced in parallel by any of the three polyhydric alcohols, d-arabitol, d-mannitol, and d-sorbitol. A common stereoconfiguration of the inducers for these enzymes is suggested. The parallel evolution of substrate specificity and inducer specificity is discussed with respect to the functional advantage that such a selective process might offer. PMID:13992484

  11. Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets

    PubMed Central

    Hiasa, Miki; Togawa, Natsuko; Miyaji, Takaaki; Omote, Hiroshi; Yamamoto, Akitsugu; Moriyama, Yoshinori

    2014-01-01

    Abstract Nucleotides are stored in the dense granules of platelets. The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation. However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood. The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized. We hypothesized that the vesicular nucleotide transporter (VNUT) is also involved in the vesicular storage of nucleotides in platelets. In this article, we present three lines of evidence that VNUT is responsible for the vesicular storage of nucleotides in platelets and that vesicular ATP transport is crucial for platelet function, detection and characterization of VNUT activity in platelets isolated from healthy humans and MEG‐01 cells, RNA interference experiments on MEG‐01 cells, and studies on nucleotide transport and release with a selective inhibitor. PMID:24907298

  12. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

    NASA Astrophysics Data System (ADS)

    Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

    2017-02-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  13. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

    NASA Astrophysics Data System (ADS)

    Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

    2017-09-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  14. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

    PubMed

    Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

    2017-02-03

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  15. [Interaction of succinate dehydrogenase and oxaloacetate].

    PubMed

    Kotliar, A B; Vinogradov, A D

    1984-04-01

    The equilibrium and rate constants for interaction of the reduced and oxidized membrane-bound succinate dehydrogenase (EC 1.3.99.1) with oxaloacetate were determined. The 10-fold decrease in the oxaloacetate affinity for the reduced enzyme was shown to be due to the 10-fold increase of the enzyme-inhibitor complex dissociation rate, which occurs upon its reduction. The rate of dissociation induced by succinate is 10 times higher than that induced by malonate in the submitochondrial particles, being equal in the soluble enzyme preparations. The rates of dissociation induced by malonate excess, or by the enzyme irreversibly utilizing oxaloacetate (transaminase in the presence of glutamate) are also equal. The data obtained suggest that succinate dehydrogenase interaction with succinate and oxaloacetate results from the competition for a single dicarboxylate-specific site. In submitochondrial particles all succinate dehydrogenase molecules are in redox equilibrium provided for by endogenous ubiquinone. No electronic equilibrium between the individual enzyme molecules exists, when succinate dehydrogenase is solubilized.

  16. The Role of Gene Duplication in the Evolution of Purine Nucleotide Salvage Pathways

    NASA Astrophysics Data System (ADS)

    Becerra, Arturo; Lazcano, Antonio

    1998-10-01

    Purine nucleotides are formed de novo by a widespread biochemical route that may be of monophyletic origin, or are synthesized from preformed purine bases and nucleosides through different salvage pathways. Three monophyletic sets of purine salvage enzymes, each of which catalyzes mechanistically similar reactions, can be identified: (a) adenine-, xanthine-, hypoxanthine- and guanine-phosphoribosyltransferases, which are all homologous among themselves, as well as to nucleoside phosphorylases; (b) adenine deaminase, adenosine deaminase, and adenosine monophophate deaminase; and (c) guanine reductase and inosine monophosphate dehydrogenase. These homologies support the idea that substrate specificity is the outcome of gene duplication, and that the purine nucleotide salvage pathways were assembled by a patchwork process that probably took place before the divergence of the three cell domains (Bacteria, Archaea, and Eucarya). Based on the ability of adenine PRTase to catalyze the condensation of PRPP with 4-aminoimidazole-5-carboxamide (AICA), a simpler scheme of purine nucleotide biosynthesis is presented. This hypothetical route requires the prior evolution of PRPP biosynthesis. Since it has been argued that PRPP, nucleosides, and nucleotides are susceptible to hydrolysis, they are very unlikely prebiotic compounds. If this is the case, it implies that many purine salvage pathways appeared only after the evolution of phosphorylated sugar biosynthetic pathways made ribosides available.

  17. Nucleotide Selectivity of Antibiotic Kinases▿

    PubMed Central

    Shakya, Tushar; Wright, Gerard D.

    2010-01-01

    Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3′)-IIIa, APH(2″)-Ib, and MPH(2′)-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3′)-IIIa exclusively uses ATP, MPH(2′)-I exclusively uses GTP, and APH(2″)-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance. PMID:20231391

  18. Automated Identification of Nucleotide Sequences

    NASA Technical Reports Server (NTRS)

    Osman, Shariff; Venkateswaran, Kasthuri; Fox, George; Zhu, Dian-Hui

    2007-01-01

    STITCH is a computer program that processes raw nucleotide-sequence data to automatically remove unwanted vector information, perform reverse-complement comparison, stitch shorter sequences together to make longer ones to which the shorter ones presumably belong, and search against the user s choice of private and Internet-accessible public 16S rRNA databases. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] In STITCH, a template 16S rRNA sequence is used to position forward and reverse reads. STITCH then automatically searches known 16S rRNA sequences in the user s chosen database(s) to find the sequence most similar to (the sequence that lies at the smallest edit distance from) each spliced sequence. The result of processing by STITCH is the identification of the most similar well-described bacterium. Whereas previously commercially available software for analyzing genetic sequences operates on one sequence at a time, STITCH can manipulate multiple sequences simultaneously to perform the aforementioned operations. A typical analysis of several dozen sequences (length of the order of 103 base pairs) by use of STITCH is completed in a few minutes, whereas such an analysis performed by use of prior software takes hours or days.

  19. Nucleotide selectivity of antibiotic kinases.

    PubMed

    Shakya, Tushar; Wright, Gerard D

    2010-05-01

    Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3')-IIIa, APH(2'')-Ib, and MPH(2')-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3')-IIIa exclusively uses ATP, MPH(2')-I exclusively uses GTP, and APH(2'')-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.

  20. Nucleotide excision repair in humans.

    PubMed

    Spivak, Graciela

    2015-12-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

    PubMed

    Shirasu, Naoto; Kuroki, Masahide

    2014-01-01

    We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

  2. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

    PubMed

    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  3. RATES OF GDP-INDUCED AND GTP-INDUCED DEPOLYMERIZATION AND ISOMERIZATION OF THE BOVINE LIVER GLUTAMATE DEHYDROGENASE-COENZYME COMPLEX: A POSSIBLE CONTROLLING FACTOR IN METABOLIC REGULATION*

    PubMed Central

    Huang, Charles Y.; Frieden, Carl

    1969-01-01

    The rate of the depolymerization of beef liver glutamate dehydrogenase induced by coenzyme and the purine nucleotides guanosine 5′-diphosphate and guanosine 5′-triphosphate, which are potent inhibitors of enzymatic activity, has been measured by rapid light scattering techniques and by absorbancy changes with stop flow. It is shown that the rate constant for this process may vary from several milliseconds to several seconds depending upon the nucleotides used. The widely varying rate constants for the nucleotide-induced depolymerization may serve a role in determining the nature of the regulation of enzyme activity by nucleotides. Depolymerization induced by guanosine 5′-diphosphate in the presence of diphosphopyridine nucleotide is slower than in the presence of triphosphopyridine nucleotide as coenzyme, and this difference is apparently due to the isomerization of the enzyme as a result of diphosphopyridine nucleotide binding to a second, nonactive site. This binding, as well as binding of the coenzyme to the active site, may be conveniently measured by a purine nucleotide-induced spectral shift in the coenzyme absorption spectrum. It is also shown that complete depolymerization of the enzyme in the presence of guanosine 5′-triphosphate is accomplished by about half saturation of the coenzyme active sites (6-8 active “monomer”). PMID:4312753

  4. Development of an amine dehydrogenase for synthesis of chiral amines.

    PubMed

    Abrahamson, Michael J; Vázquez-Figueroa, Eduardo; Woodall, Nicholas B; Moore, Jeffrey C; Bommarius, Andreas S

    2012-04-16

    A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

  5. Calculations of hydrogen tunnelling and enzyme catalysis: a comparison of liver alcohol dehydrogenase, methylamine dehydrogenase and soybean lipoxygenase

    NASA Astrophysics Data System (ADS)

    Tresadern, Gary; McNamara, Jonathan P.; Mohr, Matthias; Wang, Hong; Burton, Neil A.; Hillier, Ian H.

    2002-06-01

    Although the potential energy barrier for hydrogen transfer is similar for the enzymes liver alcohol dehydrogenase, methylamine dehydrogenase and soybean lipoxygenase, the degree of tunnelling is predicted to differ greatly, and is reflected by their primary kinetic isotope effects.

  6. [Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels].

    PubMed

    Kulis, Iu Iu

    1979-03-01

    The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.

  7. Long-range correlations in nucleotide sequences

    NASA Astrophysics Data System (ADS)

    Peng, C.-K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-03-01

    DNA SEQUENCES have been analysed using models, such as an it-step Markov chain, that incorporate the possibility of short-range nucleotide correlations1. We propose here a method for studying the stochastic properties of nucleotide sequences by constructing a 1:1 map of the nucleotide sequence onto a walk, which we term a 'DNA walk'. We then use the mapping to provide a quantitative measure of the correlation between nucleotides over long distances along the DNA chain. Thus we uncover in the nucleotide sequence a remarkably long-range power law correlation that implies a new scale-invariant property of DNA. We find such long-range correlations in intron-containing genes and in nontranscribed regulatory DNA sequences, but not in complementary DNA sequences or intron-less genes.

  8. Long-range correlations in nucleotide sequences

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-01-01

    DNA sequences have been analysed using models, such as an n-step Markov chain, that incorporate the possibility of short-range nucleotide correlations. We propose here a method for studying the stochastic properties of nucleotide sequences by constructing a 1:1 map of the nucleotide sequence onto a walk, which we term a 'DNA walk'. We then use the mapping to provide a quantitative measure of the correlation between nucleotides over long distances along the DNA chain. Thus we uncover in the nucleotide sequence a remarkably long-range power law correlation that implies a new scale-invariant property of DNA. We find such long-range correlations in intron-containing genes and in nontranscribed regulatory DNA sequences, but not in complementary DNA sequences or intron-less genes.

  9. Long-range correlations in nucleotide sequences

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-01-01

    DNA sequences have been analysed using models, such as an n-step Markov chain, that incorporate the possibility of short-range nucleotide correlations. We propose here a method for studying the stochastic properties of nucleotide sequences by constructing a 1:1 map of the nucleotide sequence onto a walk, which we term a 'DNA walk'. We then use the mapping to provide a quantitative measure of the correlation between nucleotides over long distances along the DNA chain. Thus we uncover in the nucleotide sequence a remarkably long-range power law correlation that implies a new scale-invariant property of DNA. We find such long-range correlations in intron-containing genes and in nontranscribed regulatory DNA sequences, but not in complementary DNA sequences or intron-less genes.

  10. Properties of formate dehydrogenase in Methanobacterium formicicum

    SciTech Connect

    Schauer, N.L.; Ferry, J.G.

    1982-04-01

    Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50..mu..mol of methyl viologen per min per mg of protein and 8.2 ..mu..mol of coenzyme F/sub 420/ per min per mg of protein. The apparent K/sub m/ for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F/sub 420/, was 10-fold greater (63 ..mu..M) than for coenzyme F/sub 420/ (6 ..mu..M). The purified enzyme also reduced flavin mononucleotide (K/sub m/ = 13 ..mu..M) and flavin adenine dinucleotide (K/sub m/ = 25 ..mu..M) with formate, but did not reduce NAD/sup +/ or NADP/sup +/. The reduction of NADP/sup +/ with formate required formate dehydrogenase, coenzyme F/sub 420/, and coenzyme F/sub 420/:NADP/sup +/ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F/sub 420/. The optimal reaction rate occurred at 55/sup 0/C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (K/sub i/ = 6 ..mu..M), azide (K/sub i/ = 39 ..mu..M),..cap alpha..,..cap alpha..-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.

  11. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    SciTech Connect

    Girio, F.M.; Amaral-Collaco, M.T.; Pelica, F.

    1996-01-01

    The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD{sup +} were 16.5 and 0.55 mM, respectively. Ca{sup 2+}, Mg{sup 2+}, and Mn{sup 2+} did not affect the enzyme activity. Conversely, Zn{sup 2+}, Cd{sup 2+}, and Co{sup 2+} strongly inhibited the enzyme activity. It was concluded that NAD{sup +}-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K{sub m} value for xylitol, and therefore should be named L-iditol:NAD{sup +}-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K for xylitol, which is low enough to assure its fast oxidation by NAD{sup +}-xylitol dehydrogenase. However, a higher K{sub m} value of xylitol dehydrogenase for NAD{sup +} compared to the K{sub m} values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields. 22 refs., 4 figs., 2 tabs.

  12. Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

    PubMed

    Inose, Ken; Fujikawa, Masako; Yamazaki, Tomohiko; Kojima, Katsuhiro; Sode, Koji

    2003-02-21

    We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea.

  13. Nucleotide diversity among natural populations of a North American poplar (Populus balsamifera, Salicaceae).

    PubMed

    Breen, Amy L; Glenn, Elise; Yeager, Adam; Olson, Matthew S

    2009-01-01

    Poplars (Populus spp.) comprise an important component of circumpolar boreal forest ecosystems and are the model species for tree genomics. In this study, we surveyed genetic variation and population differentiation in three nuclear genes among populations of balsam poplar (Populus balsamifera) in North America. We examined nucleotide sequence variation in alcohol dehydrogenase 1 (Adh1) and glyceraldehyde 3-phosphate dehydrogenase (G3pdh), two well-studied nuclear loci in plants, and abscisic acid insensitivity 1B (ABI1B), a locus coincident with timing of seasonal dormancy in quantitative trait locus (QTL) studies of hybrid poplars. We compared estimates of baseline population genetic parameters for these loci with those obtained in studies of other poplar species, particularly European aspen (Populus tremula). Average pairwise nucleotide diversity (pi(tot) = 0.00216-0.00353) was equivalent to that in Populus trichocarpa, but markedly less than that in P. tremula. Elevated levels of population structure were observed in ABI1B between the northern and southern regions (F(CT) = 0.184, P < 0.001) and among populations (F(ST) = 0.256, P < 0.001). These results suggest that geographic or taxonomic factors are important for understanding patterns of variation throughout the genus Populus. Our findings have the potential to aid in the design of sampling regimes for conservation and breeding stock and contribute to historical inferences regarding the factors that shaped the genetic diversity of boreal plant species.

  14. Cloning and Expression of a Xylitol-4-Dehydrogenase Gene from Pantoea ananatis

    PubMed Central

    Aarnikunnas, J. S.; Pihlajaniemi, A.; Palva, A.; Leisola, M.; Nyyssölä, A.

    2006-01-01

    The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to l-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5′ region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD+ as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the d-form of xylulose. Xylitol was converted to l-xylulose with a high yield (>80%) by the resting recombinant cells, and the l-xylulose was secreted into the medium. No evidence of d-xylulose being synthesized by the recombinant cells was found. PMID:16391066

  15. Biochemical properties of rat liver mitochondrial aldehyde dehydrogenase with respect to oxidation of formaldehyde.

    PubMed

    Cinti, D L; Keyes, S R; Lemelin, M A; Denk, H; Schenkman, J B

    1976-03-25

    The oxidation of formaldehyde by rat liver mitochondria in the presence of 50 mM phosphate was enhanced 2-fold by exogenous NAD+. Absolute requirement of NAD+ for formaldehyde oxidation was demonstrated by depleting the mitochondria of their NAD+ content (4.6 nmol/mg of protein), followed by reincorporation of the NAD+ into the depleted mitochondria. Aldehyde (formaldehyde) dehydrogenase activity was completely abolished in the depleted mitochondria, but the enzyme activity was restored to control levels following reincorporation of the pyridine nucleotide. Phosphate stimulation of formaldehyde oxidation could not be explained fully by the phosphate-induced swelling which enhances membrane permeability to NAD+, since stimulation of the enzyme activity by increased phosphate concentrations was still observed in the absence of exogenous NAD+. The Km for formaldehyde oxidation by the mitochondria was found to be 0.38 nM, a value similar to that obtained with varying concentrations of NAD+; both Vmax values were very similar, giving a value of 70 to 80 nmol/min/mg of protein. The pH optimum for the mitochondrial enzyme was 8.0. Inhibition of the enzyme activity by anaerobiosis was apparently due to the inability of the respiratory chain to oxidize the generated NADH. The inhibition of mitochondrial formaldehyde oxidation by succinate was found to be due to a lowering of the NAD+ level in the mitochondria. Succinate also inhibited acetaldehyde oxidation by the mitochondria. Malonate, a competitive inhibitor of succinic dehydrogenase, blocked the inhibitory effect of succinate. The respiratory chain inhibitors, rotenone, and antimycin A plus succinate, strongly inhibited formaldehyde oxidation by apparently the same mechanism, although the crude enzyme preparation (freed from the membrane) was slightly sensitive to rotenone. The mitochondria were subfractionated, and 85% of the enzyme activity was found in the inner membrane fraction (mitoplast). Furthermore, separation

  16. Novel Psychrophilic and Thermolabile l-Threonine Dehydrogenase from Psychrophilic Cytophaga sp. Strain KUC-1

    PubMed Central

    Kazuoka, Takayuki; Takigawa, Shouhei; Arakawa, Noriaki; Hizukuri, Yoshiyuki; Muraoka, Ikuo; Oikawa, Tadao; Soda, Kenji

    2003-01-01

    A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent l-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other l-threonine dehydrogenases that have thus far been studied. l-Threonine and dl-threo-3-hydroxynorvaline were the substrates, and NAD+ and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The Km for l-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD+ binds to an enzyme prior to l-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, l-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. l-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the

  17. Nucleotide capacitance calculation for DNA sequencing

    SciTech Connect

    Lu, Jun-Qiang; Zhang, Xiaoguang

    2008-01-01

    Using a first-principles linear response theory, the capacitance of the DNA nucleotides, adenine, cytosine, guanine and thymine, are calculated. The difference in the capacitance between the nucleotides is studied with respect to conformational distortion. The result suggests that although an alternate current capacitance measurement of a single-stranded DNA chain threaded through a nano-gap electrodes may not sufficient to be used as a stand alone method for rapid DNA sequencing, the capacitance of the nucleotides should be taken into consideration in any GHz-frequency electric measurements and may also serve as an additional criterion for identifying the DNA sequence.

  18. Plant cyclic nucleotide signalling: facts and fiction.

    PubMed

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc; Maathuis, Frans Jm

    2007-11-01

    The presence of the cyclic nucleotides 3',5'-cyclic adenyl monophosphate (cAMP) and 3',5'-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed.

  19. "Enzymogenesis": classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line.

    PubMed Central

    Danielsson, O; Jörnvall, H

    1992-01-01

    Analysis of the activity and structure of lower vertebrate alcohol dehydrogenases reveals that relationships between the classical liver and yeast enzymes need not be continuous. Both the ethanol activity of class I-type alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and the glutathione-dependent formaldehyde activity of the class III-type enzyme [formaldehyde:NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1] are present in liver down to at least the stage of bony fishes (cod liver: ethanol activity, 3.4 units/mg of protein in one enzyme; formaldehyde activity, 4.5 units/mg in the major form of another enzyme). Structural analysis of the latter protein reveals it to be a typical class III enzyme, with limited variation from the mammalian form and therefore with stable activity and structure throughout much of the vertebrate lineage. In contrast, the classical alcohol dehydrogenase (the class I enzyme) appears to be the emerging form, first in activity and later also in structure. The class I activity is present already in the piscine line, whereas the overall structural-type enzyme is not observed until amphibians and still more recent vertebrates. Consequently, the class I/III duplicatory origin appears to have arisen from a functional class III form, not a class I form. Therefore, ethanol dehydrogenases from organisms existing before this duplication have origins separate from those leading to the "classical" liver alcohol dehydrogenases. The latter now often occur in isozyme forms from further gene duplications and have a high rate of evolutionary change. The pattern is, however, not simple and we presently find in cod the first evidence for isozymes also within a class III alcohol dehydrogenase. Overall, the results indicate that both of these classes of vertebrate alcohol dehydrogenase are important and suggest a protective metabolic function for the whole enzyme system. Images PMID:1409630

  20. Single Nucleotide Polymorphisms and Osteoarthritis

    PubMed Central

    Wang, Ting; Liang, Yuting; Li, Hong; Li, Haibo; He, Quanze; Xue, Ying; Shen, Cong; Zhang, Chunhua; Xiang, Jingjing; Ding, Jie; Qiao, Longwei; Zheng, Qiping

    2016-01-01

    Abstract Osteoarthritis (OA) is a complex disorder characterized by degenerative articular cartilage and is largely attributed to genetic risk factors. Single nucleotide polymorphisms (SNPs) are common DNA variants that have shown promising and efficiency, compared with positional cloning, to map candidate genes of complex diseases, including OA. In this study, we aim to provide an overview of multiple SNPs from a number of genes that have recently been linked to OA susceptibility. We also performed a comprehensive meta-analysis to evaluate the association of SNP rs7639618 of double von Willebrand factor A domains (DVWA) gene with OA susceptibility. A systematic search of studies on the association of SNPs with susceptibility to OA was conducted in PubMed and Google scholar. Studies subjected to meta-analysis include human and case-control studies that met the Hardy–Weinberg equilibrium model and provide sufficient data to calculate an odds ratio (OR). A total of 9500 OA cases and 9365 controls in 7 case-control studies relating to SNP rs7639618 were included in this study and the ORs with 95% confidence intervals (CIs) were calculated. Over 50 SNPs from different genes have been shown to be associated with either hip (23), or knee (20), or both (13) OA. The ORs of these SNPs for OA and the subtypes are not consistent. As to SNP rs7639618 of DVWA, increased knee OA risk was observed in all genetic models analyzed. Specifically, people from Asian with G-allele showed significantly increased risk of knee OA (A versus G: OR = 1.28, 95% CI 1.13–1.46; AA versus GG: OR = 1.60, 95% CI 1.25–2.05; GA versus GG: OR = 1.31, 95% CI 1.18–1.44; AA versus GA+GG: OR = 1.34, 95% CI 1.12–1.61; AA+GA versus GG: OR = 1.40, 95% CI 1.19–1.64), but not in Caucasians or with hip OA. Our results suggest that multiple SNPs play different roles in the pathogenesis of OA and its subtypes; SNP rs7639618 of DVWA gene is associated with a significantly increased

  1. Structure of NADP(+)-dependent glutamate dehydrogenase from Escherichia coli--reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases.

    PubMed

    Sharkey, Michael A; Oliveira, Tânia F; Engel, Paul C; Khan, Amir R

    2013-09-01

    Glutamate dehydrogenases (GDHs; EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate, using NAD(+) and/or NADP(+) as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding domain I followed by a nucleotide-binding domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the GDH family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5-Å resolution. Modelling of NADP(+) in domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine that follows the P7 aspartate is presumed to form a hydrogen bond with the 2'-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP(+) recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD(+)-dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution to the coordination of the 2'-ribose phosphate of NADP(+). These studies clarify the sequence-structure relationships in bacterial GDHs, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual-specificity GDHs of animals.

  2. Structure of NADP+-dependent glutamate dehydrogenase from Escherichia coli - reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

    SciTech Connect

    Sharkey, Michael A.; Oliveira, Tânia F.; Engel, Paul C.; Khan, Amir R.

    2013-09-05

    Glutamate dehydrogenases catalyse the oxidative deamination of L-glutamate to α-ketoglutarate, using NAD+ and/or NADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding domain I followed by a nucleotide-binding domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the GDH family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5-Å resolution. Modelling of NADP+ in domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine that follows the P7 aspartate is presumed to form a hydrogen bond with the 2'-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP+ recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD+-dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution to the coordination of the 2'-ribose phosphate of NADP+. These studies clarify the sequence–structure relationships in bacterial GDHs, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual-specificity GDHs of animals.

  3. Purification of arogenate dehydrogenase from Phenylobacterium immobile.

    PubMed

    Mayer, E; Waldner-Sander, S; Keller, B; Keller, E; Lingens, F

    1985-01-07

    Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The Mr of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The Km values were 0.09 mM for arogenate and 0.02 mM for NAD+. The enzyme has a high specificity with respect to its substrate arogenate.

  4. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility.

  5. Inosine 5'-monophosphate dehydrogenase binds nucleic acids in vitro and in vivo.

    PubMed Central

    McLean, Jeremy E; Hamaguchi, Nobuko; Belenky, Peter; Mortimer, Sarah E; Stanton, Martin; Hedstrom, Lizbeth

    2004-01-01

    Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain. PMID:14766016

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

    PubMed

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

    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 IC50 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.

  7. Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

    PubMed Central

    Garcia, G E; Stadtman, T C

    1991-01-01

    The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon. PMID:1825826

  8. Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

    PubMed

    Garcia, G E; Stadtman, T C

    1991-03-01

    The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.

  9. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    SciTech Connect

    Smithgall, T.E.

    1986-01-01

    Carcinogenic activation of polycyclic aromatic hydrocarbons by microsomal monoxygenases proceeds through trans-dihydrodiol metabolites to diol-epoxide ultimate carcinogens. This thesis directly investigated the role of dihydrodiol dehydrogenase, a cytosolic NAD(P)-linked oxidoreductase, in the detoxification of polycyclic aromatic trans-dihydrodiols. A wide variety of non-K-region trans-dihydrodiols were synthesized and shown to be substrates for the homogeneous rat liver dehydrogenase, including several potent proximate carcinogens derived from 7,12-dimethylbenz(a)anthracene, 5-methylchrysene, and benzo(a)pyrene. Since microsomal activation of polycyclic aromatic hydrocarbons is highly stereospecific, the stereochemical course of enzymatic trans-dihydrodiol oxidation was monitored using circular dichroism spectropolarimetry. The major product formed from the dehydrogenase-catalyzed oxidation of the trans-1,2-dihydrodiol of naphthalene was characterized using UV, IR, NMR, and mass spectroscopy, and appears to be 4-hydroxy-1,2-naphthoquinone. Mass spectral analysis suggests that an analogous hydroxylated o-quinone is formed as the major product of benzo(a)pyrene-7,8-dihydrodiol oxidation. Enzymatic oxidation of trans-dihydrodiols was shown to be potently inhibited by all of the major classes of the nonsteroidal antiinflammatory drugs. Enhancement of trans-dihydrodiol proximate carcinogen oxidation may protect against possible adverse effects of the aspirin-like drugs, and help maintain the balance between activation and detoxification of polycyclic aromatic hydrocarbons.

  10. Relationships within the aldehyde dehydrogenase extended family.

    PubMed Central

    Perozich, J.; Nicholas, H.; Wang, B. C.; Lindahl, R.; Hempel, J.

    1999-01-01

    One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project. PMID:10210192

  11. Advances in targeting cyclic nucleotide phosphodiesterases

    PubMed Central

    Maurice, Donald H.; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C.

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

  12. Regulatory region with putA gene of proline dehydrogenase that links to the lum and the lux operons in Photobacterium leiognathi.

    PubMed

    Lin, J W; Yu, K Y; Chen, H Y; Weng, S F

    1996-02-27

    Nucleotide sequence of regulatory region (R & R) with putA gene (EMBL Accession No. U39227) from Photobacterium leiognathi PL741 has been determined, and the putA gene encoded amino acid sequence of proline dehydrogenase is deduced. Alignment and comparison of proline dehydrogenase of P. leiognathi with the proline dehydrogenase domain in the PutA protein of Escherichia coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that regulatory region with the putA gene is linked to the lum and lux operons in genome; the gene order is <--putA--R & R(I)<--ter-lumQ-lumP-R & R-luxC-luxD-luxA-luxB-luxE--> (R & R: regulatory region; ter:transcriptional terminator), whereas the R & R is the regulatory region for the lum and the lux operons, ter is the transcriptional terminator for the lum operon, and R & R(I) apparently is the regulatory region for the putA and related genes. Nucleotide sequence analysis illustrates the specific inverted repeat (SIR), cAMP-CRP consensus sequence, canonical -10/-35 promoter, putative operator and Shine-Dalgarno (SD) sequence on the regulatory region R & R(I) for the putA and related genes; it suggests that the putA and related genes are simply linked to the lum and the lux operons in genome, the regulatory region R & R(I) is independent for the putA and related genes.

  13. Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

    PubMed Central

    Koenig, K; Andreesen, J R

    1990-01-01

    The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases. Images PMID:2170335

  14. A type of nucleotide motif that distinguishes tobamovirus species more efficiently than nucleotide signatures.

    PubMed

    Gibbs, A J; Armstrong, J S; Gibbs, M J

    2004-10-01

    The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.

  15. Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

    PubMed

    Nimmesgern, E; Black, J; Futer, O; Fulghum, J R; Chambers, S P; Brummel, C L; Raybuck, S A; Sintchak, M D

    1999-11-01

    Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.

  16. Isolation and Expression of Lactate Dehydrogenase Genes from Rhizopus oryzae

    PubMed Central

    Skory, Christopher D.

    2000-01-01

    Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, ldhA and ldhB, which code for NAD+-dependent l-lactate dehydrogenases (LDH) (EC 1.1.1.27), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts from ldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize that ldhB encodes a second NAD+-dependent LDH that is capable of converting l-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. Both ldhA and ldhB restored fermentative growth to Escherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid. PMID:10831409

  17. First Crystal Structure of l-Lysine 6-Dehydrogenase as an NAD-dependent Amine Dehydrogenase*

    PubMed Central

    Yoneda, Kazunari; Fukuda, Junya; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2010-01-01

    A gene encoding an l-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable l-lysine dehydrogenase yet described, with a half-life of 180 min at 100 °C. The product of the enzyme's catalytic activity is Δ1-piperideine-6-carboxylate, which makes this enzyme an l-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the ϵ- amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 Å in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of l-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase. PMID:20056607

  18. Cloning and expression in Escherichia coli of a gene coding for a secondary alcohol dehydrogenase from Candida parapsilosis.

    PubMed

    Yamamoto, H; Kawada, N; Matsuyama, A; Kobayashi, Y

    1999-06-01

    A gene encoding a stereo-specific secondary alcohol dehydrogenase (CpSADH) that catalyzed the oxidation of (S)-1,3-BDO to 4-hydroxy-2-butanone was cloned from Candida parapsilosis. This CpSADH-gene consisted of 1,009 nucleotides coding for a protein with M(r) 35,964. A recombinant Escherichia coli JM109 strain harboring the expression plasmid, pKK-CPA1, produced (R)-1,3-BDO (93.5% ee., 94.7% yield) from the racemate without any additive to regenerate NAD+ from NADH.

  19. Dehydrogenase and Oxoreductase Activities of Porcine Placental 11Beta-Hydroxysteroid Dehydrogenase

    DTIC Science & Technology

    2016-06-07

    activity (p < .001). There were positive linear associations (p < . 01) between net dehydrogenase activity (dehydrogenase minus oxoreductase) and...Fragments ( ~ 3 grams ) of placentae from 7-8 fetuses from each of three gilts were removed and placed in ice cold sterile Eagle’s Minimum Essential...Females (n) Males Fetal weight ( grams ) 12 272.7 ± 20.7b 10 302.5 ± 12.8b Fetal length (mm) 12 185.9 ± 5.4 b 10 196.4± 4.8b Placental weight ( grams

  20. SVOP Is a Nucleotide Binding Protein

    PubMed Central

    Yao, Jia; Bajjalieh, Sandra M.

    2009-01-01

    Background Synaptic Vesicle Protein 2 (SV2) and SV2-related protein (SVOP) are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF) family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1). In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties. Methodology/Principal Findings We performed in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[γ] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9–12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A. Conclusions/Significance SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately. PMID:19390693

  1. Guanine nucleotide-induced polymerization of actin in electropermeabilized human neutrophils

    PubMed Central

    1989-01-01

    The effects of exogenous guanine nucleotides on the polymerization of actin in human neutrophils were tested in an electropermeabilized cell preparation. Close to 40% permeabilization was achieved with a single electric discharge as measured by nucleic acid staining with ethidium bromide or propidium iodide with minimal (less than 2%) release of the cytoplasmic marker lactate dehydrogenase. In addition, electropermeabilized neutrophils retained their capacity to produce superoxide anions and to sustain a polymerization of actin in response to surface-receptor dependent stimuli such as chemotactic factors. Electropermeabilization produced a rapid and transient permeabilization that allowed the entry of guanine nucleotides into the cells. GTP and, to a larger extent, its nonhydrolyzable analog guanosine 5'-O-2- thiotriphosphate (GTP[S]), induced a time- and concentration-dependent polymerization of actin, as determined by increased staining with 7- nitrobenz-2-oxa-1,3-diazolylphallacidin. The effects of the aforementioned guanine nucleotides were antagonized by GDP[S], but were insensitive to pertussis toxin. Cholera toxin potentiated to a small degree the amount of actin polymerization induced by GTP[S]. These results provided direct evidence for the involvement of GTP-binding proteins in the regulation of the organization of the cytoskeleton of neutrophils, an event that is of crucial importance to the performance of the defense-oriented functions of these cells. PMID:2768336

  2. Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

    PubMed

    Maurice, Donald H; Wilson, Lindsay S; Rampersad, Sarah N; Hubert, Fabien; Truong, Tammy; Kaczmarek, Milosz; Brzezinska, Paulina; Freitag, Silja I; Umana, M Bibiana; Wudwud, Alie

    2014-04-01

    The cyclic nucleotide second messengers cAMP and cGMP each affect virtually all cellular processes. Although these hydrophilic small molecules readily diffuse throughout cells, it is remarkable that their ability to activate their multiple intracellular effectors is spatially and temporally selective. Studies have identified a critical role for compartmentation of the enzymes which hydrolyse and metabolically inactivate these second messengers, the PDEs (cyclic nucleotide phosphodiesterases), in this specificity. In the present article, we describe several examples from our work in which compartmentation of selected cAMP- or cGMP-hydrolysing PDEs co-ordinate selective activation of cyclic nucleotide effectors, and, as a result, selectively affect cellular functions. It is our belief that therapeutic strategies aimed at targeting PDEs within these compartments will allow greater selectivity than those directed at inhibiting these enzymes throughout the cells.

  3. Functional characterization and transcriptional analysis of icd2 gene encoding an isocitrate dehydrogenase of Xanthomonas campestris pv. campestris.

    PubMed

    Chiang, Ying-Chuan; Liao, Chao-Tsai; Du, Shin-Chiao; Hsiao, Yi-Min

    2017-08-01

    Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. In the genome of Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, two putative IDH genes, icd1 and icd2, have been annotated. Their physiological roles in X. campestris pv. campestris are unclear. In this study, the icd2 gene from X. campestris pv. campestris was characterized in detail. We demonstrated genetically that icd2 gene encodes a functional IDH, and is involved in virulence as well as bacterial attachment. Furthermore, the icd2 transcription initiation site was mapped at nucleotide G, 127 nucleotide upstream of the icd2 translation start codon. In addition, promoter analysis revealed that icd2 expression exhibits a distinct expression profile under different culture conditions, is subjected to catabolite repression, and is affected by acetate. This is the first time that the function and transcription of icd2 have been characterized in the crucifer pathogen X. campestris pv. campestris.

  4. Inhibitory effect of disulfiram (Antabuse) on alcohol dehydrogenase activity.

    PubMed

    Carper, W R; Dorey, R C; Beber, J H

    1987-10-01

    We investigated the effect of disulfiram (Antabuse) on the activity of alcohol dehydrogenase (EC 1.1.1.1) in vitro. We observed a time-dependent inhibition of this dehydrogenase by disulfiram and diethyldithiocarbamate similar to that obtained for aldehyde dehydrogenase (EC 1.2.1.3). These results suggest a possible explanation for various side effects observed in the clinical use of Antabuse.

  5. Proofreading of misincorporated nucleotides in DNA transcription

    NASA Astrophysics Data System (ADS)

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B.

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighboring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction.

  6. Proofreading of misincorporated nucleotides in DNA transcription

    NASA Astrophysics Data System (ADS)

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B.

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighbouring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction.

  7. Inhibition of membrane-bound succinate dehydrogenase by disulfiram.

    PubMed

    Jay, D

    1991-04-01

    The effect of disulfiram on succinate oxidase and succinate dehydrogenase activities of beef heart submitochondrial particles was studied. Results show that disulfiram inhibits both functions. Succinate and malonate suppress the inhibitory action of disulfiram when succinate dehydrogenase is stabilized in an active conformation. Disulfiram is not able to inhibit the enzyme when succinate dehydrogenase is inactivated by oxaloacetate. The inhibitory effect of disulfiram is reverted by the addition of dithiothreitol. From these results, it is proposed that disulfiram inhibits the utilization of succinate by a direct modification of an -SH group located in the catalytically active site of succinate dehydrogenase.

  8. A lipoamide dehydrogenase from Neisseria meningitidis has a lipoyl domain.

    PubMed

    Bringas, R; Fernandez, J

    1995-04-01

    A protein of molecular weight of 64 kDa (p64k) found in the outer membrane of Neisseria meningitidis shows a high degree of homology with both the lipoyl domain of the acetyltransferase and the entire sequence of the lipoamide dehydrogenase, the E2 and E3 components of the dehydrogenase multienzyme complexes, respectively. The alignment of the p64k with lipoyl domains and lipoamide dehydrogenases from different species is presented. The possible implications of this protein in binding protein-dependent transport are discussed. This is the first lipoamide dehydrogenase reported to have a lipoyl domain.

  9. Placental glucose dehydrogenase polymorphism in Koreans.

    PubMed

    Kim, Y J; Paik, S G; Park, H Y

    1994-12-01

    The genetic polymorphism of placental glucose dehydrogenase (GDH) was investigated in 300 Korean placentae using horizontal starch gel electrophoresis. The allele frequencies for GDH1, GDH2 and GDH3 were 0.537, 0.440 and 0.005, respectively, which were similar to those in Japanese. We also observed an anodal allele which was similar to the GDH4 originally reported in Chinese populations at a low frequency of 0.015. An additional new cathodal allele (named GDH6) was observed in the present study with a very low frequency of 0.003.

  10. Spectra of glutamate dehydrogenase with diethylstilbestrol.

    PubMed

    Hillar, M

    1978-02-01

    Glutamate dehydrogenase displays hyperchromicity at 256 nm and at 276 nm upon binding of diethylstilbestrol. Increase in absorbancy is linear at both regions up to 250 micrometer DES, and becomes parabolic at higher concentration of DES. ADP in the presence of DES causes decrease in absorbancy at 256 nm; absorbancy at 276 nm increased by DES is not affected by ADP. DES prevents spectral effects produced by GTP (decrease in absorbancy at 254 nm and at 276 nm). ADP still decreases absorbancy at 254 nm, leaving the 276 nm region unchanged. ADP enhances spectral effects produced by GTP. GTP, however, prevents changes produced by ADP.

  11. Hydrogenases and formate dehydrogenases of Syntrophobacter fumaroxidans.

    PubMed

    de Bok, F A M; Roze, E H A; Stams, A J M

    2002-08-01

    The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases. All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm. These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations. These findings suggest that both H2 and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate.

  12. Identity of the subunits and the stoicheiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase.

    PubMed Central

    Kasprzak, A A; Papas, E J; Steenkamp, D J

    1983-01-01

    Trimethylamine dehydrogenases from bacterium W3A1 and Hyphomicrobium X and the dimethylamine dehydrogenase from Hyphomicrobium X were found to contain only one kind of subunit. The millimolar absorption coefficient of a single [4Fe-4S] cluster in trimethylamine dehydrogenase from bacterium W3A1 was estimated to be 14.8 mM-1 . cm-1 at 443 nm. From this value a 1:1 stoicheiometry of the prosthetic groups, 6-S-cysteinyl-FMN and the [4Fe-4S] cluster, was established. Millimolar absorption coefficients of the three enzymes were in the range 49.4-58.7 mM-1 . cm-1 at approx. 440 nm. This range of values is consistent with the presence of two [4Fe-4S] clusters and two flavin residues, for which the millimolar absorption coefficient had earlier been found to be 12.3 mM-1 . cm-1 at 437 nm. The N-terminal amino acid was alanine in each of the three enzymes. Sequence analysis of the first 15 residues from the N-terminus of dimethylamine dehydrogenase indicated a single unique sequence. Two identical subunits, each containing covalently bound 6-S-cysteinyl-FMN and a [4Fe-4S] cluster, in each of the enzymes are therefore indicated. Images Fig. 1. PMID:6882357

  13. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules

    PubMed Central

    Sesma, Juliana I.; Kreda, Silvia M.; Okada, Seiko F.; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C.; O'Neal, Wanda K.; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori

    2013-01-01

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins. PMID:23467297

  14. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

    PubMed

    Sesma, Juliana I; Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C; O'Neal, Wanda K; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori; Lazarowski, Eduardo R

    2013-05-15

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

  15. Expansion of the aspartate [beta]-semialdehyde dehydrogenase family: the first structure of a fungal ortholog

    SciTech Connect

    Arachea, B.T.; Liu, X.; Pavlovsky, A.G.; Viola, R.E.

    2010-08-13

    The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a critical transformation that produces the first branch-point intermediate in an essential microbial amino-acid biosynthetic pathway. The first structure of an ASADH isolated from a fungal species (Candida albicans) has been determined as a complex with its pyridine nucleotide cofactor. This enzyme is a functional dimer, with a similar overall fold and domain organization to the structurally characterized bacterial ASADHs. However, there are differences in the secondary-structural elements and in cofactor binding that are likely to cause the lower catalytic efficiency of this fungal enzyme. Alterations in the dimer interface, through deletion of a helical subdomain and replacement of amino acids that participate in a hydrogen-bonding network, interrupt the intersubunit-communication channels required to support an alternating-site catalytic mechanism. The detailed functional information derived from this new structure will allow an assessment of ASADH as a possible target for antifungal drug development.

  16. Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei.

    PubMed

    Díaz, Susana; Pérez-Pomares, Francisco; Pire, Carmen; Ferrer, Juan; Bonete, María-José

    2006-04-01

    The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.

  17. Inhibition effects of some metal ions on the rat liver 6-phosphogluconate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Adem, Şevki; Kayhan, Naciye

    2016-04-01

    6-phosphogluconate dehydrogenase is an enzyme in the pentose phosphate path. The main functions of the pathway are the manufacture of the reduced coenzyme NADPH and the formation of ribose 5-phosphate for nucleic acid synthesis and nucleotide. Both NADPH and ribose 5-phosphate involve a critical biochemical process. Metals have been recognized as important toxic agents for living for a long time. It has been considered that they lead to in the emergence of many diseases. To evaluate whether metals is effect towards rat liver 6PGD, we apply various concentrations of metals and enzyme inhibition was analyzed using enzyme activity assays. The IC50 values of Pb+2, Cr+3, Co+2, Ni+2, Cd+2, and Va+2, metals on rat liver 6PGD were calculated as 138,138, 169, 214, 280, and 350 µM, respectively.

  18. Nucleotide Metabolism in Salt-Stressed Zea mays L. Root Tips: I. Adenine and Uridine Nucleotides.

    PubMed

    Peterson, T A; Nieman, R H; Clark, R A

    1987-12-01

    Corn plants (Zea mays L. cv Pioneer 3906) were grown in a glass house on control and saline nutrient solutions, in winter and summer. There were two saline treatments, both with osmotic potential = -0.4 megapascal but with different Ca(2+)/Na(+) ratios: 0.03 and 0.73. Root tips and shoot meristems (culm tissue) of 26 day-old plants were analyzed for nucleotides to ascertain if there were correlations between nucleotide pool size and the reduced growth on saline cultures. Several other cell components also were determined. Plants grown in winter were only half as large as those grown in summer mainly because of the lower light intensity and lower temperature. But the relative yield reduction on salt treatment compared to the control was similar in winter and summer. The two different salt treatments caused similar yield reductions. Neither salt treatment affected nucleotide pools in culm tissue, with the possible exception of UDPG in winter. In the case of root tips, salt treatment had little or no effect on nucleotide pool sizes in winter when many already seemed near a critical minimum, but in summer it reduced several pools including ATP, total adenine nucleotide, UTP, total uridine nucleotide, and UDP-glucose. The reductions were greatest on the salt treatment with low Ca(2+)/Na(+). There was no simple correlation between the effects of salt stress on growth and on nucleotide pool size. The nucleotide pools of culm tissue indicated that in some respects this tissue was effectively insulated from the salt stress. Roots that were in direct contact with the saline solution indicated significant reductions in nucleotide pools only in the summer whereas growth was reduced both summer and winter. It is possible that the nucleotide concentrations of root cells in winter were already near a critical minimum so that nucleotide synthesis and growth were tightly linked. Significant reductions in nucleotide pools that would be expected to affect growth were more evident in

  19. The International Nucleotide Sequence Database Collaboration

    PubMed Central

    Cochrane, Guy; Karsch-Mizrachi, Ilene; Takagi, Toshihisa; Sequence Database Collaboration, International Nucleotide

    2016-01-01

    The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org) comprises three global partners committed to capturing, preserving and providing comprehensive public-domain nucleotide sequence information. The INSDC establishes standards, formats and protocols for data and metadata to make it easier for individuals and organisations to submit their nucleotide data reliably to public archives. This work enables the continuous, global exchange of information about living things. Here we present an update of the INSDC in 2015, including data growth and diversification, new standards and requirements by publishers for authors to submit their data to the public archives. The INSDC serves as a model for data sharing in the life sciences. PMID:26657633

  20. The International Nucleotide Sequence Database Collaboration.

    PubMed

    Nakamura, Yasukazu; Cochrane, Guy; Karsch-Mizrachi, Ilene

    2013-01-01

    The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org), one of the longest-standing global alliances of biological data archives, captures, preserves and provides comprehensive public domain nucleotide sequence information. Three partners of the INSDC work in cooperation to establish formats for data and metadata and protocols that facilitate reliable data submission to their databases and support continual data exchange around the world. In this article, the INSDC current status and update for the year of 2012 are presented. Among discussed items of international collaboration meeting in 2012, BioSample database and changes in submission are described as topics.

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

    SciTech Connect

    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

    2015-01-09

    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. As a result, these findings offer a potential strategy for further ligand optimization.

  2. The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation

    PubMed Central

    Kiss, Gergely; Konrad, Csaba; Doczi, Judit; Starkov, Anatoly A.; Kawamata, Hibiki; Manfredi, Giovanni; Zhang, Steven F.; Gibson, Gary E.; Beal, M. Flint; Adam-Vizi, Vera; Chinopoulos, Christos

    2013-01-01

    A decline in α-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20–48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ∼30% higher ADP-ATP exchange rates compared to those obtained from DLST+/− or DLD+/− littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on “in-house” mitochondrial ATP reserves.—Kiss, G., Konrad, C., Doczi, J., Starkov, A. A., Kawamata, H., Manfredi, G., Zhang, S. F., Gibson, G. E., Beal, M. F., Adam-Vizi, V., Chinopoulos, C. The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation. PMID:23475850

  3. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  4. Kinetic mechanism of chicken liver xanthine dehydrogenase.

    PubMed Central

    Bruguera, P; Lopez-Cabrera, A; Canela, E I

    1988-01-01

    The kinetic behaviour of chicken-liver xanthine dehydrogenase (xanthine/NAD+ oxidoreductase; EC 1.2.1.37) has been studied. Steady-state results, obtained from a wide range of concentrations of substrates and products, were fitted by rational functions of degree 1:1, 1:2, 2:2 and 3:3 with respect to substrates, and 0:1, 1:1, 0:2 and 1:2 with regard to products, using a non-linear regression program which guarantees the fit. The goodness of fit was improved using a computer program that combines model discrimination, parameter refinement and sequential experimental design. The AIC and F tests were also used for model discrimination. For comparative purposes, the xanthine/oxygen oxidoreductase reaction was also studied. From the functions which give the maximum improvement, the complete rate equation was deduced. The significance of the terms was stated by the above methods. It was concluded that xanthine dehydrogenase requires a minimum mechanism of degree 1:1 for xanthine, 2:2 for NAD+, 1:1 for uric acid and 1:2 for NADH in the xanthine/NAD+ oxidoreductase reaction. These are the minimum degrees required but a rate equation of higher degree is not excluded. PMID:3422556

  5. Properties of a Purified Halophilic Malic Dehydrogenase

    PubMed Central

    Holmes, P. K.; Halvorson, H. Orin

    1965-01-01

    Holmes, P. K. (University of Illinois, Urbana), and H. Orin Halvorson. Properties of a purified halophilic malic dehydrogenase. J. Bacteriol. 90:316–326. 1965.—The malic dehydrogenase (MDH) from Halobacterium salinarium required high concentrations of monovalent ions for stability and activity. Studies of inactivation rates at different salt concentrations suggested that approximately 25% NaCl (w/v) is required to stabilize MDH. From 50 to 100% reactivation, depending on the salt concentration present during inactivation, could occur in 2.5 to 5 m NaCl or KCl. The optimal salt concentration for activity of MDH was a function of the pH, and ranged from 1 to 3 m NaCl or KCl. The effect of salt concentration on the pH-activity curves occurred chiefly below pH 7.0. Inactivation of MDH with heat or thiol reagents showed that the enzyme was more labile in the state induced by absence of salt. The activation of MDH by salts was attributed to a decreased rate of dissociation of MDH and reduced nicotinamide adenine dinucleotide (NADH2). The inactivation of the enzyme in the absence of salt could be largely prevented by the presence of NADH2. The S20.w of MDH decreased threefold at low salt concentrations. The enzyme was assumed to be in its native compact configuration only in the presence of a high concentration of salt. PMID:14329442

  6. Structure of glycerol dehydrogenase from Serratia.

    PubMed

    Musille, Paul; Ortlund, Eric

    2014-02-01

    The 1.90 Å resolution X-ray crystal structure of glycerol dehydrogenase derived from contaminating bacteria present during routine Escherichia coli protein expression is presented. This off-target enzyme showed intrinsic affinity for Ni(2+)-Sepharose, migrated at the expected molecular mass for the target protein during gel filtration and was crystallized before it was realised that contamination had occurred. In this study, it is shown that liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) can efficiently identify the protein composition of crystals in a crystallization experiment as part of a structure-determination pipeline for an unknown protein. The high-resolution X-ray data enabled sequencing directly from the electron-density maps, allowing the source of contamination to be placed within the Serratia genus. Incorporating additional protein-identity checks, such as tandem LC-MS/MS, earlier in the protein expression, purification and crystallization workflow may have prevented the unintentional structure determination of this metabolic enzyme, which represents the first enterobacterial glycerol dehydrogenase reported to date.

  7. Crystallization and preliminary X-ray diffraction analysis of Leishmania major dihydroorotate dehydrogenase

    SciTech Connect

    Cordeiro, Artur T.; Feliciano, Patricia R.; Nonato, M. Cristina

    2006-10-01

    Dihydroorotate dehydrogenase from L. major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitant agent. A complete data set from a native crystal has been collected to 2.0 Å resolution using an in-house rotating-anode generator. Dihydroorotate dehydrogenases (DHODHs) are flavin-containing enzymes that catalyze the oxidation of l-dihydroorotate to orotate, the fourth step in the de novo pyrimidine nucleotide synthesis pathway. In this study, DHODH from Leishmania major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitating agent. The crystals belong to space group P6{sub 1}, with unit-cell parameters a = 143.7, c = 69.8 Å. X-ray diffraction data were collected to 2.0 Å resolution using an in-house rotating-anode generator. Analysis of the solvent content and the self-rotation function indicate the presence of two molecules in the asymmetric unit. The structure has been solved by the molecular-replacement technique.

  8. The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation.

    PubMed

    Kiss, Gergely; Konrad, Csaba; Doczi, Judit; Starkov, Anatoly A; Kawamata, Hibiki; Manfredi, Giovanni; Zhang, Steven F; Gibson, Gary E; Beal, M Flint; Adam-Vizi, Vera; Chinopoulos, Christos

    2013-06-01

    A decline in α-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20-48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ~30% higher ADP-ATP exchange rates compared to those obtained from DLST(+/-) or DLD(+/-) littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on "in-house" mitochondrial ATP reserves.

  9. Catecholamine regulation of lactate dehydrogenase in rat brain cell culture

    SciTech Connect

    Kumar, S.; McGinnis, J.F.; de Vellis, J.

    1980-03-25

    The mechanism of catecholamine induction of the soluble cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27) was studied in the rat glial tumor cell line, C6. Lactate dehydrogenase was partially purified from extracts of (/sup 3/H)leucine-labeled cells by affinity gel chromatography and quantitatively immunoprecipitated with anti-lactate dehydrogenase-5 IgG and with antilactate dehydrogenase-1 IgG. The immunoprecipitates were dissociated and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Using this methodology, the increased enzyme activity of lactate dehydrogenase in norepinephrine-treated C6 cells was observed to be concomitant with the increased synthesis of enzyme molecules. Despite the continued presence of norepinephrine, the specific increase in the rate of synthesis of lactate dehydrogenase was transient. It was first detected at 4 h, was maximum at 9 h, and returned to basal levels by 24 h. The half-life of lactate dehydrogenase enzyme activity was 36 h during the induction and 40 h during deinduction. The half-life for decay of /sup 3/H-labeled lactate dehydrogenase was 41 h. These observations suggest that the increase in lactate dehydrogenase activity in norepinephrine-treated cells does not involve any change in the rate of degradation. Norepinephrine increased the specific rate of synthesis of both lactate dehydrogenase-5 (a tetramer of four M subunits) and lactate dehydrogenase-1 (a tetramer of four H subunits), although to different extents. Since these subunits are coded for by two separate genes on separate chromosomes, it suggests that the regulatory mechanism involves at least two separate sites of action.

  10. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. A chemical proteomic probe for detecting dehydrogenases: catechol rhodanine.

    PubMed

    Ge, Xia; Sem, Daniel S

    2012-01-01

    Inherent complexity of the proteome often demands that it be studied as manageable subsets, termed subproteomes. A subproteome can be defined in a number of ways, although a pragmatic approach is to define it based on common features in an active site that lead to binding of a common small molecule ligand (e.g., a cofactor or a cross-reactive drug lead). The subproteome, so defined, can be purified using that common ligand tethered to a resin, with affinity chromatography. Affinity purification of a subproteome is described in the next chapter. That subproteome can then be analyzed using a common ligand probe, such as a fluorescent common ligand that can be used to stain members of the subproteome in a native gel. Here, we describe such a fluorescent probe, based on a catechol rhodanine acetic acid (CRAA) ligand that binds to dehydrogenases. The CRAA ligand is fluorescent and binds to dehydrogenases at pH > 7, and hence can be used effectively to stain dehydrogenases in native gels to identify what subset of proteins in a mixture are dehydrogenases. Furthermore, if one is designing inhibitors to target one or more of these dehydrogenases, the CRAA staining can be performed in a competitive assay format, with or without inhibitor, to assess the selectivity of the inhibitor for the targeted dehydrogenase. Finally, the CRAA probe is a privileged scaffold for dehydrogenases, and hence can easily be modified to increase affinity for a given dehydrogenase.

  12. Toxic Neuronal Death by Glyeraldehyde-3-Phosphate Dehydrogenase and Mitochondria

    DTIC Science & Technology

    2003-08-01

    Neuroreport, 10(5), 1149-1153. Sioud, M., & Jespersen, L. (1996). Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase...1996) Enhancemen t of hammerhead r ibozyme cata lysis by glycera ldehyde-3- phospha te dehydrogenase. J Mol Biol 257:775–789. Sirover MA (1997) Role of

  13. Regulation of innate immunity by extracellular nucleotides

    PubMed Central

    Gorini, Stefania; Gatta, Lucia; Pontecorvo, Laura; Vitiello, Laura; la Sala, Andrea

    2013-01-01

    Extracellular ATP (eATP) is the most abundant among extracellular nucleotides and is commonly considered as a classical danger signal, which stimulates immune responses in the presence of tissue injury. In fact, increased nucleotide concentration in the extracellular space is generally closely associated with tissue stress or damage. However non-lytic nucleotide release may also occur in many cell types under a variety of conditions. Extracellular nucleotides are sensed by a class of plasma membrane receptors called P2 purinergic receptors (P2Rs). P2 receptors are expressed by all immunological cells and their activation elicits different responses. Extracellular ATP can act as an initiator or terminator of immune responses being able to induce different effects on immune cells depending on the pattern of P2 receptors engaged, the duration of the stimulus and its concentration in the extracellular milieu. Millimolar (high) concentrations of extracellular ATP, induce predominantly proinflammatory effects, while micromolar (low) doses exert mainly tolerogenic/immunosuppressive action. Moreover small, but significant differences in the pattern of P2 receptor expression in mice and humans confer diverse capacities of ATP in regulating the immune response. PMID:23358447

  14. Crystal structures of type III{sub H} NAD-dependent D-3-phosphoglycerate dehydrogenase from two thermophiles

    SciTech Connect

    Kumar, S.M.; Pampa, K.J.; Manjula, M.; Hemantha Kumar, G.; Kunishima, Naoki; Lokanath, N.K.

    2014-08-15

    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{sub 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{sub H} and such PGDHs structures having this type are reported for the first time.

  15. Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

    PubMed Central

    Gilbert, H J; Lowe, C R; Drabble, W T

    1979-01-01

    Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. PMID:44191

  16. Conformations of Diphosphopyridine Coenzymes upon Binding to Dehydrogenases

    PubMed Central

    Lee, Chi-Yu; Eichner, Ronald D.; Kaplan, Nathan O.

    1973-01-01

    The binding of oxidized as well as reduced coenzyme to some dehydrogenases has been studied under different concentration ratios and temperatures by nuclear magnetic resonance spectroscopy. A significant difference in the spectral behavior between DPN+ and DPNH upon binding is interpreted in terms of fast and slow on-off rates relative to the nuclear magnetic resonance time scale in the binding of these two coenzymes. Significant downfield shifts of DPN+ were observed upon binding, comparable in magnitude to those expected upon opening (destacking) of the coenzymes in the case of chicken-muscle and lobster-tail lactate dehydrogenase (EC 1.1.1.27) and yeast alchol dehydrogenase (EC 1.1.1.1.). A preliminary survey of several other dehydrogenases is consistent with these findings. In the case of 3-phosphoglyceraldehyde dehydrogenase, there is a possibility that the coenzyme exists in the folded form. PMID:4351183

  17. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  18. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

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

    PubMed Central

    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 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. PMID:24970175

  20. The dihydroorotate dehydrogenases: Past and present.

    PubMed

    Reis, Renata A G; Calil, Felipe Antunes; Feliciano, Patricia Rosa; Pinheiro, Matheus Pinto; Nonato, M Cristina

    2017-06-27

    The flavoenzyme dihydroorotate dehydrogenase catalyzes the stereoselective oxidation of (S)-dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine metabolism by selectively targeting DHODHs has been exploited in the development of new therapies against cancer, immunological disorders, bacterial and viral infections, and parasitic diseases. Through a chronological narrative, this review summarizes the efforts of the scientific community to achieve our current understanding of structural and biochemical properties of DHODHs. It also attempts to describe the latest advances in medicinal chemistry for therapeutic development based on the selective inhibition of DHODH, including an overview of the experimental techniques used for ligand screening during the process of drug discovery. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. NADH electrochemical sensor coupled with dehydrogenase enzymes

    SciTech Connect

    Yamanaka, Hideko; Mascini, Marco )

    1992-06-01

    A graphite electrode assembled in a flow cell has shown to be a good detector for NADH. Current is linearly dependent on concentration in the range 10{sup {minus}7}-10{sup {minus}3} M without any mediator at the potential applied of 300 mV vs Ag/AgCl. Lactate and alcohol dehydrogenases were immobilized near to the electrode surface or in a reactor to obtain an NADH-based biosensor for lactate or ethanol. With lactate the authors succeeded to obtain a response only if the reactor was used and for alcohol a current proportional to the concentration was obtained either if the enzyme was immobilized in a membrane and placed near the electrode surface or when the enzyme was immobilized in a reactor form. By FIA procedures fast responses and recoveries were obtained, but with a short linear range.

  2. Fast internal dynamics in alcohol dehydrogenase

    SciTech Connect

    Monkenbusch, M.; Stadler, A. Biehl, R.; Richter, D.; Ollivier, J.; Zamponi, M.

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  3. Lactate dehydrogenase isoenzyme patterns in cetaceans.

    PubMed

    Reidarson, T H; McBain, J; Dalton, L M

    1999-06-01

    Serum lactate dehydrogenase (LDH) isoenzyme activity was analyzed in cetaceans. Animals that were treated by i.m. injection and others that received azole therapy had distinctly different LDH isoenzyme profiles. A third distinctive pattern was occasionally observed in clinically normal animals with elevations in total transaminase and LDH activity levels. DH isoenzyme activity patterns were not affected by mild or moderate hemolysis, refrigeration after 24 hr, or freezing for 24 hr with subsequent thawing. However, severe hemolysis produced artifactual changes similar to those observed in individuals that received injections but of a lesser magnitude. DH isoenzyme activity patterns may provide useful corroboration of other clinical findings when diagnostic modalities are limited, especially to differentiate nonspecific enzyme elevation from nonpathologic elevations in serum enzyme concentrations due to i.m. injections or azole therapy.

  4. Stability of immobilized yeast alcohol dehydrogenase

    SciTech Connect

    Ooshima, H.; Genko, Y.; Harano, Y.

    1981-12-01

    The effects of substrate on stabilities of native (NA) and three kinds of immobilized yeast alcohol dehydrogenase (IMA), namely PGA (the carrier; porous glass), SEA (agarose gel) prepared covalently, and AMA (anion-exchange resin) prepared ionically, were studied. The following results were obtained. 1) The deactivations of NA and IMA free from the substrate or in the presence of ethanol obey the first-order kinetics, whereas, in the presence of butyraldehyde, their deactivation behaviors are explained on the basis of coexistence of two components of YADHs, namely the labile E1 and the comparatively stable E2, with different first-order deactivation constants. (2) A few attempts for stabilization of IMA were carried out from the viewpoint of the effects of crosslinkages among the subunits of YADH for PGA and the multibonding between the carrier and enzyme for SEA. The former is effective for the stabilization, whereas the latter is not. (Refs. 19).

  5. [Glucose-6-phosphate dehydrogenase deficiency in Japan].

    PubMed

    Kanno, Hitoshi; Ogura, Hiromi

    2015-07-01

    In the past 10 years, we have diagnosed congenital hemolytic anemia in 294 patients, approximately 33% of whom were found to have glucose-6-phosphate dehydrogenase (G6PD) deficiency. It is becoming more common for Japanese to marry people of other ethnic origins, such that G6PD deficiency is becoming more prevalent in Japan. Japanese G6PD deficiency tends to be diagnosed in the neonatal period due to severe jaundice, while G6PD-deficient patients with foreign ancestors tend to be diagnosed at the onset of an acute hemolytic crisis before the age of six. It is difficult to predict the clinical course of each patient by G6PD activity, reduced glutathione content, or the presence/absence of severe neonatal jaundice. We propose that both neonatal G6PD screening and systematic analyses of G6PD gene mutations may be useful for personalized management of patients with G6PD-deficient hemolytic anemia.

  6. Mitochondrial aldehyde dehydrogenase and cardiac diseases

    PubMed Central

    Chen, Che-Hong; Sun, Lihan; Mochly-Rosen, Daria

    2010-01-01

    Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular ‘aldehydic load’. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases. PMID:20558439

  7. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  8. Fast internal dynamics in alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Monkenbusch, M.; Stadler, A.; Biehl, R.; Ollivier, J.; Zamponi, M.; Richter, D.

    2015-08-01

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  9. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase in salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.

  10. Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene.

    PubMed

    De Zoysa, P A; Connerton, I F; Watson, D C; Johnston, J R

    1991-08-01

    The cloned NADP-specific glutamate dehydrogenase (GDH) genes of Aspergillus nidulans (gdhA) and Neurospora crassa (am) have been shown to hybridize under reduced stringency conditions to genomic sequences of the yeast Schwanniomyces occidentalis. Using 5' and 3' gene-specific probes, a unique 5.1 kb BclI restriction fragment that encompasses the entire Schwanniomyces sequence has been identified. A recombinant clone bearing the unique BclI fragment has been isolated from a pool of enriched clones in the yeast/E. coli shuttle vector pWH5 by colony hybridization. The identity of the plasmid clone was confirmed by functional complementation of the Saccharomyces cerevisiae gdh-1 mutation. The nucleotide sequence of the Schw. occidentalis GDH gene, which consists of 1380 nucleotides in a continuous reading frame of 459 amino acids, has been determined. The predicted amino acid sequence shows considerable homology with GDH proteins from other fungi and significant homology with all other available GDH sequences.

  11. Accessibility of adenine binding sites in dehydrogenases to small molecules studied by fluorescence quenching.

    PubMed

    Gafni, A

    1979-04-17

    Quenching of the fluorescence of ethenoadenine derivatives by iodide ions and by methionine was studied in solution and when the nucleotides were bound to several dehydrogenases. The fluorescence of epsilonADPR in neutral aqueous solution is dynamically quenched by both quenching agents. The quenching of free epsilonNAD+ by methionine was found to be predominantly static and was satisfactorily described to result from complex formation between quencher and dinucleotide. The rat constant for quenching by iodide of epsilonNAD+ in the ternary complex with LADH and pyrazole is comparable to that of free epsilonADPR or epsilonADP. it is concluded that the bound epsilon-adenine ring is partially exposed to the solvent. The opening, to the solvent, of the adenine binding site is not large enough to allow free methionine diffusion since the rate constant for quenching of bound coenzyme by this quenching agent is relatively small. The difference between the rate constants for quenching of free and enzyme bound nucleotide was used to evaluate the binding constants of epsilonADPR to GPDH, epsilonNAD+ to LDH, and oxalate to the LDH:epsilonNAD+ complex. This technique may prove to be particularly useful when the binding of a fluorescent ligand to a protein is not accompanied by significant changes in its fluorescence.

  12. Cloning, characterization, and regulation of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.A.; Huberman, E. |

    1997-01-01

    Human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. Regulated IMPDH activity is associated with cellular proliferation, transformation, and differentiation. The authors cloned and sequenced the entire gene for type II IMPDH and here provide details regarding the organization of the gene and the characterization of its promoter. The gene spans approximately 5 kb and is disrupted by 12 introns. The transcriptional start sites were determined by S1 nuclease mapping to be somewhat heterogeneous but predominated at 102 and 85 nucleotides from the translational initiation codon. Through the use of heterologous gene constructs and transient transfection assays, a minimal promoter from {minus}206 to {minus}85 was defined. This promoter is TATA-less and contains several transcription factor motifs including four potential Sp 1 binding sites. The minimal promoter is GC-rich (69%) and resembles a CpG island. Through the use of gel mobility shift assays, nuclear proteins were shown to specifically interact with this minimal promoter. Stable transfectants were used to demonstrate that the down-regulation of IMPDH gene expression in response to reduced cellular proliferation occurs by a transcriptional mechanism.

  13. The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

    PubMed Central

    Dalziel, K.; Dickinson, F. M.

    1965-01-01

    1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH2 of high purity, the maximal specific rates were similar to those obtained with NAD and NADH2, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH2, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH2 do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH2. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH2 than NAD and NADH2. 5. Phosphate behaves as a competitive inhibitor towards NADP. PMID:14340079

  14. Kinetic studies of dogfish liver glutamate dehydrogenase.

    PubMed Central

    Electricwala, A H; Dickinson, F M

    1979-01-01

    Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme. PMID:35153

  15. Variants of glycerol dehydrogenase having D-lactate dehydrogenase activity and uses thereof

    DOEpatents

    Wang, Qingzhao; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2017-08-29

    The present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide. Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.

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

    PubMed Central

    2009-01-01

    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. PMID:19164088

  17. Deficiency of dihydrolipoamide dehydrogenase due to two mutant alleles (E340K and G101del). Analysis of a family and prenatal testing.

    PubMed

    Hong, Y S; Kerr, D S; Liu, T C; Lusk, M; Powell, B R; Patel, M S

    1997-12-31

    A male child with metabolic acidosis was diagnosed as having dihydrolipoamide dehydrogenase (E3) deficiency. E3 activity of the proband's cultured fibroblasts and blood lymphocytes was 3-9% of normal, while in the parent's lymphocytes it was about 60% of normal. The proband's pyruvate dehydrogenase complex (PDC) and the alpha-ketoglutarate dehydrogenase complex activities from cultured skin fibroblasts were 12% and 6% of normal, respectively. PDC activity in the parents cultured fibroblasts was 25-31% of normal. Western and Northern blot analyses showed similar quantities of E3 protein and mRNA in cultured fibroblasts from the proband and his parents. DNA sequencing of cloned full-length E3 cDNAs, from the proband and the parents, showed two mutations on different alleles of proband were inherited from the parents. One mutation is a three nucleotide (AGG) deletion, from the mother, resulting in deletion of Gly101 in the FAD binding domain. The other mutation is a nucleotide substitution (G to A), from the father, leading to substitution of Lys for Glu340 in the central domain. The same deletion mutation was found in E3 cDNA from a chorionic villus sample and cultured fibroblasts obtained from the mother's subsequent offspring. This finding illustrates the possibility of successful prenatal diagnosis of E3 deficiency utilizing mutations characterized prior to initiation of pregnancy.

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

    PubMed

    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.

  19. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes.

  20. Nucleotide sequence of papaya mosaic virus RNA.

    PubMed

    Sit, T L; Abouhaidar, M G; Holy, S

    1989-09-01

    The RNA genome of papaya mosaic virus is 6656 nucleotides long [excluding the poly(A) tail] with six open reading frames (ORFs) more than 200 nucleotides long. The four nearest the 5' end each overlap with adjacent ORFs and could code for proteins with Mr 176307, 26248, 11949 and 7224 (ORFs 1 to 4). The fifth ORF produces the capsid protein of Mr 23043 and the sixth ORF, located completely within ORF1, could code for a protein with Mr 14113. The translation products of ORFs 1 to 3 show strong similarity with those of other potexviruses but the ORF 4 protein has only limited similarity with the other potexvirus ORF 4 proteins of 7K to 11K.

  1. Radiation and thermal stabilities of adenine nucleotides.

    PubMed

    Demidov, V V; Potaman, V N; Solyanina, I P; Trofimov, V I

    1995-03-01

    We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

  2. Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

    PubMed Central

    Lazo, P A; Sols, A

    1980-01-01

    1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA. PMID:7193456

  3. Mass spectrometry analysis of nucleosides and nucleotides.

    PubMed

    Dudley, Ed; Bond, Liz

    2014-01-01

    Mass spectrometry has been widely utilised in the study of nucleobases, nucleosides and nucleotides as components of nucleic acids and as bioactive metabolites in their own right. In this review, the application of mass spectrometry to such analysis is overviewed in relation to various aspects regarding the analytical mass spectrometric and chromatographic techniques applied and also the various applications of such analysis. © 2013 Wiley Periodicals, Inc.

  4. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    SciTech Connect

    Shin, Joong-Won; Bernstein, Elliot R.

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  5. Isolation of fast purine nucleotide synthase ribozymes.

    PubMed

    Lau, Matthew W L; Cadieux, Kelly E C; Unrau, Peter J

    2004-12-08

    Here we report the in vitro selection of fast ribozymes capable of promoting the synthesis of a purine nucleotide (6-thioguanosine monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine ((6S)Gua). The two most proficient purine synthases have apparent efficiencies of 284 and 230 M(-1) min(-1) and are both significantly more efficient than pyrimidine nucleotide synthase ribozymes selected previously by a similar approach. Interestingly, while both ribozymes showed good substrate discrimination, one ribozyme had no detectable affinity for 6-thioguanine while the second had a K(m) of approximately 80 muM, indicating that these ribozymes use considerably different modes of substrate recognition. The purine synthases were isolated after 10 rounds of selection from two high-diversity RNA pools. The first pool contained a long random sequence region. The second pool contained random sequence elements interspersed with the mutagenized helical elements of a previously characterized 4-thiouridine synthase ribozyme. While nearly all of the ribozymes isolated from this biased pool population appeared to have benefited from utilizing one of the progenitor's helical elements, little evidence for more complicated secondary structure preservation was evident. The discovery of purine synthases, in addition to pyrimidine synthases, demonstrates the potential for nucleotide synthesis in an 'RNA World' and provides a context from which to study small molecule RNA catalysis.

  6. Evolution of functional six-nucleotide DNA.

    PubMed

    Zhang, Liqin; Yang, Zunyi; Sefah, Kwame; Bradley, Kevin M; Hoshika, Shuichi; Kim, Myong-Jung; Kim, Hyo-Joong; Zhu, Guizhi; Jiménez, Elizabeth; Cansiz, Sena; Teng, I-Ting; Champanhac, Carole; McLendon, Christopher; Liu, Chen; Zhang, Wen; Gerloff, Dietlind L; Huang, Zhen; Tan, Weihong; Benner, Steven A

    2015-06-03

    Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

  7. Nucleotide excision repair in Escherichia coli.

    PubMed Central

    Van Houten, B

    1990-01-01

    One of the best-studied DNA repair pathways is nucleotide excision repair, a process consisting of DNA damage recognition, incision, excision, repair resynthesis, and DNA ligation. Escherichia coli has served as a model organism for the study of this process. Recently, many of the proteins that mediate E. coli nucleotide excision have been purified to homogeneity; this had led to a molecular description of this repair pathway. One of the key repair enzymes of this pathway is the UvrABC nuclease complex. The individual subunits of this enzyme cooperate in a complex series of partial reactions to bind to and incise the DNA near a damaged nucleotide. The UvrABC complex displays a remarkable substrate diversity. Defining the structural features of DNA lesions that provide the specificity for damage recognition by the UvrABC complex is of great importance, since it represents a unique form of protein-DNA interaction. Using a number of in vitro assays, researchers have been able to elucidate the action mechanism of the UvrABC nuclease complex. Current research is devoted to understanding how these complex events are mediated within the living cell. PMID:2181258

  8. [Connective tissue dysplasia, magnesium, and nucleotide polymorphisms].

    PubMed

    Torshin, I Iu; Gromova, O A

    2008-01-01

    Undifferentiated connective tissue dysplasia (UCTD) is one of most common diseases of the connective tissue. High frequency of UCTD in population along with the fact that it can provoke a number of other diseases make UCTD an important object of the modern biomedical research in the areas of cardiology, neurology, rheumatology and pulmonology. Modern diagnostics and determination of the predisposition to UCTD allow elaboration of personalized therapy. In particular, Mg-containing supplements and medications can be effectively used in the therapy of UCTD. In one of our previous works we have analyzed possible molecular mechanisms of UCTD etiology as well as therapeutic action of magnesium. The use of data on nucleotide polymorphisms as complementation of standard medical diagnostics is one of perspective trends of the post-genomic medical research. The present work suggest a number of nucleotide polymorphisms that can be used in genetic association analyses of the UCTD as of well as therapeutic efficiency of magnesium treatment. Selection and analysis of the polymorphisms was done on the base of molecular mechanisms we had proposed earlier, comprehensive analysis of published data and also with the use of an integral approach to analysis of the functional effects of the nucleotide polymorphisms and corresponding amino acid substitutions.

  9. [Nucleotide receptors in learning and neuronal plasticity].

    PubMed

    Czajkowski, Rafał

    2014-01-01

    Nucleotide signalling plays an important role in neuronal plasticity and learning. Nucleotides are released at the synaptic terminals and may act pre- and postsynaptically by activating Pland P2 receptors. The A1 receptor, activated tonically by resting concentration of adenosine regulates basal neurotransmission. The A2A receptor is activated by increased adenosine levels and participates in plastic changes. ATP may act as an independent neurotransmitter on the P2X1 receptor, or via P2X3 subtype as a neuromodulator that affects NMDA receptor signalling. The G protein coupled P2Y receptors also evoke neuromodulatory effect on the neuronal plasticity, inhibiting LTD in prefrontal cortex. P2X7 receptor is responsible for communication between astrocytes and for synchronizing their activity. ATP and adenosine released by astrocytes act as neuromodulators both at the release site and heterosynaptically. Taken together, these multiple actions of nucleotides constitute a mechanism regulating homeostatic processes that are necessary for proper brain functioning: synaptic scaling and metaplasticity.

  10. Nucleotide-Dependent Bioautocatalytic Timer Reaction.

    PubMed

    Chen, Ting-Ru; Hsu, Ching-Fong; Chen, Chih-Lin; Witek, Henryk A; Urban, Pawel L

    2016-09-16

    We describe a biochemical timer composed of three biocatalytic reactions involving three types of adenylate nucleotides: adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). The timer is triggered by a small amount of ATP or ADP. An abrupt increase in the ATP concentration (following numerous amplification cycles) leads to a sudden increase of luminescence from the reaction mixture. The time point when the luminescence appears is found to be a function of the initial concentration of the triggering nucleotide (5.0 × 10(-8)-1.0 × 10(-6) M), even in the presence of a complex biological matrix. The mechanism of the observed dependence of the time of luminescence increase on the concentration has been confirmed with simple kinetic models. Due to the biocompatibility of the proposed trienzymatic reaction scheme (sensitivity to common nucleotides and occurrence in a neutral pH aqueous environment), the scheme can be used in bioengineered systems that require modulation of the response time (light emission) by concentration.

  11. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    NASA Astrophysics Data System (ADS)

    Shin, Joong-Won; Bernstein, Elliot R.

    2014-01-01

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5'-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C-C and C-O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  12. Priapism and glucose-6-phosphate dehydrogenase deficiency: An underestimated correlation?

    PubMed

    De Rose, Aldo Franco; Mantica, Guglielmo; Tosi, Mattia; Bovio, Giulio; Terrone, Carlo

    2016-10-05

    Priapism is a rare clinical condition characterized by a persistent erection unrelated to sexual excitement. Often the etiology is idiopathic. Three cases of priapism in glucose-6-phosphate dehydrogenase (G6PD) deficiency patients have been described in literature. We present the case of a 39-year-old man with glucose- 6-phosphate dehydrogenase deficiency, who reached out to our department for the arising of a non-ischemic priapism without arteriolacunar fistula. We suggest that the glucose-6-phosphate dehydrogenase deficiency could be an underestimated risk factor for priapism.

  13. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

    PubMed Central

    Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

    2016-01-01

    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. PMID:26899032

  14. Evidence for Balancing Selection from Nucleotide Sequence Analyses of Human G6PD

    PubMed Central

    Verrelli, Brian C.; McDonald, John H.; Argyropoulos, George; Destro-Bisol, Giovanni; Froment, Alain; Drousiotou, Anthi; Lefranc, Gerard; Helal, Ahmed N.; Loiselet, Jacques; Tishkoff, Sarah A.

    2002-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) mutations that result in reduced enzyme activity have been implicated in malarial resistance and constitute one of the best examples of selection in the human genome. In the present study, we characterize the nucleotide diversity across a 5.2-kb region of G6PD in a sample of 160 Africans and 56 non-Africans, to determine how selection has shaped patterns of DNA variation at this gene. Our global sample of enzymatically normal B alleles and A, A−, and Med alleles with reduced enzyme activities reveals many previously uncharacterized silent-site polymorphisms. In comparison with the absence of amino acid divergence between human and chimpanzee G6PD sequences, we find that the number of G6PD amino acid polymorphisms in human populations is significantly high. Unlike many other G6PD-activity alleles with reduced activity, we find that the age of the A variant, which is common in Africa, may not be consistent with the recent emergence of severe malaria and therefore may have originally had a historically different adaptive function. Overall, our observations strongly support previous genotype-phenotype association studies that proposed that balancing selection maintains G6PD deficiencies within human populations. The present study demonstrates that nucleotide sequence analyses can reveal signatures of both historical and recent selection in the genome and may elucidate the impact that infectious disease has had during human evolution. PMID:12378426

  15. Nucleotide sequence and expression of the Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewer's yeast.

    PubMed Central

    Sone, H; Fujii, T; Kondo, K; Shimizu, F; Tanaka, J; Inoue, T

    1988-01-01

    The nucleotide sequence of a 1.4-kilobase DNA fragment containing the alpha-acetolactate decarboxylase gene of Enterobacter aerogenes was determined. The sequence contains an entire protein-coding region of 780 nucleotides which encodes an alpha-acetolactate decarboxylase of 260 amino acids. The DNA sequence coding for alpha-acetolactate decarboxylase was placed under the control of the alcohol dehydrogenase I promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both S. cerevisiae and Escherichia coli. Brewer's yeast cells transformed by this plasmid showed alpha-acetolactate decarboxylase activity and were used in laboratory-scale fermentation experiments. These experiments revealed that the diacetyl concentration in wort fermented by the plasmid-containing yeast strain was significantly lower than that in wort fermented by the parental strain. These results indicated that the alpha-acetolactate decarboxylase activity produced by brewer's yeast cells degraded alpha-acetolactate and that this degradation caused a decrease in diacetyl production. PMID:3278689

  16. Proline biosynthesis augments tumor cell growth and aerobic glycolysis: involvement of pyridine nucleotides.

    PubMed

    Liu, Wei; Hancock, Chad N; Fischer, Joseph W; Harman, Meredith; Phang, James M

    2015-11-24

    The metabolism of the nonessential amino acid proline contributes to tumor metabolic reprogramming. Previously we showed that MYC increases proline biosynthesis (PB) from glutamine. Here we show MYC increases the expression of the enzymes in PB at both protein and mRNA levels. Blockade of PB decreases tumor cell growth and energy production. Addition of Δ(1)-pyrroline-5-carboxylate (P5C) or proline reverses the effects of P5C synthase knockdown but not P5C reductases knockdown. Importantly, the reversal effect of proline was blocked by concomitant proline dehydrogenase/oxidase (PRODH/POX) knockdown. These findings suggest that the important regulatory contribution of PB to tumor growth derives from metabolic cycling between proline and P5C rather than product proline or intermediate P5C. We further document the critical role of PB in maintaining pyridine nucleotide levels by connecting the proline cycle to glycolysis and to the oxidative arm of the pentose phosphate pathway. These findings establish a novel function of PB in tumorigenesis, linking the reprogramming of glucose, glutamine and pyridine nucleotides, and may provide a novel target for antitumor therapy.

  17. Cloning, expression, purification and preliminary crystallographic analysis of the short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica

    SciTech Connect

    Harmer, Nicholas J.; King, Jerry D.; Palmer, Colin M.; Preston, Andrew; Maskell, Duncan J.; Blundell, Tom L.

    2007-08-01

    The expression, purification, and crystallisation of the short-chain dehydrogenases WbmF, WbmG and WbmH from B. bronchiseptica are described. Native diffraction data to 1.5, 2.0, and 2.2 Å were obtained for the three proteins, together with complexes with nucleotides. The short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica were cloned into Escherichia coli expression vectors, overexpressed and purified to homogeneity. Crystals of all three wild-type enzymes were obtained using vapour-diffusion crystallization with high-molecular-weight PEGs as a primary precipitant at alkaline pH. Some of the crystallization conditions permitted the soaking of crystals with cofactors and nucleotides or nucleotide sugars, which are possible substrate compounds, and further conditions provided co-complexes of two of the proteins with these compounds. The crystals diffracted to resolutions of between 1.50 and 2.40 Å at synchrotron X-ray sources. The synchrotron data obtained were sufficient to determine eight structures of the three enzymes in complex with a variety of cofactors and substrate molecules.

  18. Recognizing nucleotides by cross-tunneling currents for DNA sequencing

    NASA Astrophysics Data System (ADS)

    Bagci, V. M. K.; Kaun, Chao-Cheng

    2011-07-01

    Using first-principles calculations, we study electron transport through nucleotides inside a rectangular nanogap formed by two pairs of gold electrodes which are perpendicular and parallel to the nucleobase plane. We propose that this setup will enhance the nucleotide selectivity of tunneling signals to a great extent. Information from three electrical probing processes offers full nucleotide recognition, which survives the noise from neighboring nucleotides and configuration fluctuations.

  19. HPLC purification of RNA aptamers up to 59 nucleotides with single-nucleotide resolution.

    PubMed

    Huang, Zhen; Lin, Chi-Yen; Jaremko, William; Niu, Li

    2015-01-01

    An RNA sample is usually heterogeneous. RNA heterogeneity refers to difference in length or size (i.e., number of nucleotides [nt]), sequence, or alternative but coexisting conformations. Separation and purification of RNA is generally required for investigating the structure and function of RNA, such as RNA catalysis and RNA structure determination by nuclear magnetic resonance or crystallography. Separation and purification of RNA is also required for using RNAs as functional probes and therapeutics as well as building blocks for RNA nanoparticles. Previously established protocols are limited in separating RNAs longer than 25 nt by single-nucleotide resolution. When the length of RNAs becomes longer, single-nucleotide separation of RNAs becomes more challenging. Here we describe protocols, by the use of ion-pair, reverse-phase high-performance liquid chromatography (HPLC), to extend our ability to separate regular RNAs up to 59 nt with single-nucleotide resolution. For chemically modified RNAs at 2' positions on the ribose, we can resolve RNAs of similar sizes even with a 26 Da difference. This is much less than 320 Da, an average single-nucleotide molecular weight difference.

  20. Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols.

    PubMed

    Inoue, Kousuke; Makino, Yoshihide; Dairi, Tohru; Itoh, Nobuya

    2006-02-01

    The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).

  1. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as acute...

  2. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as acute...

  3. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as acute...

  4. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  5. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  6. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  7. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  8. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  9. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  10. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  11. Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase

    PubMed Central

    Dean, Antony M.; Golding, G. Brian

    1997-01-01

    Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) first evolved from an NAD-dependent precursor about 3.5 billion years ago. Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis. Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and the distantly related Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase. Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes. Reconstructed ancestral sequences indicate that these replacements are ancestral. Hence the adaptive history of molecular evolution is amenable to experimental investigation. PMID:9096353

  12. Glucose oxidation and PQQ-dependent dehydrogenases in Gluconobacter oxydans.

    PubMed

    Hölscher, Tina; Schleyer, Ute; Merfort, Marcel; Bringer-Meyer, Stephanie; Görisch, Helmut; Sahm, Hermann

    2009-01-01

    Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343). Copyright (c) 2008 S. Karger AG, Basel.

  13. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  14. Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology.

    PubMed Central

    Parés, X; Cederlund, E; Moreno, A; Hjelmqvist, L; Farrés, J; Jörnvall, H

    1994-01-01

    The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I. PMID:8127901

  15. Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases.

    PubMed

    Smilda, T; Kamminga, A H; Reinders, P; Baron, W; van Hylckama Vlieg, J E; Beintema, J J

    2001-05-01

    Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar.

  16. Quinohemoprotein alcohol dehydrogenases: structure, function, and physiology.

    PubMed

    Toyama, Hirohide; Mathews, F Scott; Adachi, Osao; Matsushita, Kazunobu

    2004-08-01

    Quino(hemo)protein alcohol dehydrogenases (ADH) that have pyrroloquinoline quinone (PQQ) as the prosthetic group are classified into 3 groups, types I, II, and III. Type I ADH is a simple quinoprotein having PQQ as the only prosthetic group, while type II and type III ADHs are quinohemoprotein having heme c as well as PQQ in the catalytic polypeptide. Type II ADH is a soluble periplasmic enzyme and is widely distributed in Proteobacteria such as Pseudomonas, Ralstonia, Comamonas, etc. In contrast, type III ADH is a membrane-bound enzyme working on the periplasmic surface solely in acetic acid bacteria. It consists of three subunits that comprise a quinohemoprotein catalytic subunit, a triheme cytochrome c subunit, and a third subunit of unknown function. The catalytic subunits of all the quino(hemo)protein ADHs have a common structural motif, a quinoprotein-specific superbarrel domain, where PQQ is deeply embedded in the center. In addition, in the type II and type III ADHs this subunit contains a unique heme c domain. Various type II ADHs each have a unique substrate specificity, accepting a wide variety of alcohols, as is discussed on the basis of recent X-ray crystallographic analyses. Electron transfer within both type II and III ADHs is discussed in terms of the intramolecular reaction from PQQ to heme c and also from heme to heme, and in terms of the intermolecular reaction with azurin and ubiquinone, respectively. Unique physiological functions of both types of quinohemoprotein ADHs are also discussed.

  17. Human liver aldehyde dehydrogenase: coenzyme binding

    SciTech Connect

    Kosley, L.L.; Pietruszko, R.

    1987-05-01

    The binding of (U-/sup 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-/sup 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.

  18. Elusive transition state of alcohol dehydrogenase unveiled.

    PubMed

    Roston, Daniel; Kohen, Amnon

    2010-05-25

    For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2 degrees kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2 degrees KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate.

  19. Malic dehydrogenase locus of Paramecium tetraurelia.

    PubMed

    Williams, T J; Smith-Sonneborn, J

    1980-04-01

    A search was undertaken for naturally occurring genetic markers for use in clonal aging studies of Paramecium tetraurelia. Clonal age is defined as the number of cell divisions since the last sexual process. Autogamy (self-fertilization) is a sexual process which can occur in aging lines, resulting in homozygosity and initiation of the next generation. Such "illicit" autogamies must be detected and eliminated from the aged clone. With codominant alleles, heterozygous aging lines can be established which will express a phenotype distinguishable from that of either parental type and autogamy can then be monitored by the appearance of either segregant homozygous phenotype. However, very few codominant alleles are available in this species. Electrophoretic mobilities of malic dehydrogenase (MDH) were assayed in 11 stocks of Paramecium tetraurelia by polyacrylamide gel electrophoresis. Nine stocks showed a single-banded "stock 51" type, while stock 174 and stock 29 each exhibited unique mobility. Crosses between stock 51 and the deviant stocks revealed distinct three-banded patterns indicative of heterozygosity of the F1 generation. In the autogamous F2 generation, 1:1 segregation of the parental types were recovered. The pattern of inheritance is consistent with codominant alleles and Mendelian inheritance. These naturally occurring biochemical markers are stable with increasing clonal age and are therefore useful genetic markers for studies of cellular aging.

  20. Targeting isocitrate dehydrogenase (IDH) in cancer.

    PubMed

    Fujii, Takeo; Khawaja, Muhammad Rizwan; DiNardo, Courtney D; Atkins, Johnique T; Janku, Filip

    2016-05-01

    Isocitrate dehydrogenase (IDH) is an essential enzyme for cellular respiration in the tricarboxylic acid (TCA) cycle. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma and chondrosarcoma. The mutated IDH1 and IDH2 proteins have a gain-of-function, neomorphic activity, catalyzing the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) by NADPH. Cancer-associated IDH mutations block normal cellular differentiation and promote tumorigenesis via the abnormal production of the oncometabolite 2-HG. High levels of 2-HG have been shown to inhibit α-KG dependent dioxygenases, including histone and deoxyribonucleic acid (DNA) demethylases, which play a key role in regulating the epigenetic state of cells. Current targeted inhibitors of IDH1 (AG120, IDH305), IDH2 (AG221), and pan-IDH1/2 (AG881) selectively inhibit mutant IDH protein and induce cell differentiation in in vitro and in vivo models. Preliminary results from phase I clinical trials with IDH inhibitors in patients with advanced hematologic malignancies have demonstrated an objective response rate ranging from 31% to 40% with durable responses (>1 year) observed. Furthermore, the IDH inhibitors have demonstrated early signals of activity in solid tumors with IDH mutations, including cholangiocarcinomas and low grade gliomas.

  1. Iodination of glyceraldehyde 3-phosphate dehydrogenase

    PubMed Central

    Thomas, Jean O.; Harris, J. Ieuan

    1970-01-01

    1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[125I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed. ImagesPLATE 1PLATE 2 PMID:5530750

  2. Carbon Monoxide Dehydrogenase Activity in Bradyrhizobium japonicum

    PubMed Central

    Lorite, María J.; Tachil, Jörg; Sanjuán, Juán; Meyer, Ortwin; Bedmar, Eulogio J.

    2000-01-01

    Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)2 subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein. PMID:10788353

  3. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

    PubMed Central

    Chen, Che-Hong; Ferreira, Julio Cesar Batista; Gross, Eric R.; Mochly-Rosen, Daria

    2014-01-01

    A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme. PMID:24382882

  4. Elusive transition state of alcohol dehydrogenase unveiled

    PubMed Central

    Roston, Daniel; Kohen, Amnon

    2010-01-01

    For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2° kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the “tunneling ready state” (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate. PMID:20457944

  5. Herbicidal Activity of an Isopropylmalate Dehydrogenase Inhibitor.

    PubMed Central

    Wittenbach, V. A.; Teaney, P. W.; Hanna, W. S.; Rayner, D. R.; Schloss, J. V.

    1994-01-01

    Isopropylmalate dehydrogenase (IPMDH) is the third enzyme specific to leucine biosynthesis. It catalyzes the oxidative decarboxylation of 3-isopropylmalate (3-IPM) to 2-ketoisocaproic acid. The partially purified enzyme from pea (Pisum sativum L.) shows a broad pH optimum of 7.8 to 9.1 and has Km values for 3-IPM and NAD of 18 and 40 [mu]M, respectively. O-Isobutenyl oxalylhydroxamate (O-IbOHA) has been discovered to be an excellent inhibitor of the pea IPMDH, with an apparent inhibitor constant of 5 nM. As an herbicide, O-IbOHA showed only moderate activity on a variety of broadleaf and grass species. We characterized the herbicidal activity of O-IbOHA on corn (Zea mays L.), a sensitive species; giant foxtail (Setaria faberi) and morning glory (Ipomoea purpurea [L.] Roth), moderately tolerant species; and soybean [Glycine max L. Merr.), a tolerant species. Differences in tolerance among the species were not due to differences in the sensitivity of IPMDH. Studies with [14C]O-IbOHA suggested that uptake and translocation were not major limitations for herbicidal activity, nor were they determinants of tolerance. Moreover, metabolism could not account for the difference in tolerance of corn, foxtail, and morning glory, although it might account for the tolerance of soybean. Herbicidal activity on all four species was correlated with the accumulation of 3-IPM in the plants. PMID:12232331

  6. Glucose-6-phosphate dehydrogenase deficiency in Chinese

    PubMed Central

    Lai, H. C.; Lai, Michael P. Y.; Leung, Kevin S. N.

    1968-01-01

    In a Chinese population 1,000 full-term male neonates and a further 117 jaundiced neonates of both sexes were studied in an investigation of the frequency of deficiency of erythrocyte glucose-6-phosphate dehydrogenase (G6PD). This enzyme was found to be deficient in 3·6% of male neonates. Correlation of the results with the birthplace of the 602 mothers who were known to come from Kwangtung province showed no significant differences in the frequency of the deficiency between certain parts of the province. The deficiency of G6PD in hemizygous males is profound but it is not associated with erythrocyte acid monophosphoesterase deficiency in Chinese in Hong Kong. The G6PD deficiency accounts for 15·4% of all the 117 cases of neonatal jaundice. The relative importance of G6PD deficiency as a cause of neonatal jaundice does not differ materially in male and female mutants. Neonatal jaundice can occur in all genotypes of G6PD mutation in Chinese. PMID:5697334

  7. Halophile aldehyde dehydrogenase from Halobacterium salinarum.

    PubMed

    Kim, Hyo-Jeong; Joo, Won-A; Cho, Chang-Won; Kim, Chan-Wha

    2006-01-01

    Halobacterium salinarum is a member of the halophilic archaea. In the present study, H. salinarum was cultured at various NaCl concentrations (3.5, 4.3, and 6.0 M NaCl), and its proteome was determined and identificated via proteomics technique. We detected 14 proteins which were significantly down-regulated in 3.5 M and/or 6 M NaCl. Among the identified protein spots, aldehyde dehydrogenase (ALDH) was selected for evaluation with regard to its potential applications in industry. The most effective metabolism function exhibited by ALDH is the oxidation of aldehydes to carboxylic acids. The ALDH gene from H. salinarum (1.5 kb fragment) was amplified by PCR and cloned into the E. coli strain, BL21 (DE3), with the pGEX-KG vector. We subsequently analyzed the enzyme activity of the recombinant ALDH (54 kDa) at a variety of salt concentrations. The purified recombinant ALDH from H. salinarum exhibited the most pronounced activity at 1 M NaCl. Therefore, the ALDH from H.salinarum is a halophilic enzyme, and may prove useful for applications in hypersaline environments.

  8. Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum.

    PubMed

    Lorite, M J; Tachil, J; Sanjuán, J; Meyer, O; Bedmar, E J

    2000-05-01

    Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.

  9. [Alcohol dehydrogenase and aldehyde dehydrogenase as tumour markers and factors intensifying carcinogenesis in colorectal cancer].

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Kedra, Bogusław; Szmitkowski, Maciej

    2008-06-01

    Numerous experiments have shown that alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are present in cells of various cancers and play role in carcinogenesis. The aim of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity, between colorectal cancer and normal colonic mucosa. We have also investigated the serum activity of these enzymes in colorectal cancer patients as potential tumour markers. The activities of ADH isoenzymes and ALDH were measured in the: cancer tissue, healthy colonic mucosa and serum of 42 patients with colorectal cancer. For the measurement of the activity of class I ADH isoenzyme and ALDH activity the fluorometric methods was employed. The total ADH activity and activity of class III and IV isoenzymes was measured by the photometric method. The activity of total alcohol dehydrogenase and class I of ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH had higher activities in cancer tissue but the differences were not statistically significant. The activity of ALDH was significantly lower in the cancer cells. The activities of all tested enzymes and isoenzymes in colorectal cancer tissue were not significantly higher in drinkers than in non-drinkers. Additionally we observed statistically significant increasing activity of class I ADH isoenzymes in the sera of patients with colorectal cancer. For this reason the total ADH activity was also significantly increased. The activities of ADH III and ADH IV isoenzymes and ALDH were unchanged in the sera of patients. There were no marked differences in activities of all tested enzymes and isoenzymes between drinkers and non-drinkers (with colorectal cancer). The differences in activities of total ADH and class I ADH isoenzymes between colorectal cancer tissues and healthy mucosa might be a factor of ethanol metabolism disorders, which can intensify carcinogenesis. The increased total

  10. Succinate Dehydrogenase Loss in Familial Paraganglioma: Biochemistry, Genetics, and Epigenetics

    PubMed Central

    Her, Yeng F.; Maher, L. James

    2015-01-01

    It is counterintuitive that metabolic defects reducing ATP production can cause, rather than protect from, cancer. Yet this is precisely the case for familial paraganglioma, a form of neuroendocrine malignancy caused by loss of succinate dehydrogenase in the tricarboxylic acid cycle. Here we review biochemical, genetic, and epigenetic considerations in succinate dehydrogenase loss and present leading models and mysteries associated with this fascinating and important tumor. PMID:26294907

  11. Enzymatic Transformation of Morphine by Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni

    PubMed Central

    Liras, Paloma; Kasparian, Stephen S.; Umbreit, Wayne W.

    1975-01-01

    Enzyme preparations from Pseudomonas testosteroni containing α- and β-hydroxysteroid dehydrogenases catalyzed the oxidation of morphine and codeine by nicotinamide adenine dinucleotide. Morphine was converted in relatively low yield into 14-hydroxymorphinone probably via morphinone as an intermediate. Codeine was converted to codeinone and 14-hydroxycodeinone. Only the conversions at the 6-position were carried out by the hydroxysteroid dehydrogenase. Hydroxylation at the 14-position did occur spontaneously (or enzymatically with a contaminating enzyme) after oxidation at the 6-position. PMID:172013

  12. Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

    PubMed Central

    Tsai, C S

    1978-01-01

    Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues. PMID:697732

  13. Evolution of Functional Six-Nucleotide DNA

    PubMed Central

    Zhang, Liqin; Yang, Zunyi; Sefah, Kwame; Bradley, Kevin M.; Hoshika, Shuichi; Kim, Myong-Jung; Kim, Hyo-Joong; Zhu1, Guizhi; Jiménez, Elizabeth; Cansiz, Sena; Teng, I-Ting; Champanhac, Carole; McLendon, Christopher; Liu, Chen; Zhang, Wen; Gerloff, Dietlind L.

    2015-01-01

    Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add additional functional groups, not found in natural DNA but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via strict Watson-Crick geometry. These were added to lies in a ibrarlaboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection systems, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Z’s and P’s. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system, and that GACTZP libraries are richer reservoir of functionality than standard libraries. PMID:25966323

  14. In Vitro Selection Using Modified or Unnatural Nucleotides

    PubMed Central

    Stovall, Gwendolyn M.; Bedenbaugh, Robert S.; Singh, Shruti; Meyer, Adam J.; Hatala, Paul J.; Ellington, Andrew D.; Hall, Bradley

    2014-01-01

    Incorporation of modified nucleotides into in vitro RNA or DNA selections offer many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions. PMID:25606981

  15. Nucleotide sequence alignment using sparse coding and belief propagation.

    PubMed

    Roozgard, Aminmohammad; Barzigar, Nafise; Wang, Shuang; Jiang, Xiaoqian; Ohno-Machado, Lucila; Cheng, Samuel

    2013-01-01

    Advances in DNA information extraction techniques have led to huge sequenced genomes from organisms spanning the tree of life. This increasing amount of genomic information requires tools for comparison of the nucleotide sequences. In this paper, we propose a novel nucleotide sequence alignment method based on sparse coding and belief propagation to compare the similarity of the nucleotide sequences. We used the neighbors of each nucleotide as features, and then we employed sparse coding to find a set of candidate nucleotides. To select optimum matches, belief propagation was subsequently applied to these candidate nucleotides. Experimental results show that the proposed approach is able to robustly align nucleotide sequences and is competitive to SOAPaligner [1] and BWA [2].

  16. The Genetics of Alcohol Metabolism: Role of Alcohol Dehydrogenase and Aldehyde Dehydrogenase Variants

    PubMed Central

    Edenberg, Howard J.

    2007-01-01

    The primary enzymes involved in alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Both enzymes occur in several forms that are encoded by different genes; moreover, there are variants (i.e., alleles) of some of these genes that encode enzymes with different characteristics and which have different ethnic distributions. Which ADH or ALDH alleles a person carries influence his or her level of alcohol consumption and risk of alcoholism. Researchers to date primarily have studied coding variants in the ADH1B, ADH1C, and ALDH2 genes that are associated with altered kinetic properties of the resulting enzymes. For example, certain ADH1B and ADH1C alleles encode particularly active ADH enzymes, resulting in more rapid conversion of alcohol (i.e., ethanol) to acetaldehyde; these alleles have a protective effect on the risk of alcoholism. A variant of the ALDH2 gene encodes an essentially inactive ALDH enzyme, resulting in acetaldehyde accumulation and a protective effect. It is becoming clear that noncoding variants in both ADH and ALDH genes also may influence alcohol metabolism and, consequently, alcoholism risk; the specific nature and effects of these variants still need further study. PMID:17718394

  17. Effect of fermented sea tangle on the alcohol dehydrogenase and acetaldehyde dehydrogenase in Saccharomyces cerevisiae.

    PubMed

    Cha, Jae-Young; Jeong, Jae-Jun; Yang, Hyun-Ju; Lee, Bae-Jin; Cho, Young-Su

    2011-08-01

    Sea tangle, a kind of brown seaweed, was fermented with Lactobacillus brevis BJ-20. The gamma-aminobutyric acid (GABA) content in fermented sea tangle (FST) was 5.56% (w/w) and GABA in total free amino acid of FST was 49.5%. The effect of FST on the enzyme activities and mRNA protein expression of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) involved in alcohol metabolism in Saccharomyces cerevisiae was investigated. Yeast was cultured in YPD medium supplemented with different concentrations of FST powder [0, 0.4, 0.8, and 1.0% (w/v)] for 18 h. FST had no cytotoxic effect on the yeast growth. The highest activities and protein expressions of ADH and ALDH from the cell-free extracts of S. cerevisiae were evident with the 0.4% and 0.8% (w/v) FST-supplemented concentrations, respectively. The highest concentrations of GABA as well as minerals (Zn, Ca, and Mg) were found in the cell-free extracts of S. cerevisiae cultured in medium supplemented with 0.4% (w/v) FST. The levels of GABA, Zn, Ca, and Mg in S. cerevisiae were strongly correlated with the enzyme activities of ADH and ALDH in yeast. These results indicate that FST can enhance the enzyme activities and protein expression of ADH and ALDH in S. cerevisiae.

  18. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

  19. Structural Studies of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

  20. Yeast Alcohol Dehydrogenase Structure and Catalysis

    PubMed Central

    2015-01-01

    Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure. PMID:25157460

  1. Succinate dehydrogenase gene mutations in cardiac paragangliomas.

    PubMed

    Martucci, Victoria L; Emaminia, Abbas; del Rivero, Jaydira; Lechan, Ronald M; Magoon, Bindiya T; Galia, Analyza; Fojo, Tito; Leung, Steve; Lorusso, Roberto; Jimenez, Camilo; Shulkin, Barry L; Audibert, Jennifer L; Adams, Karen T; Rosing, Douglas R; Vaidya, Anand; Dluhy, Robert G; Horvath, Keith A; Pacak, Karel

    2015-06-15

    Pheochromocytomas and paragangliomas are chromaffin cell tumors arising from neuroendocrine cells. At least 1/3 of paragangliomas are related to germline mutations in 1 of 17 genes. Although these tumors can occur throughout the body, cardiac paragangliomas are very rare, accounting for <0.3% of mediastinal tumors. The purpose of this study was to determine the clinical characteristics of patients with cardiac paragangliomas, particularly focusing on their genetic backgrounds. A retrospective chart analysis of 15 patients with cardiac paragangliomas was performed to determine clinical presentation, genetic background, diagnostic workup, and outcomes. The average age at diagnosis was 41.9 years. Typical symptoms of paraganglioma (e.g., hypertension, sweating, palpitations, headache) were reported at initial presentation in 13 patients (86.7%); the remaining 2, as well as 4 symptomatic patients, initially presented with cardiac-specific symptoms (e.g., chest pain, dyspnea). Genetic testing was done in 13 patients (86.7%); 10 (76.9%) were positive for mutations in succinate dehydrogenase (SDHx) subunits B, C, or D. Thirteen patients (86.7%) underwent surgery to remove the paraganglioma with no intraoperative morbidity or mortality; 1 additional patient underwent surgical resection but experienced intraoperative complications after removal of the tumor due to co-morbidities and did not survive. SDHx mutations are known to be associated with mediastinal locations and malignant behavior of paragangliomas. In this report, the investigators extend the locations of predominantly SDHx-related paragangliomas to cardiac tumors. In conclusion, cardiac paragangliomas are frequently associated with underlying SDHx germline mutations, suggesting a need for genetic testing of all patients with this rare tumor. Published by Elsevier Inc.

  2. Structural Studies of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

  3. The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris.

    PubMed

    Hadj-Saïd, Jessica; Pandelia, Maria-Eirini; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

    2015-12-01

    Ni-containing Carbon Monoxide Dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO₂and are involved in energy conservation and carbon fixation. These homodimeric enzymes house two NiFeS active sites (C-clusters) and three accessory [4Fe-4S] clusters. The Desulfovibrio vulgaris (Dv) genome contains a two-gene CODH operon coding for a CODH (cooS) and a maturation protein (cooC) involved in nickel insertion in the active site. According to the literature, the question of the precise function of CooC as a chaperone folding the C-cluster in a form which accommodates free nickel or as a mere nickel donor is not resolved. Here, we report the biochemical and spectroscopic characterization of two recombinant forms of the CODH, produced in the absence and in the presence of CooC, designated CooS and CooS(C), respectively. CooS contains no nickel and cannot be activated, supporting the idea that the role of CooC is to fold the C-cluster so that it can bind nickel. As expected, CooS(C) is Ni-loaded, reversibly converts CO and CO₂, displays the typical Cred1 and Cred2 EPR signatures of the C-cluster and activates in the presence of methyl viologen and CO in an autocatalytic process. However, Ni-loaded CooS(C) reaches maximum activity only upon reductive treatment in the presence of exogenous nickel, a phenomenon that had not been observed before. Surprisingly, the enzyme displays the Cred1 and Cred2 signatures whether it has been activated or not, showing that this activation process of the Ni-loaded Dv CODH is not associated with structural changes at the active site.

  4. Succinate Dehydrogenase Gene Mutations in Cardiac Paragangliomas

    PubMed Central

    Martucci, Victoria L.; Emaminia, Abbas; del Rivero, Jaydira; Lechan, Ronald M.; Magoon, Bindiya T.; Galia, Analyza; Fojo, Tito; Leung, Steve; Lorusso, Roberto; Jimenez, Camilo; Shulkin, Barry L.; Audibert, Jennifer L.; Adams, Karen T.; Rosing, Douglas R.; Vaidya, Anand; Dluhy, Robert G.; Horvath, Keith A.; Pacak, Karel

    2015-01-01

    Pheochromocytomas and paragangliomas are chromaffin cell tumors arising from neuroendocrine cells. At least one third of paragangliomas are related to germline mutations in one of 17 genes. While these tumors can occur throughout the body, cardiac paragangliomas are very rare, accounting for less than 0.3% of mediastinal tumors. The purpose of this study was to determine the clinical characteristics of patients with cardiac paragangliomas, particularly focusing on their genetic backgrounds. A retrospective chart analysis of fifteen patients with cardiac paraganglioma was performed to determine clinical presentation, genetic background, diagnostic work-up, and outcomes. The average age at diagnosis was 41.9 years. Typical symptoms of paraganglioma (e.g., hypertension, sweating, palpitations, headache) were reported at initial presentation in 13 patients (86.7%); the remaining 2, as well as 4 symptomatic patients, initially presented with cardiac-specific symptoms (e.g., chest pain, dyspnea). Genetic testing was done in 13 cases (86.7%); 10 (76.9%) were positive for mutations in succinate dehydrogenase (SDHx) subunits B, C, or D. Thirteen cases (86.7%) underwent surgery to remove the paraganglioma with no intraoperative morbidity or mortality; one additional patient underwent surgical resection but experienced intraoperative complications after removal of the tumor due to comorbities and did not survive. SDHx mutations are known to be associated with mediastinal locations and malignant behavior of paragangliomas. In this report, we extend the locations of predominantly SDHx-related paragangliomas to cardiac tumors. In conclusion, cardiac paragangliomas are frequently associated with underlying SDHx germline mutations, suggesting a need for genetic testing of all patients with this rare tumor. PMID:25896150

  5. Regulation of dihydropyrimidine dehydrogenase in colorectal cancer.

    PubMed

    Johnston, S J; Ridge, S A; Cassidy, J; McLeod, H L

    1999-09-01

    Dihydropyrimidine dehydrogenase (DPD) is responsible for degradation of the pyrimidines uracil and thymine and the inactivation of the chemotherapeutic agent 5-fluorouracil. DPD activity is highly variable in cancer populations, and this variation may influence the antitumor efficacy of 5-fluorouracil. However, little is known about the regulation of DPD mRNA expression in any tissues. Using a reverse transcription competitive PCR assay, we quantified DPD mRNA levels in 10 matched colorectal tumors and adjacent normal mucosae and 7 colorectal liver metastases and adjacent normal livers. Lower levels of DPD mRNA expression were observed in colorectal tumor compared with adjacent normal colon mucosa (median, 0.01 versus 0.37 amole/microg total RNA, P = 0.02). DPD mRNA expression was also lower in metastases than adjacent normal liver tissue (median, 0.11 versus 1.17 amole/microg total RNA, P = 0.001). DPD mRNA expression was higher in normal liver than normal colonic mucosa (median, 1.17 versus 0.37 amole/microg total RNA, P = 0.02). A significant relationship was observed between DPD mRNA and catalytic activity (r(s) = 0.66, P<0.001). The tumor:normal ratio for DPD mRNA, protein, and activity was relatively stable in liver (0.25, 0.55, and 0.51, respectively) but varied considerably in colon (0.085, 0.9, and 1.25, respectively), consistent with enhanced translation of DPD transcript in primary colorectal tumor. This suggests that DPD can be regulated at the levels of both transcription and translation.

  6. Isocitrate dehydrogenase mutations in myeloid malignancies

    PubMed Central

    Medeiros, B C; Fathi, A T; DiNardo, C D; Pollyea, D A; Chan, S M; Swords, R

    2017-01-01

    Alterations to genes involved in cellular metabolism and epigenetic regulation are implicated in the pathogenesis of myeloid malignancies. Recurring mutations in isocitrate dehydrogenase (IDH) genes are detected in approximately 20% of adult patients with acute myeloid leukemia (AML) and 5% of adults with myelodysplastic syndromes (MDS). IDH proteins are homodimeric enzymes involved in diverse cellular processes, including adaptation to hypoxia, histone demethylation and DNA modification. The IDH2 protein is localized in the mitochondria and is a critical component of the tricarboxylic acid (also called the ‘citric acid' or Krebs) cycle. Both IDH2 and IDH1 (localized in the cytoplasm) proteins catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Mutant IDH enzymes have neomorphic activity and catalyze reduction of α-KG to the (R) enantiomer of 2-hydroxyglutarate, which is associated with DNA and histone hypermethylation, altered gene expression and blocked differentiation of hematopoietic progenitor cells. The prognostic significance of mutant IDH (mIDH) is controversial but appears to be influenced by co-mutational status and the specific location of the mutation (IDH1-R132, IDH2-R140, IDH2-R172). Treatments specifically or indirectly targeted to mIDH are currently under clinical investigation; these therapies have been generally well tolerated and, when used as single agents, have shown promise for inducing responses in some mIDH patients when used as first-line treatment or in relapsed or refractory AML or MDS. Use of mIDH inhibitors in combination with drugs with non-overlapping mechanisms of action is especially promising, as such regimens may address the clonal heterogeneity and the multifactorial pathogenic processes involved in mIDH myeloid malignancies. Advances in mutational analysis have made testing more rapid and convenient, and less expensive; such testing should become part of routine diagnostic workup and repeated at

  7. Variance estimation for nucleotide substitution models.

    PubMed

    Chen, Weishan; Wang, Hsiuying

    2015-09-01

    The current variance estimators for most evolutionary models were derived when a nucleotide substitution number estimator was approximated with a simple first order Taylor expansion. In this study, we derive three variance estimators for the F81, F84, HKY85 and TN93 nucleotide substitution models, respectively. They are obtained using the second order Taylor expansion of the substitution number estimator, the first order Taylor expansion of a squared deviation and the second order Taylor expansion of a squared deviation, respectively. These variance estimators are compared with the existing variance estimator in terms of a simulation study. It shows that the variance estimator, which is derived using the second order Taylor expansion of a squared deviation, is more accurate than the other three estimators. In addition, we also compare these estimators with an estimator derived by the bootstrap method. The simulation shows that the performance of this bootstrap estimator is similar to the estimator derived by the second order Taylor expansion of a squared deviation. Since the latter one has an explicit form, it is more efficient than the bootstrap estimator.

  8. Estimation of evolutionary distances between nucleotide sequences.

    PubMed

    Zharkikh, A

    1994-09-01

    A formal mathematical analysis of the substitution process in nucleotide sequence evolution was done in terms of the Markov process. By using matrix algebra theory, the theoretical foundation of Barry and Hartigan's (Stat. Sci. 2:191-210, 1987) and Lanave et al.'s (J. Mol. Evol. 20:86-93, 1984) methods was provided. Extensive computer simulation was used to compare the accuracy and effectiveness of various methods for estimating the evolutionary distance between two nucleotide sequences. It was shown that the multiparameter methods of Lanave et al.'s (J. Mol. Evol. 20:86-93, 1984), Gojobori et al.'s (J. Mol. Evol. 18:414-422, 1982), and Barry and Hartigan's (Stat. Sci. 2:191-210, 1987) are preferable to others for the purpose of phylogenetic analysis when the sequences are long. However, when sequences are short and the evolutionary distance is large, Tajima and Nei's (Mol. Biol. Evol. 1:269-285, 1984) method is superior to others.

  9. Cyclic nucleotide imaging and cardiovascular disease.

    PubMed

    Berisha, Filip; Nikolaev, Viacheslav O

    2017-02-16

    The universal second messengers cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) play central roles in cardiovascular function and disease. They act in discrete, functionally relevant subcellular microdomains which regulate, for example, calcium cycling and excitation-contraction coupling. Such localized cAMP and cGMP signals have been difficult to measure using conventional biochemical techniques. Recent years have witnessed the advent of live cell imaging techniques which allow visualization of these functionally relevant second messengers with unprecedented spatial and temporal resolution at cellular, subcellular and tissue levels. In this review, we discuss these new imaging techniques and give examples how they are used to visualize cAMP and cGMP in physiological and pathological settings to better understand cardiovascular function and disease. Two primary techniques include the use of Förster resonance energy transfer (FRET) based cyclic nucleotide biosensors and nanoscale scanning ion conductance microscopy (SICM). These methods can provide deep mechanistic insights into compartmentalized cAMP and cGMP signaling.

  10. [Identification of single nucleotide polymorphisms in centenarians].

    PubMed

    Gambini, Juan; Gimeno-Mallench, Lucía; Inglés, Marta; Olaso, Gloria; Abdelaziz, Kheira Mohamed; Avellana, Juan Antonio; Belenguer, Ángel; Cruz, Raquel; Mas-Bargues, Cristina; Borras, Consuelo; Viña, José

    2016-01-01

    Longevity is determined by genetic and external factors, such as nutritional, environmental, social, etc. Nevertheless, when living conditions are optimal, longevity is determined by genetic variations between individuals. In a same population, with relative genotypic homogeneity, subtle changes in the DNA sequence affecting a single nucleotide can be observed. These changes, called single nucleotide polymorphisms (SNP) are present in 1-5% of the population. A total of 92 subjects were recruited, including 28 centenarians and 64 controls, in order to find SNP that maybe implicated in the extreme longevity, as in the centenarians. Blood samples were collected to isolate and amplify the DNA in order to perform the analysis of SPN by Axiom™ Genotyping of Affymetrix technology. Statistical analyses were performed using the Plink program and libraries SNPassoc and skatMeta. Our results show 12 mutations with a p<.001, where 5 of these (DACH1, LOC91948, BTB16, NFIL3 y HDAC4) have regulatory functions of the expressions of others genes. Therefore, these results suggest that the genetic variation between centenarians and controls occurs in five genes that are involved in the regulation of gene expression to adapt to environmental changes better than controls. Copyright © 2015 SEGG. Published by Elsevier Espana. All rights reserved.

  11. Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequence analysis and evolutionary implications.

    PubMed

    Pavesi, A; Ficarelli, A; Tassi, F; Restivo, F M

    2000-04-01

    Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction). The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB. Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only. Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH). A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A. officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots. Codon usage analysis showed a bias towards A/T ending codons. This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons. Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated. The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi. Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.

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

    PubMed Central

    2009-01-01

    Background Microsatellite instability (MSI) refers to mutations in short motifs of tandemly repeated nucleotides resulting from replication errors and deficient mismatch repair (MMR). Colorectal cancer with MSI has characteristic biology and chemosensitivity, however the molecular basis remains unclarified. The association of MSI and MMR status with outcome and with thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression in colorectal cancer were evaluated. Methods MSI in five reference loci, MMR enzymes (hMSH2, hMSH6, hMLH1 and hPMS2), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression were assessed in paraffin embedded tumor specimens, and associated with outcome in 340 consecutive patients completely resected for colorectal cancer stages II-IV and subsequently receiving adjuvant 5-fluorouracil therapy. Results MSI was found in 43 (13.8%) tumors. Absence of repair protein expression was assessed in 52 (17.0%) tumors, which had primarily lost hMLH1 in 39 (12.7%), hMSH2 in 5 (1.6%), and hMSH6 in 8 (2.6%) tumors. In multivariate analysis MSI (instable) compared to MSS (stable) tumors were significantly associated with lower risk of recurrence (hazard ratio (HR) = 0.3; 95% CI: 0.2–0.7; P = 0.0007) and death (HR = 0.4; 95% CI: 0.2–0.9; P = 0.02) independently of the TS and DPD expressions. A direct relationship between MSI and TS intensity (P = 0.001) was found, while there was no significant association with DPD intensity (P = 0.1). Conclusion The favourable outcome of MSI colorectal carcinomas is ascribed mainly to the tumor biology and to a lesser extent to antitumor response to 5-fluorouracil therapy. There is no evidence that differential TS or DPD expression may account for these outcome characteristics. PMID:19154585

  13. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    PubMed

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  14. Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

    PubMed Central

    Chalmers, R M; Fewson, C A

    1989-01-01

    Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism. Images Fig. 1. PMID:2597133

  15. The Growth-Promoting Effect of Dietary Nucleotides in Fish Is Associated with an Intestinal Microbiota-Mediated Reduction in Energy Expenditure.

    PubMed

    Guo, Xiaoze; Ran, Chao; Zhang, Zhen; He, Suxu; Jin, Min; Zhou, Zhigang

    2017-03-29

    Background: Nucleotides have been used as functional nutrients to improve the growth and health of animals, including fish. The mechanism involved in the growth-promotion effect of nucleotides is still unclear.Objective: We investigated the bioenergetic mechanism underlying the growth-promotion effect of nucleotides in zebrafish and the associated roles played by the intestinal microbiota.Methods: Larval zebrafish were fed a control or a 0.1% mixed nucleotides-supplemented diet for 2 wk. Standard metabolic rate, the minimal rate of energy expenditure by animals at rest, was evaluated by oxygen consumption with the use of a respirometer. The expressions of fasting-induced adipose factor (Fiaf), inflammatory cytokines, and genes involved in fatty acid (FA) oxidation were tested by quantitative reverse transcriptase-polymerase chain reaction. The intestinal microbiota from the nucleotide-fed fish (NT fish) or control fish was transferred to 3-d postfertilization germ-free zebrafish in which oxygen consumption and expression of cytokines and fiaf were evaluated.Results: Compared with controls, nucleotide supplementation at 0.1% increased the weight and energy gains of zebrafish by 10% and 25%, respectively (P < 0.01). Standard metabolic rate was 28% lower in NT fish than in controls (P < 0.001). Nucleotide supplementation downregulated the inflammatory tone in the head kidney of the fish. Moreover, NT fish had a 51% lower intestinal expression of fiaf than did controls (P < 0.05), which was consistent with decreased expression of key genes involved in FA oxidation [carnitine:palmitoyl transferase 1a (cpt1a) and medium-chain acyl coenzyme A dehydrogenase (mcad)] in liver and muscle. Germ-free zebrafish colonized with microbiota from NT fish had a 25% lower standard metabolic rate than did those colonized by control microbiota (P < 0.01), whereas direct nucleotide feeding of germ-free zebrafish did not affect standard metabolic rate relative to germ-free controls that

  16. Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

    PubMed

    Lorenz, Robin; Moon, Eui-Whan; Kim, Jeong Joo; Schmidt, Sven H; Sankaran, Banumathi; Pavlidis, Ioannis V; Kim, Choel; Herberg, Friedrich W

    2017-07-06

    Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  17. Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg²⁺, Nucleotides, and RNA.

    PubMed

    Frankel, Erica A; Bevilacqua, Philip C; Keating, Christine D

    2016-03-01

    Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid-liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5'-mono-, di-, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg(2+) and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg(2+) concentration of 10 mM had 1-5 M Mg(2+) in the coacervates, and RNA random sequence (N54) partitioned ∼10,000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg(2+), and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg(2+) concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments.

  18. Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?

    PubMed

    Srivastava, D K; Smolen, P; Betts, G F; Fukushima, T; Spivey, H O; Bernhard, S A

    1989-09-01

    Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that

  19. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

    PubMed Central

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L.

    2016-01-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells. PMID:27273042

  20. [Nucleotide receptors and actin cytoskeleton dynamics].

    PubMed

    Kłopocka, Wanda; Korczyński, Jarosław

    2014-01-01

    Signaling cascades evoked by P2Y2 receptor plays an important role in the phenomena dependent on the actin cytoskeleton dynamics endocy-tosis, cell division, adhesion, intracellular transport and migration. P2Y2R coupled with G proteins, in response to ATP or UTP activates Rac1 and RhoA proteins important factors in actin cytoskeletal reorganization and regulates the level of phosphatidylinositol-4,5-bisphosphate (PIP2) that binds directly to a variety of actin regulatory proteins and modulates their function. The P2Y2 nucleotide receptor contains the integrin-binding domain enables it to interact selectively with α(v)β3 and α(v)β5 integrins and is required for G0-mediated Rac1 activation. Interaction with α(v)β5 is necessary for coupling the P2Y2 receptor to G12 and subsequent activation of RhoA.

  1. Renal inner medullary choline dehydrogenase activity: characterization and modulation.

    PubMed

    Grossman, E B; Hebert, S C

    1989-01-01

    Betaine belongs to the trimethylamine class of osmolytes (osmotically active substances believed to play an important role in cell volume homeostasis) and has recently been identified in the inner medulla of the mammalian kidney. Trimethylamines accumulate in the renal inner medulla during hypertonic stress, and betaine content in the inner medulla has been shown recently to increase during hypernatremia, yet the mechanisms governing the modulation of trimethylamine content and, in particular, of betaine content are not well understood. In this study, we demonstrate the presence of choline dehydrogenase activity in the renal inner medullas of three separate rat strains. Choline dehydrogenase is the enzyme that catalyzes the first of two successive oxidation steps in the biosynthetic conversion of choline to betaine. The presence of choline dehydrogenase activity in the inner medulla suggests that betaine accumulation in the inner medulla may result, at least in part, through in situ synthesis. The Km and Vmax of the reaction in the inner medullas of Long-Evans rats are 4.7 +/- 0.5 mM and 36.9 +/- 5.0 nmol.mg protein-1.min-1, respectively. These values are similar to the characteristics of choline dehydrogenase in mammalian liver. During hypernatremia, when betaine content of the inner medulla has been shown to increase 1.5-fold, choline dehydrogenase activity remains unchanged (or slightly increased), whereas enzyme activity in the cortex increases approximately 50%. Possible mechanisms of inner medullary betaine accumulation are discussed.

  2. Dehydrogenase activity of forest soils depends on the assay used

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; Długa, Joanna; Socha, Jarosław

    2015-01-01

    Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

  3. Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity.

    PubMed

    Henderson, B

    1976-08-25

    A system has been developed for the quantitative measurment of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, gylceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonsatration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose 1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the intergrity of the Embden-Meyerhof pathway.

  4. Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians.

    PubMed

    Humanes, L; López-Ruiz, A; Merino, M T; Roldán, J M; Diez, J

    1997-09-01

    Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.

  5. Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians.

    PubMed Central

    Humanes, L; López-Ruiz, A; Merino, M T; Roldán, J M; Diez, J

    1997-01-01

    Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect. PMID:9292989

  6. The Conformation of NAD+ Bound to Lactate Dehydrogenase Determined by Nuclear Magnetic Resonance with Suppression of Spin Diffusion

    NASA Astrophysics Data System (ADS)

    Vincent, Sebastien J. F.; Zwahlen, Catherine; Post, Carol Beth; Burgner, John W.; Bodenhausen, Geoffrey

    1997-04-01

    We have reinvestigated the conformation of NAD+ bound to dogfish lactate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distances between pairs of protons, without perturbation of spin-diffusion effects from other protons belonging either to the cofactor or to the binding pocket of the enzyme. The analysis indicates that the structure of bound NAD+ is in accord with the conformation determined in the solid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate conformational averaging about the glycosidic bond of the nicotinamide nucleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD+ that we infer from solution-state NMR, we suggest that LDH binds the cofactor in both syn and anti conformations, but that binding interactions in the syn conformation are not catalytically productive.

  7. Cloning and sequencing of the gene coding for the large subunit of methylamine dehydrogenase from Thiobacillus versutus.

    PubMed Central

    Huitema, F; van Beeumen, J; van Driessche, G; Duine, J A; Canters, G W

    1993-01-01

    The gene that codes for the alpha-subunit of methylamine dehydrogenase from Thiobacillus versutus, madA, was cloned and sequenced. It codes for a protein of 395 amino acids preceded by a leader sequence of 31 amino acids. The derived amino acid sequence was confirmed by partial amino acid sequencing. The start of the mature protein could not be determined by direct sequencing, since the N terminus appeared to be blocked. Instead, it was determined by electrospray mass spectrometry. Confirmation of the results was obtained by sequencing the N terminus after pyroglutamate aminopeptidase digestion. The sequence is homologous to the Paracoccus denitrificans nucleotide sequence. A second open reading frame, called open reading frame 3, is located immediately downstream of madA. PMID:8407797

  8. Uniparental Isodisomy of Chromosome 1 Unmasking an Autosomal Recessive 3-Beta Hydroxysteroid Dehydrogenase Type II-Related Congenital Adrenal Hyperplasia

    PubMed Central

    Panzer, Karin; Ekhaguere, Osayame A.; Darbro, Benjamin; Cook, Jennifer; Shchelochkov, Oleg A.

    2017-01-01

    Steroid 3-beta hydroxysteroid dehydrogenase type II (3β-HSD2) deficiency is a rare autosomal recessive form of congenital adrenal hyperplasia (CAH). We report the genetic basis of 3β-HSD2 deficiency arising from uniparental isodisomy (UPD) of chromosome 1. We describe a term undervirilized male whose newborn screen indicated borderline CAH. The patient presented on the 7th day of life in salt-wasting adrenal crisis. Steroid hormone testing revealed a complex pattern suggestive of 3β-HSD deficiency. Chromosomal microarray and single nucleotide polymorphism analysis revealed complete UPD of chromosome 1. Sanger sequencing of HSD3B2 revealed a previously described missense mutation, c.424G>A (p.E142K) in homozygous state, thus confirming the diagnosis of 3β-HSD2 deficiency. We provide evidence of the existence of an uncommon mechanism for HSD3B2 gene-related CAH arising from UPD of chromosome 1. PMID:27796263

  9. Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium.

    PubMed

    Raices, M; Paifer, E; Cremata, J; Montesino, R; Ståhlberg, J; Divne, C; Szabó, I J; Henriksson, G; Johansson, G; Pettersson, G

    1995-08-07

    The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5' end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide-binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.

  10. Cyclic Nucleotide Signaling in Polycystic Kidney Disease

    PubMed Central

    Wang, Xiaofang; Ward, Christopher J.; Harris, Peter C.; Torres, Vicente E.

    2013-01-01

    Increased levels of 3’–5’-cyclic adenosine monophosphate (cAMP) stimulate cell proliferation and fluid secretion in polycystic kidney disease (PKD). Since hydrolytic capacity of phosphodiesterases (PDE) far exceeds maximum rate of synthesis by adenylyl cyclases (AC), cellular levels of cAMP are more sensitive to PDE inhibition than to AC activity changes. We have used enzymatic, western blot, immunohistochemistry, PCR and biochemical assays to study activity and expression of PDE families and isoforms and expression of downstream effectors of cAMP signaling in wildtype and PKD rat and mouse kidneys. The results indicate: 1) Species specific differences in PDE expression; higher PDE activity in kidneys from mice compared to rats; higher contribution of PDE1, relative to PDE4 and PDE3, to total PDE activity of kidney lysate and lower PDE1, PDE3 and PDE4 activities in murine cystic compared to wildtype kidneys. 2) Reduced levels of several PDE1, PDE3 and PDE4 proteins despite mRNA upregulation, possibly due to increased protein degradation. 3) Increased cGMP levels in polycystic kidneys, suggesting in vivo downregulation of PDE1 activity. 4) Additive stimulatory effect of cAMP and cGMP on cystogenesis in vitro. 5) Upregulation of cAMP-dependent protein kinase (PKA) subunits Iα and IIβ, PKare, CREB-1 mRNA, and CREM, ATF-1 and ICER proteins in cystic compared to wildtype kidneys. In summary, the results of this study suggest that alterations in cyclic nucleotide catabolism may render the cystic epithelium particularly susceptible to factors acting on Gs coupled receptors, account at least in part for the upregulation of cyclic nucleotide signaling in PKD, and contribute substantially to the progression of this disease. PMID:19924104

  11. Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

    SciTech Connect

    Park, Yun-Hee; Patel, Mulchand S.

    2010-05-07

    Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.

  12. Participation of phosphofructokinase, malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa.

    PubMed

    Breininger, E; Dubois, D; Pereyra, V E; Rodriguez, P C; Satorre, M M; Cetica, P D

    2017-10-01

    The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10(10) spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/10(10) and 2.22 ± 0.14 U/10(10) spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10(10) spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa. © 2017 Blackwell Verlag GmbH.

  13. Association of nucleotides with homoionic clays. [catalysis of prebiotic materials

    NASA Technical Reports Server (NTRS)

    Odom, D. G.; Rao, M.; Oro, J.; Lawless, J. G.

    1979-01-01

    The binding of nucleotides to homoionic clays is studied as a possible mechanism for the concentration and catalysis of biological or prebiotic materials on the prebiotic earth. Samples of radioactively labeled adenosine and thymidine nucleotides were mixed in solutions with bentonite, kaolinite or Dowex-50 particles in which all exchangeable sites were occupied by Na, Mg, Ca, Mn, Fe, Cu or Zn ions. The binding of nucleotides to homoionic clays is observed, with adenosine nucleotides favored over thymidine, bentonite as the best absorber, and greater binding to clays homoionic in transition metal ions. Results indicate that the oligomerization of nucleotides may be possible by this mechanism, however difficulties in nucleotide variability and base pairing may arise due to the observed preference for purines at the adsorption sites.

  14. [Nucleotide receptors--structure and function, history and perspectives].

    PubMed

    Barańska, Jolanta

    2014-01-01

    First nucleotide receptors were discovered by Geoffrey Burnstock in 70ties of the last century, as a purinoreceptors activated by ATP. It was further found that they may be activated both by purine and pyrimidine nucleotides and their name was changed to nucleotide receptors. They are divided into two fsamilies: P1, activated by adenosine and P2, activated by nucleotides which are further divided into P2X and P2Y subfamilies. P2X are ionotropic receptors activated by ATP, P2Y (as the P1) are metabotropic receptors coupled with protein G. P2Y receptors are activated by ATP, ADP, UTP, UDP and UDP-sugar derivatives. This review describes early history of extracellular nucleotide signaling studies and presents current knowledge of the particular nucleotide receptors subtypes. The article also describes the structure and functional roles of these receptors and speculates about future research and therapeutic directions in this field.

  15. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    SciTech Connect

    Couto, Sheila G.; Cristina Nonato, M.

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  16. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  17. Structures of a DNA Polymerase Inserting Therapeutic Nucleotide Analogues.

    PubMed

    Schaich, Matthew A; Smith, Mallory R; Cloud, Ashley S; Holloran, Sean M; Freudenthal, Bret D

    2017-09-01

    Members of the nucleoside analogue class of cancer therapeutics compete with canonical nucleotides to disrupt numerous cellular processes, including nucleotide homeostasis, DNA and RNA synthesis, and nucleotide metabolism. Nucleoside analogues are triphosphorylated and subsequently inserted into genomic DNA, contributing to the efficacy of therapeutic nucleosides in multiple ways. In some cases, the altered base acts as a mutagen, altering the DNA sequence to promote cellular death; in others, insertion of the altered nucleotide triggers DNA repair pathways, which produce lethal levels of cytotoxic intermediates such as single and double stranded DNA breaks. As a prerequisite to many of these biological outcomes, the modified nucleotide must be accommodated in the DNA polymerase active site during nucleotide insertion. Currently, the molecular contacts that mediate DNA polymerase insertion of modified nucleotides remain unknown for multiple therapeutic compounds, despite decades of clinical use. To determine how modified bases are inserted into duplex DNA, we used mammalian DNA polymerase β (pol β) to visualize the structural conformations of four therapeutically relevant modified nucleotides, 6-thio-2'-deoxyguanosine-5'-triphosphate (6-TdGTP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (5-FdUTP), 5-formyl-deoxycytosine-5'-triphosphate (5-FodCTP), and 5-formyl-deoxyuridine-5'-triphosphate (5-FodUTP). Together, the structures reveal a pattern in which the modified nucleotides utilize Watson-Crick base pairing interactions similar to that of unmodified nucleotides. The nucleotide modifications were consistently positioned in the major groove of duplex DNA, accommodated by an open cavity in pol β. These results provide novel information for the rational design of new therapeutic nucleoside analogues and a greater understanding of how modified nucleotides are tolerated by polymerases.

  18. The druggability of intracellular nucleotide-degrading enzymes.

    PubMed

    Rampazzo, Chiara; Tozzi, Maria Grazia; Dumontet, Charles; Jordheim, Lars Petter

    2016-05-01

    Nucleotide metabolism is the target of a large number of anticancer drugs including antimetabolites and specific enzyme inhibitors. We review scientific findings that over the last 10-15 years have allowed the identification of several intracellular nucleotide-degrading enzymes as cancer drug targets, and discuss further potential therapeutic applications for Rcl, SAMHD1, MTH1 and cN-II. We believe that enzymes involved in nucleotide metabolism represent potent alternatives to conventional cancer chemotherapy targets.

  19. cDNA, genomic sequence cloning and overexpression of glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) from the Giant Panda.

    PubMed

    Hou, Wan-Ru; Hou, Yi-Ling; Du, Yu-Jie; Zhang, Tian; Hao, Yan-Zhe

    2010-01-01

    GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence of some diseases. The cDNA and the genomic sequence of GAPDH were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mu musculus, Rattus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could be used for further functional studies.

  20. Radial immunodiffusion and immunoelectrophoresis compared for identifying autoantibodies to lactate dehydrogenase in human serum.

    PubMed

    Harff, G A; Backer, E T

    1990-12-14

    Variant electrophoretic patterns of lactate dehydrogenase isoenzymes were studied. By radial immunodiffusion and immunoelectrophoresis, immunoglobulin and light chain class of autoantibodies to lactate dehydrogenase were identified in nine sera: seven of these sera demonstrated IgG (5 lambda, 2 kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase. We conclude that radial immunodiffusion and immunoelectrophoresis are equally effective for identifying auto-antibodies to lactate dehydrogenase in serum. Radial immunodiffusion, however, is easier to perform than immunoelectrophoresis.

  1. L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

    PubMed

    Deutch, Charles E

    2013-11-01

    The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

  2. Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases.

    PubMed

    Lange, L G; Vallee, B L

    1976-10-19

    A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.

  3. Asymmetric oxidoreductions catalyzed by alcohol dehydrogenase in organic solvents

    SciTech Connect

    Grunwald, J.; Wirz, B.; Scollar, M.P.; Klibanov, A.M.

    1986-10-15

    A methodology is developed for the use of alcohol dehydrogenase (and other NAD/sup +//NADH-dependent enzymes) as catalysts in organic solvents. The enzyme and the cofactor are deposited onto the surface of glass beads which are then suspended in a water-immiscible organic solvent containing the substrate. Both NADH and NAD/sup +/ are efficiently regenerated in such a system with alcohol dehydrogenase-catalyzed oxidation of ethanol and reduction of isobutyraldehyde, respectively; cofactor turnover numbers of 10/sup 5/ to greater than 10/sup 6/ have been obtained. With use of asymmetric oxidoreductions catalyzed by horse liver alcohol dehydrogenase in isopropyl ether, optically active (ee of 95 to 100%) alcohols and ketones have been prepared on a 1 to 10 mmol scale.

  4. Cloning and heterologous expression of two aryl-aldehyde dehydrogenases from the white-rot basidiomycete Phanerochaete chrysosporium

    SciTech Connect

    Nakamura, Tomofumi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-04-09

    We identified two aryl-aldehyde dehydrogenase proteins (PcALDH1 and PcALDH2) from the white-rot basidiomycete Phanerochaete chrysosporium. Both PcALDHs were translationally up-regulated in response to exogenous addition of vanillin, one of the key aromatic compounds in the pathway of lignin degradation by basidiomycetes. To clarify the catalytic functions of PcALDHs, we isolated full-length cDNAs encoding these proteins and heterologously expressed the recombinant enzymes using a pET/Escherichia coli system. The open reading frames of both PcALDH1 and PcALDH2 consisted of 1503 nucleotides. The deduced amino acid sequences of both proteins showed high homologies with aryl-aldehyde dehydrogenases from other organisms and contained ten conserved domains of ALDHs. Moreover, a novel glycine-rich motif 'GxGxxxG' was located at the NAD{sup +}-binding site. The recombinant PcALDHs catalyzed dehydrogenation reactions of several aryl-aldehyde compounds, including vanillin, to their corresponding aromatic acids. These results strongly suggested that PcALDHs metabolize aryl-aldehyde compounds generated during fungal degradation of lignin and various aromatic xenobiotics.

  5. Regulation of NAD+- and NADP+-linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6.

    PubMed

    Hofmann, K H; Babel, W

    1980-01-01

    Cell-free extracts of the obligate methanol-utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to alpha-ketoglutarate in the presence of NAD+ and NADP+. After electro-focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+-linked isocitrate dehydrogenase (NAD+-IDH) and of NADP+-linked isocitrate dehydrogenase (NADP+-IDH) could be observed. The NAD+-IDH was completely separated from the NADP+-IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G-A. The NAD+-IDH was inhibited by a high energy charge, whereas the NADP+-IDH was found to be independent of energy charge. Consequently the NAD+-IDH showed the control behaviour of an enzyme of an energy-generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and alpha-ketoglutarate differences between NAD+-IDH and NADP+-IDH were also found. Only the NADP+-linked enzyme exhibited a feedback inhibition by its reaction products alpha-ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+-IDH. These results confer an amphibolic character to the sequence from citrate to alpha-ketoglutarate in the incomplete citric-acid cycle of Pseudomonas W6.

  6. Homologues of yeast and bacterial rotenone-insensitive NADH dehydrogenases in higher eukaryotes: two enzymes are present in potato mitochondria.

    PubMed

    Rasmusson, A G; Svensson, A S; Knoop, V; Grohmann, L; Brennicke, A

    1999-10-01

    Two different cDNAs, homologous to genes for rotenone-insensitive NADH dehydrogenases of bacteria and yeast, were isolated from potato. The encoded proteins, called NDA and NDB, have calculated molecular masses of 55 and 65 kDa, respectively. The N-terminal parts show similarity to mitochondrial targeting peptides and the polypeptides are in vitro imported into potato mitochondria. Import processing to a smaller polypeptide is seen for the NDA but not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the intermembrane space. Imported proteins are associated to membranes upon digitonin permeabilization. On expression in Escherichia coli, NDB is released from the bacterial membrane in the absence of divalent cations whereas detergents are necessary for solubilization of NDA. Both deduced amino-acid sequences contain the dual motifs for nucleotide binding with the characteristics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydro- genases, the NDB amino-acid sequence contains a non-conserved insert, which is similar to EF-hand motifs for calcium binding. Phylogenetic analyses group the rotenone-insensitive NADH dehydrogenases largely by species, but suggest ancient gene duplications.

  7. Molecular basis for thermoprotection in Bemisia: structural differences between whitefly ketose reductase and other medium-chain dehydrogenases/reductases.

    PubMed

    Wolfe, G R; Smith, C A; Hendrix, D L; Salvucci, M E

    1999-02-01

    The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) accumulates sorbitol as a thermoprotectant in response to elevated temperature. Sorbitol synthesis in this insect is catalyzed by an unconventional ketose reductase (KR) that uses NADPH to reduce fructose. A cDNA encoding the NADPH-KR from adult B. argentifolii was cloned and sequenced to determine the primary structure of this enzyme. The cDNA encoded a protein of 352 amino acids with a calculated molecular mass of 38.2 kDa. The deduced amino acid sequence of the cDNA shared 60% identity with sheep NAD(+)-dependent sorbitol dehydrogenase (SDH). Residues in SDH involved in substrate binding were conserved in the whitefly NADPH-KR. An important structural difference between the whitefly NADPH-KR and NAD(+)-SDHs occurred in the nucleotide-binding site. The Asp residue that coordinates the adenosyl ribose hydroxyls in NAD(+)-dependent dehydrogenases (including NAD(+)-SDH), was replaced by an Ala in the whitefly NADPH-KR. The whitefly NADPH-KR also contained two neutral to Arg substitutions within four residues of the Asp to Ala substitution. Molecular modeling indicated that addition of the Arg residues and loss of the Asp decreased the electric potential of the adenosine ribose-binding pocket, creating an environment favorable for NADPH-binding. Because of the ability to use NADPH, the whitefly NADPH-KR synthesizes sorbitol under physiological conditions, unlike NAD(+)-SDHs, which function in sorbitol catabolism.

  8. Structural Characterization of CalS8, a TDP-α-D-Glucose Dehydrogenase Involved in Calicheamicin Aminodideoxypentose Biosynthesis.

    PubMed

    Singh, Shanteri; Michalska, Karolina; Bigelow, Lance; Endres, Michael; Kharel, Madan K; Babnigg, Gyorgy; Yennamalli, Ragothaman M; Bingman, Craig A; Joachimiak, Andrzej; Thorson, Jon S; Phillips, George N

    2015-10-23

    Classical UDP-glucose 6-dehydrogenases (UGDHs; EC 1.1.1.22) catalyze the conversion of UDP-α-d-glucose (UDP-Glc) to the key metabolic precursor UDP-α-d-glucuronic acid (UDP-GlcA) and display specificity for UDP-Glc. The fundamental biochemical and structural study of the UGDH homolog CalS8 encoded by the calicheamicin biosynthetic gene is reported and represents one of the first studies of a UGDH homolog involved in secondary metabolism. The corresponding biochemical characterization of CalS8 reveals CalS8 as one of the first characterized base-permissive UGDH homologs with a >15-fold preference for TDP-Glc over UDP-Glc. The corresponding structure elucidations of apo-CalS8 and the CalS8·substrate·cofactor ternary complex (at 2.47 and 1.95 Å resolution, respectively) highlight a notably high degree of conservation between CalS8 and classical UGDHs where structural divergence within the intersubunit loop structure likely contributes to the CalS8 base permissivity. As such, this study begins to provide a putative blueprint for base specificity among sugar nucleotide-dependent dehydrogenases and, in conjunction with prior studies on the base specificity of the calicheamicin aminopentosyltransferase CalG4, provides growing support for the calicheamicin aminopentose pathway as a TDP-sugar-dependent process.

  9. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints.

  10. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

    PubMed Central

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  11. Frequency and Correlation of Nearest Neighboring Nucleotides in Human Genome

    NASA Astrophysics Data System (ADS)

    Jin, Neng-zhi; Liu, Zi-xian; Qiu, Wen-yuan

    2009-02-01

    Zipf's approach in linguistics is utilized to analyze the statistical features of frequency and correlation of 16 nearest neighboring nucleotides (AA, AC, AG, ..., TT) in 12 human chromosomes (Y, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, and 12). It is found that these statistical features of nearest neighboring nucleotides in human genome: (i) the frequency distribution is a linear function, and (ii) the correlation distribution is an inverse function. The coefficients of the linear function and inverse function depend on the GC content. It proposes the correlation distribution of nearest neighboring nucleotides for the first time and extends the descriptor about nearest neighboring nucleotides.

  12. Metabolism of glycyrrhetic acid by rat liver microsomes: glycyrrhetinate dehydrogenase.

    PubMed

    Akao, T; Akao, T; Kobashi, K

    1990-02-06

    Glycyrrhetic acid, derived from a main component of liquorice, was converted to 3-ketoglycyrrhetic acid reversibly by rat liver homogenates in the presence of NADPH or NADP+. Glycyrrhetic acid-oxidizing and 3-ketoglycyrrhetic acid-reducing activities were localized in microsomes among the subcellular fractions of rat liver. Glycyrrhetic acid-oxidizing activity and 3-ketoglycyrrhetic acid-reducing activities showed pH optima at 6.3 and 8.5, respectively, and required NADP+ or NAD+ and NADPH or NADH, respectively, indicating that these activities were due to glycyrrhetinate dehydrogenase. The dehydrogenase was not solubilized from the membranes by the treatment with 1 M NaCl or sonication, indicating that the enzyme is a membrane component. The dehydrogenase was solubilized with detergents such as Emalgen 913, Triton X-100 and sodium cholate, and then separated from 3 beta-hydroxysteroid dehydrogenase (5 beta-androstan-3 beta-ol-17-one-oxidizing activity) by butyl-Toyopearl 650 M column chromatography. Partially purified enzyme catalyzed the reversible reaction between glycyrrhetic acid and 3-ketoglycyrrhetic acid, but was inactive toward 3-epiglycyrrhetic acid and other steroids having the 3 beta-hydroxyl group. The enzyme required NADP+ and NADPH for the highest activities of oxidation and reduction, respectively, and NAD+ and NADH for considerable activities, similar to the results with microsomes. From these results the enzyme is defined as glycyrrhetinate dehydrogenase, being quite different from 3 beta-hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestine, which is active for both glycyrrhetic acid and steroids having the 3 beta-hydroxyl group.

  13. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    SciTech Connect

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C.

    2013-09-18

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases that includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal differences in

  14. Prostaglandin dehydrogenase and the initiation of labor.

    PubMed

    Challis, J R; Patel, F A; Pomini, F

    1999-01-01

    In summary, these studies have suggested that prostaglandin dehydrogenase may have a central role to play in the mechanisms which determine biologically active prostaglandin concentrations within human fetal membranes and placenta at the time of labor, at term or preterm. Moreover, our studies indicate that the regulation of PGDH may by multifactorial (figure 3). In certain regions of the membranes, we suggest that PGDH expression may be influenced by levels of anti-inflammatory and pro-inflammatory cytokines. In other regions of the membranes, we suggest that PGDH may be regulated at a transcriptional level by competing activities of progesterone and cortisol. The action of progesterone could be effected through systemically-derived steroid, or by locally synthesized steroid, acting in a paracrine and/or autocrine fashion. The effects of cortisol in placenta must be due to glucocorticoid derived from the maternal or fetal compartment, since the placenta lacks the hydroxylases required for endogenous cortisol production. However, metabolism of cortisol by 11 beta-HSD-2 reduces the potency of this glucocorticoid in placental tissue. In chorion however, cortisol may be formed locally, from cortisone, in addition to its being derived from the maternal circulation and/or from the amniotic fluid. Our current studies do not allow us to delineate whether the effects of progesterone and cortisol on PGDH are exerted through the glucocorticoid receptor (GR) or progesterone receptor (PR) or both. It is possible that through pregnancy, PGDH activity is maintained by progesterone acting either through low levels of PR in membranes, or, more likely, acting through GR. At term, elevated levels of cortisol compete with and displace progesterone from GR, resulting in inhibition of PGDH transcription and activity. In this way, local withdrawal of progesterone action would be effected within human intrauterine tissues, without requiring changes in systemic, circulating progesterone

  15. The α-ketoglutarate dehydrogenase complex in cancer metabolic plasticity.

    PubMed

    Vatrinet, Renaud; Leone, Giulia; De Luise, Monica; Girolimetti, Giulia; Vidone, Michele; Gasparre, Giuseppe; Porcelli, Anna Maria

    2017-01-01

    Deregulated metabolism is a well-established hallmark of cancer. At the hub of various metabolic pathways deeply integrated within mitochondrial functions, the α-ketoglutarate dehydrogenase complex represents a major modulator of electron transport chain activity and tricarboxylic acid cycle (TCA) flux, and is a pivotal enzyme in the metabolic reprogramming following a cancer cell's change in bioenergetic requirements. By contributing to the control of α-ketoglutarate levels, dynamics, and oxidation state, the α-ketoglutarate dehydrogenase is also essential in modulating the epigenetic landscape of cancer cells. In this review, we will discuss the manifold roles that this TCA enzyme and its substrate play in cancer.

  16. Purification of xanthine dehydrogenase and sulfite oxidase from chicken liver.

    PubMed

    Ratnam, K; Brody, M S; Hille, R

    1996-05-01

    Xanthine dehydrogenase and sulfite oxidase from chicken liver are oxomolybdenum enzymes which catalyze the oxidation of xanthine to uric acid and sulfite to sulfate, respectively. Independent purification protocols have been previously described for both enzymes. Here we describe a procedure by which xanthine dehydrogenase and sulfite oxidase are purified simultaneously from the same batch of fresh chicken liver. Also, unlike the protocols described earlier, this procedure avoids the use of acetone extraction as well as a heat step, thus minimizing damage to the molybdenum centers of the enzymes.

  17. The Pyruvate Dehydrogenase Complexes: Structure-based Function and Regulation*

    PubMed Central

    Patel, Mulchand S.; Nemeria, Natalia S.; Furey, William; Jordan, Frank

    2014-01-01

    The pyruvate dehydrogenase complexes (PDCs) from all known living organisms comprise three principal catalytic components for their mission: E1 and E2 generate acetyl-coenzyme A, whereas the FAD/NAD+-dependent E3 performs redox recycling. Here we compare bacterial (Escherichia coli) and human PDCs, as they represent the two major classes of the superfamily of 2-oxo acid dehydrogenase complexes with different assembly of, and interactions among components. The human PDC is subject to inactivation at E1 by serine phosphorylation by four kinases, an inactivation reversed by the action of two phosphatases. Progress in our understanding of these complexes important in metabolism is reviewed. PMID:24798336

  18. Reversible inactivation of CO dehydrogenase with thiol compounds

    SciTech Connect

    Kreß, Oliver; Gnida, Manuel; Pelzmann, Astrid M.; Marx, Christian; Meyer-Klaucke, Wolfram; Meyer, Ortwin

    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

  19. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    PubMed Central

    Johner, Andrea; Kunz, Stefan; Linder, Markus; Shakur, Yasmin; Seebeck, Thomas

    2006-01-01

    Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were

  20. In silico model-driven assessment of the effects of single nucleotide polymorphisms (SNPs) on human red blood cell metabolism.

    PubMed

    Jamshidi, Neema; Wiback, Sharon J; Palsson B, Bernhard Ø

    2002-11-01

    The completion of the human genome project and the construction of single nucleotide polymorphism (SNP) maps have lead to significant efforts to find SNPs that can be linked to pathophysiology. In silico models of complete biochemical reaction networks relate a cell's individual reactions to the function of the entire network. Sequence variations can in turn be related to kinetic properties of individual enzymes, thus allowing an in silico model-driven assessment of the effects of defined SNPs on overall cellular functions. This process is applied to defined SNPs in two key enzymes of human red blood cell metabolism: glucose-6-phosphate dehydrogenase and pyruvate kinase. The results demonstrate the utility of in silico models in providing insight into differences between red cell function in patients with chronic and nonchronic anemia. In silico models of complex cellular processes are thus likely to aid in defining and understanding key SNPs in human pathophysiology.

  1. Genetic relationships among Aedes aegypti collections in Venezuela as determined by mitochondrial DNA variation and nuclear single nucleotide polymorphisms.

    PubMed

    Urdaneta-Marquez, Ludmel; Bosio, Christopher; Herrera, Flor; Rubio-Palis, Yasmin; Salasek, Michael; Black, William C

    2008-03-01

    A population genetic analysis of gene flow was conducted among 619 Aedes aegypti from nine collections distributed among six geographic regions of Venezuela. Genetic markers included a 387-basepair region of the mitochondrial NADH dehydrogenase 4 (ND4) gene and single nucleotide polymorphisms (SNPs) at 11 nuclear loci. Genotypes at SNP loci were identified using melting curve analysis. Six different ND4 haplotypes were detected and patterns of variation suggested that collections were isolated by distance. The variance in SNP allele frequencies was much less than the variance in haplotype frequencies and a pattern of isolation by distance was not detected. Aedes aegypti from eight collections were orally challenged with dengue 2 virus. Disseminated infection rates ranged from 77% to 95%. The percentage of mosquitoes exhibiting a midgut infection barrier ranged from 2% to 15%, and those exhibiting a midgut escape barrier ranged from 2% to 18%. Venezuelan Ae. aegypti appear to be susceptible to dengue virus infection.

  2. A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase.

    PubMed

    Futer, Olga; Sintchak, Michael D; Caron, Paul R; Nimmesgern, Elmar; DeCenzo, Maureen T; Livingston, David J; Raybuck, Scott A

    2002-01-31

    The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.

  3. Structure of glyceraldehyde-3-phosphate dehydrogenase from the archaeal hyperthermophile Methanocaldococcus jannaschii

    PubMed Central

    Malay, Ali D.; Bessho, Yoshitaka; Ellis, Mark J.; Antonyuk, Svetlana V.; Strange, Richard W.; Hasnain, S. Samar; Shinkai, Akeo; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2009-01-01

    The X-ray crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj-GAPDH) was determined to 1.81 Å resolution. The crystal belonged to space group C2221, with unit-cell parameters a = 83.4, b = 152.0, c = 118.6 Å. The structure was solved by molecular replacement and was refined to a final R factor of 17.1% (R free = 19.8%). The final structure included the cofactor NADP+ at the nucleotide-binding site and featured unoccupied inorganic and substrate phosphate-binding sites. A comparison with GAPDH structures from mesophilic sources suggested that Mj-GAPDH is stabilized by extensive electrostatic interactions between the C-terminal α-helices and various distal loop regions, which are likely to contribute to thermal stability. The key phosphate-binding residues in the active site of Mj-GAPDH are conserved in other archaeal GAPDH proteins. These residues undergo a conformational shift in response to occupancy of the inorganic phosphate site. PMID:20054117

  4. Succinate-dependent energy generation and pyruvate dehydrogenase complex activity in isolated Ascaris suum mitochondria

    SciTech Connect

    Campbell, T.A.

    1988-01-01

    Body wall muscle from the parasitic nematode, Ascaris suum, contain unique anaerobic mitochondria that preferentially utilize fumarate and branched-chain enoyl CoA's as terminal electron acceptors instead of oxygen. While electron transport in these organelles is well characterized, the role of oxygen in succinate-dependent phosphorylation is still not clearly defined. Therefore, the present study was designed to more fully characterize succinate metabolism in these organelles as well as the in vitro regulation of a key mitochondrial enzyme, the pyruvate dehydrogenase complex (PDC). In the absence of added adenine nucleotides, incubations in succinate resulted in substantial elevations in intramitochrondrial ATP levels, but ATP/ADP ratios were considerably higher in incubations with malate. The stimulation of phosphorylation in aerobic incubations with succinate was rotenone sensitive and appears to be Site I dependent. Increase substrate level phosphorylation, coupled to propionate formation, or additional sites of electron-transport associated ATP synthesis were not significant. Under aerobic conditions, {sup 14}CO{sub 2} evolution from 1,4-({sup 14}C)succinate was stimulated and NADH/NAD{sup +} ratios were elevated, but the formation of {sup 14}C propionate was unchanged.

  5. Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in Trypanosoma cruzi.

    PubMed

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

  6. Glyceraldehyde 3-Phosphate Dehydrogenase-Telomere Association Correlates with Redox Status in Trypanosoma cruzi

    PubMed Central

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. PMID:25775131

  7. H2O2 release from the very long chain acyl-CoA dehydrogenase

    PubMed Central

    Kakimoto, Pâmela A.H.B.; Tamaki, Fábio K.; Cardoso, Ariel R.; Marana, Sandro R.; Kowaltowski, Alicia J.

    2015-01-01

    Enhanced mitochondrial generation of oxidants, including hydrogen peroxide (H2O2), is related to a large number of pathological conditions, including diet-induced obesity and steatohepatosis. Indeed, we have previously shown that high fat diets increase the generation of H2O2 in liver mitochondria energized by activated fatty acids. Here, we further study fatty-acid induced H2O2 release in liver mitochondria, and determine the characteristics that regulate it. We find that this production of H2O2 is independent of mitochondrial inner membrane integrity and insensitive to purine nucleotides. On the other hand, palmitate-induced H2O2 production is strongly enhanced by high fat diets and is pH-sensitive, with a peak at a matrix pH of ~8.5. Using recombinantly expressed human very long chain acyl-CoA dehydrogenase, we are able to demonstrate that palmitate-induced H2O2 release may be ascribed to the activity of this enzyme alone, acting as an oxidase. Our results add to a number of findings indicating that sources outside of the electron transport chain can generate significant, physiopathologically relevant, amounts of oxidants in mitochondria. PMID:25728796

  8. Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications.

    PubMed

    Bunik, Victoria; Artiukhov, Artem; Aleshin, Vasily; Mkrtchyan, Garik

    2016-12-14

    Glutamate dehydrogenase (GDH) of animal cells is usually considered to be a mitochondrial enzyme. However, this enzyme has recently been reported to be also present in nucleus, endoplasmic reticulum and lysosomes. These extramitochondrial localizations are associated with moonlighting functions of GDH, which include acting as a serine protease or an ATP-dependent tubulin-binding protein. Here, we review the published data on kinetics and localization of multiple forms of animal GDH taking into account the splice variants, post-translational modifications and GDH isoenzymes, found in humans and apes. The kinetic properties of human GLUD1 and GLUD2 isoenzymes are shown to be similar to those published for GDH1 and GDH2 from bovine brain. Increased functional diversity and specific regulation of GDH isoforms due to alternative splicing and post-translational modifications are also considered. In particular, these structural differences may affect the well-known regulation of GDH by nucleotides which is related to recent identification of thiamine derivatives as novel GDH modulators. The thiamine-dependent regulation of GDH is in good agreement with the fact that the non-coenzyme forms of thiamine, i.e., thiamine triphosphate and its adenylated form are generated in response to amino acid and carbon starvation.

  9. Characterization of pH-induced transitions of Entamoeba histolytica D-phosphoglycerate dehydrogenase.

    PubMed

    Mishra, Vibhor; Kumar, Ashutosh; Ali, Vahab; Zhang, Kam Y J; Nozaki, Tomoyoshi

    2015-08-01

    Entamoeba histolytica D-phosphoglycerate dehydrogenase (EhPGDH) exists as a functionally active homodimer at pH 7. Our earlier studies have shown that ionic interactions are essentially required for the oligomeric status and activity of the protein. Present study focuses on pH associated structural modulations of EhPGDH. Far-UV CD spectra showed loss in the secondary structure of the protein as a function of low pH, however, the protein was not completely unfolded even at pH 2. Energy minimized average simulated models of EhPGDH at different pH show stable secondary structure elements in the nucleotide binding domain (NBD) however, the substrate binding domain (SBD) was more sensitive toward acidic pH and completely unfolds at pH 2. The data suggest presence of partially folded/unfolded intermediate state at pH 2. Size exclusion chromatography shows that this intermediate has larger hydrodynamic radius compared with dimer (pH 7) or monomer (pH 5). The intermediate has poor tertiary organization with significantly exposed hydrophobic patches monitored by pH-dependent fluorescence spectroscopy and molecular dynamic simulations. Collectively, the results suggest that the two domains (NBD and SBD) of EhPGDH have independent pH-dependent structural transitions with stabilization of an intermediate state at pH 2.

  10. The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression.

    PubMed Central

    Barderi, P; Campetella, O; Frasch, A C; Santomé, J A; Hellman, U; Pettersson, U; Cazzulo, J J

    1998-01-01

    NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme. PMID:9480915

  11. Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding Gene as a Useful Taxonomic Tool for Staphylococcus spp.

    PubMed Central

    Yugueros, Javier; Temprano, Alejandro; Berzal, Beatriz; Sánchez, María; Hernanz, Carmen; Luengo, José María; Naharro, Germán

    2000-01-01

    The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific. PMID:11101563

  12. Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp.

    PubMed

    Yugueros, J; Temprano, A; Berzal, B; Sánchez, M; Hernanz, C; Luengo, J M; Naharro, G

    2000-12-01

    The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.

  13. Triazole inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

    PubMed Central

    Maurya, Sushil K.; Gollapalli, Deviprasad R.; Kirubakaran, Sivapriya; Zhang, Minjia; Johnson, Corey R.; Benjamin, Nicole N.; Hedstrom, Lizbeth; Cuny, Gregory D.

    2010-01-01

    Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Since C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure-activity relationship study revealed that a small alkyl group on the alpha-position of the ether was required with the (R)-enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the 3- and/or 4-positions of the pendent phenyl ring were best and conversion of the quinoline containing inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis. PMID:19624136

  14. Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies

    PubMed Central

    Arakaki, Tracy L; Buckner, Frederick S; Gillespie, J Robert; Malmquist, Nicholas A; Phillips, Margaret A; Kalyuzhniy, Oleksandr; Luft, Joseph R; DeTitta, George T; Verlinde, Christophe L M J; Van Voorhis, Wesley C; Hol, Wim G J; Merritt, Ethan A

    2010-01-01

    Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosyn-thetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream-form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluoruracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 Å. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH. PMID:18312275

  15. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    PubMed

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients.

  16. New PCR Assay Using Glucose-6-Phosphate Dehydrogenase for Identification of Leishmania Species

    PubMed Central

    Castilho, Tiago M.; Shaw, Jeffrey Jon; Floeter-Winter, Lucile M.

    2003-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) is one of the multilocus enzymes used to identify Leishmania by zymodeme analysis. The polymorphic pattern revealed by partial characterization of the gene encoding G6PD generated molecular markers useful in the identification of different Leishmania species by PCR. Initially degenerate oligonucleotides were designed on the basis of data on the conserved active center described for other organisms. Primers for reverse transcription-PCR experiments, designed from the nucleotide sequence of the PCR product, enabled us to characterize the 5′ and 3′ untranslated regions and the G6PD open reading frame of reference strains of Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Leishmania) mexicana, and Leishmania (Leishmania) amazonensis. Sets of paired primers were designed and used in PCR assays to discriminate between the parasites responsible for tegumentar leishmaniasis of the subgenera Leishmania (Leishmania) and Leishmania (Viannia) and to distinguish L. (Viannia) braziliensis from others organisms of the subgenus Leishmania (Viannia). No amplification products were detected for the DNA of Crithidia fasciculata, Trypanosoma cruzi, or Leishmania (Sauroleishmania) tarentolae or DNA from a healthy human control. The tests proved to be specific and were sensitive enough to detect parasites in human biopsy specimens. The successful discrimination of L. (Viannia) braziliensis from other parasites of the subgenus Leishmania (Viannia) opens the way to epidemiological studies in areas where more than one species of the subgenus Leishmania (Viannia) exist, such as Amazonia, as well as follow-up studies after chemotherapy and assessment of clinical prognoses. PMID:12574243

  17. A marriage full of surprises: forty-five years living with glutamate dehydrogenase.

    PubMed

    Engel, Paul C

    2011-09-01

    Detailed kinetic studies of bovine glutamate dehydrogenase [GDH] from the 1960s revealed complexities that remain to be fully explained. In the absence of heterotropic nucleotide regulators the enzyme follows a random pathway of substrate addition but saturation with ADP enforces a compulsory-order mechanism in which glutamate is the leading substrate. The rate dependence on NAD(P)(+) concentration is complex and is probably only partly explained by negative binding cooperativity. Bovine GDH eluded successful analysis by crystallographers for 30 years but the final structural solution presented in this symposium at last provides a comprehensible framework for much of the heterotropic regulation, focussing attention on an antenna region in the C-terminal tail, a structure that is missing in the slightly smaller hexameric GDHs of lower organisms. Nonetheless, our studies with one such smaller (clostridial) GDH reveal that even without the antenna the underlying core structure still mediates homotropic cooperativity, and the ability to generate a variety of mutants has made it possible to start to dissect this machinery. In addition, this short personal review discusses a number of unresolved issues such as the significance of phospholipid inhibition and of specific interaction with mRNA, and above all the question of why it is necessary to regulate an enzyme reputedly maintaining its reactants at equilibrium and whether this might be in some way related to its coexistence with an energy-linked transhydrogenase.

  18. The role of IMP dehydrogenase 2 in Inauhzin-induced ribosomal stress.

    PubMed

    Zhang, Qi; Zhou, Xiang; Wu, RuiZhi; Mosley, Amber; Zeng, Shelya X; Xing, Zhen; Lu, Hua

    2014-10-27

    The 'ribosomal stress (RS)-p53 pathway' is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. Inosine monophosphate (IMP) dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ suppresses cancer cell growth by dually targeting SIRT1 and IMPDH2.

  19. Selective and potent urea inhibitors of Cryptosporidium parvum inosine 5′ monophosphate dehydrogenase

    PubMed Central

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Liu, Xiaoping; Sharling, Lisa; Gollapalli, Deviprasad R.; Striepen, Boris; Hedstrom, Lizbeth; Cuny, Gregory D.

    2012-01-01

    Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC50 < 2 nM) against CpIMPDH, excellent selectivity > 1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model. PMID:22950983

  20. Age-related compensatory activation of pyruvate dehydrogenase complex in rat heart.

    PubMed

    Moreau, Régis; Heath, Shi-Hua D; Doneanu, Catalin E; Harris, Robert A; Hagen, Tory M

    2004-12-03

    Mitochondrial uptake and beta-oxidation of long-chain fatty acids are markedly impaired in the aging rat heart. While these alterations would be expected to adversely affect overall pyridine nucleotides, NADH levels do not change significantly with age. This conundrum suggests that specific compensatory mechanisms occur in the aging heart. The comparison of cardiac pyruvate dehydrogenase complex (PDC) kinetics in 4- and 24- to 28-month-old F344 rats revealed a 60% significant increase in V(max) with no change in PDC expression, and a 1.6-fold decrease in the Michaelis constant (K(m)) in old compared to young rats. The observed kinetic adjustments were selective to PDC, as neither the V(max) nor K(m) of citrate synthase changed with age. PDC kinase-4 mRNA levels decreased by 57% in old vs young rat hearts and correlated with a 45% decrease in PDC phosphorylation. We conclude that PDC from old rat hearts catabolizes pyruvate more efficiently due to an adaptive change in phosphorylation.

  1. Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications

    PubMed Central

    Bunik, Victoria; Artiukhov, Artem; Aleshin, Vasily; Mkrtchyan, Garik

    2016-01-01

    Glutamate dehydrogenase (GDH) of animal cells is usually considered to be a mitochondrial enzyme. However, this enzyme has recently been reported to be also present in nucleus, endoplasmic reticulum and lysosomes. These extramitochondrial localizations are associated with moonlighting functions of GDH, which include acting as a serine protease or an ATP-dependent tubulin-binding protein. Here, we review the published data on kinetics and localization of multiple forms of animal GDH taking into account the splice variants, post-translational modifications and GDH isoenzymes, found in humans and apes. The kinetic properties of human GLUD1 and GLUD2 isoenzymes are shown to be similar to those published for GDH1 and GDH2 from bovine brain. Increased functional diversity and specific regulation of GDH isoforms due to alternative splicing and post-translational modifications are also considered. In particular, these structural differences may affect the well-known regulation of GDH by nucleotides which is related to recent identification of thiamine derivatives as novel GDH modulators. The thiamine-dependent regulation of GDH is in good agreement with the fact that the non-coenzyme forms of thiamine, i.e., thiamine triphosphate and its adenylated form are generated in response to amino acid and carbon starvation. PMID:27983623

  2. A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging.

    PubMed

    Schauer, Marianne; Kottek, Tina; Schönherr, Madeleine; Bhattacharya, Animesh; Ibrahim, Saleh M; Hirose, Misa; Köhling, Rüdiger; Fuellen, Georg; Schmitz, Ulf; Kunz, Manfred

    2015-04-20

    Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

  3. Aldehyde dehydrogenase 2 is associated with cognitive functions in patients with Parkinson’s disease

    PubMed Central

    Yu, Rwei-Ling; Tan, Chun-Hsiang; Lu, Ying-Che; Wu, Ruey-Meei

    2016-01-01

    Neurotransmitter degradation has been proposed to cause the accumulation of neurotoxic metabolites. The metabolism of these metabolites involves aldehyde dehydrogenase 2 (ALDH2). The Asian-specific single nucleotide polymorphism rs671 causes reduced enzyme activity. This study aims to explore whether Parkinson’s disease (PD) patients with reduced ALDH2 activity owing to the rs671 polymorphism are at risk for neuropsychological impairments. A total of 139 PD patients were recruited. Each participant was assessed for medical characteristics and their ALDH2 genotype. The Mini-Mental State Examination (MMSE), the Clinical Dementia Rating Scale and the Frontal Behavioral Inventory were used to measure neuropsychological functions. We found that the MMSE scores were significantly lower in patients with inactive ALDH2 (U = 1873.5, p = 0.02). The presence of cognitive impairments was significantly more frequent in the inactive ALDH2 group (46.0%) than in the active ALDH2 group (26.3%) (χ2 = 5.886, p = 0.01). The inactive group showed significant deterioration in hobbies and exhibited more severe “disorganization” and “hyper-sexuality” behaviours. The additive effects of the allele on the development of cognitive impairments in PD patients may be an important finding that provides further insight into the pathogenic mechanism of cognitive dysfunction in PD. PMID:27453488

  4. A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging

    PubMed Central

    Schauer, Marianne; Kottek, Tina; Schönherr, Madeleine; Bhattacharya, Animesh; Ibrahim, Saleh M; Hirose, Misa; Köhling, Rüdiger; Fuellen, Georg; Schmitz, Ulf; Kunz, Manfred

    2015-01-01

    Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways. PMID:25839158

  5. Pyridine nucleotide coenzymes: Chemical, biological, and medical aspects. Vol. 2, Pt. A

    SciTech Connect

    Dolphin, D.; Poulson, R.; Avramovic, O.

    1987-01-01

    This text contains the following: History of the Pyridine Nucleotides Nomenclature; Evolution of Pyridine Nucleotide; Relationship Between Biosynthesis and Evolution; Crystal Structure; Coenzyme Conformations; Protein Interactions; Optical Spectroscopy of the Pyridine Nucleotides; Excited States of Pyridine Nucleotide Coenzymes; Fluorescence and Phosphorescence; Nuclear Magnetic Resonance Spectroscopy of Pyridine Nucleotides; Mass Spectrometry of Pyridine Nucleotides; Mechanism of Action of the Pyridine Nucleotides; Chemical Stability and Reactivity of Pyridine Nucleotide Coenzymes; Stereochemistry of Fatty Acid Biosynthesis and Metabolism; Kinetics of Pyridine Nucleotide-Utilizing Enzymes; Preparation and Properties of NAD and NADP Analogs; Model Studies and Biological Activity of Analogs; and Spin-Labeled Pyridine Nucleotide Derivatives.

  6. Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase.

    PubMed

    Adroher, F J; Osuna, A; Lupiañez, J A

    1988-11-15

    The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.

  7. The pca-pob supraoperonic cluster of Acinetobacter calcoaceticus contains quiA, the structural gene for quinate-shikimate dehydrogenase.

    PubMed Central

    Elsemore, D A; Ornston, L N

    1994-01-01

    An 18-kbp Acinetobacter calcoaceticus chromosomal segment contains the pcaIJFBDKCHG operon, which is required for catabolism of protocatechuate, and pobSRA, genes associated with conversion of p-hydroxybenzoate to protocatechuate. The genetic function of the 6.5 kbp of DNA between pcaG and pobS was unknown. Deletions in this DNA were designed by removal of fragments between restriction sites, and the deletion mutations were introduced into A. calcoaceticus by natural transformation. The mutations prevented growth with either quinate or shikimate, growth substrates that depend upon qui gene function for their catabolism to protocatechuate. The location of quiA, a gene encoding quinate-shikimate dehydrogenase, was indicated by its expression in one of the deletion mutants, and the position of the gene was confirmed by determination of its 2,427-bp nucleotide sequence. The deduced amino acid sequence of QuiA confirmed that it is a member of a family of membrane-associated, pyrrolo-quinoline quinone-dependent dehydrogenases, as had been suggested by earlier biochemical investigations. Catabolism of quinate and skikimate is initiated by NAD(+)-dependent dehydrogenases in other microorganisms, so it is evident that different gene pools were called upon to provide the ancestral enzyme for this metabolic step. Images PMID:8002591

  8. Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism.

    PubMed

    Jeong, Dae-won; Cho, Il Taeg; Kim, Tae Soo; Bae, Gun Won; Kim, Ik-Hwan; Kim, Ick Young

    2006-03-01

    In order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (GPDH), we engineered a CHO dhfr(-) cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3/anti-LDH cells). LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease according to the order: cell lines GP3/anti-LDH > anti-LDH > GP3 > CHO. Intracellular ATP contents, representing the extent of respiration rate, also decreased, according to a rank order as follows: GP3 > CHO > GP3/anti-LDH > anti-LDH. We also attempted to identify and characterize any physiological changes occurring in the cells which harbored diverse metabolic pathways. First, anti-LDH cells with heightened respiration rates were found to display a higher degree of sensitivity to the prooxidant tert-butyl hydroperoxide (tBOOH), and the mitochondrial complex III inhibitor, antimycin A, than the GPDH-expressing cells (GP3 and GP3/anti-LDH), which have a lower respiration rate. Second, the anti-sense LDH-A RNA-expressing cells (anti-LDH and GP3/anti-LDH) evidenced a higher degree of resistance to apoptosis by cell-cell contact inhibition, and a faster doubling time ( approximately 19 h compared with approximately 26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO(3) (-)-free conditions to induce a steep acidification could be maintained with the anti-sense LDH-A RNA-expressing cells, but could not be maintained with the CHO and GP3 cells. Third, we observed that the most appropriate cell line for the optical production of a certain therapeutic protein (Tissue-Plasminogen Activator) was the GP3/anti-LDH cells. Collectively, our data indicate a variety of physiological roles for LDH and GPDH, including cellular acidosis, oxidoresistance, apoptosis by both acidosis and cell-cell contact inhibition, cell growth, and the generation of

  9. BOND: Basic OligoNucleotide Design

    PubMed Central

    2013-01-01

    Background DNA microarrays have become ubiquitous in biological and medical research. The most difficult problem that needs to be solved is the design of DNA oligonucleotides that (i) are highly specific, that is, bind only to the intended target, (ii) cover the highest possible number of genes, that is, all genes that allow such unique regions, and (iii) are computed fast. None of the existing programs meet all these criteria. Results We introduce a new approach with our software program BOND (Basic OligoNucleotide Design). According to Kane’s criteria for oligo design, BOND computes highly specific DNA oligonucleotides, for all the genes that admit unique probes, while running orders of magnitude faster than the existing programs. The same approach enables us to introduce also an evaluation procedure that correctly measures the quality of the oligonucleotides. Extensive comparison is performed to prove our claims. BOND is flexible, easy to use, requires no additional software, and is freely available for non-commercial use from http://www.csd.uwo.ca/∼ilie/BOND/. Conclusions We provide an improved solution to the important problem of oligonucleotide design, including a thorough evaluation of oligo design programs. We hope BOND will become a useful tool for researchers in biological and medical sciences by making the microarray procedures faster and more accurate. PMID:23444904

  10. Human molecular cytogenetics: From cells to nucleotides.

    PubMed

    Riegel, Mariluce

    2014-03-01

    The field of cytogenetics has focused on studying the number, structure, function and origin of chromosomal abnormalities and the evolution of chromosomes. The development of fluorescent molecules that either directly or via an intermediate molecule bind to DNA has led to the development of fluorescent in situ hybridization (FISH), a technology linking cytogenetics to molecular genetics. This technique has a wide range of applications that increased the dimension of chromosome analysis. The field of cytogenetics is particularly important for medical diagnostics and research as well as for gene ordering and mapping. Furthermore, the increased application of molecular biology techniques, such as array-based technologies, has led to improved resolution, extending the recognized range of microdeletion/microduplication syndromes and genomic disorders. In adopting these newly expanded methods, cytogeneticists have used a range of technologies to study the association between visible chromosome rearrangements and defects at the single nucleotide level. Overall, molecular cytogenetic techniques offer a remarkable number of potential applications, ranging from physical mapping to clinical and evolutionary studies, making a powerful and informative complement to other molecular and genomic approaches. This manuscript does not present a detailed history of the development of molecular cytogenetics; however, references to historical reviews and experiments have been provided whenever possible. Herein, the basic principles of molecular cytogenetics, the technologies used to identify chromosomal rearrangements and copy number changes, and the applications for cytogenetics in biomedical diagnosis and research are presented and discussed.

  11. Single nucleotide polymorphisms and suicidal behaviour.

    PubMed

    Pregelj, Peter

    2012-09-01

    The World Health Organization estimates that almost one million deaths each year are attributable to suicide, and suicide attempt is close to 10 times more common than suicide completion. Suicidal behaviour has multiple causes that are broadly divided into proximal stressors or triggers and predisposition such as genetic. It is also known that single nucleotide polymorphisms (SNPs) occur throughout a human DNA influencing the structure, quantity and the function of proteins and other molecules. Abnormalities of the serotonergic system were observed in suicide victims. Beside 5-HT1A and other serotonin receptors most studied are the serotonin transporter 5' functional promoter variant, and monoamine oxidase A and the tryptophan-hydroxylase 1 and 2 (TPH) polymorphisms. It seems that especially genes regulating serotoninergic system and neuronal systems involved in stress response are associated with suicidal behaviour. Most genetic studies on suicidal behaviour have considered a small set of functional polymorphisms relevant mostly to monoaminergic neurotransmission. However, genes involved in regulation of other factors such as brain-derived neurotropic factor seems to be even more relevant for further research.

  12. Davydov's solitons in a homogeneous nucleotide chain

    NASA Astrophysics Data System (ADS)

    Lakhno, Victor D.

    Charge transfer in homogeneous nucleotide chains is modeled on the basis of Holstein Hamiltonian. The path length of Davydov solitons in these chains is being studied. It is shown that in a dispersionless case, when the soliton velocity V is small, the path length grows exponentially as V decreases. In this case, the state of a moving soliton is quasisteady. In the presence of dispersion determined by the dependenceΩ2 =Ω 02 + V 02κ2, the path length in the region 0 < V < V0 is equal to infinity. In this case, the phonon environment follows the charge motion. In the region V > V0, the soliton motion is accompanied by emission of phonons which leads to a finite path length of a soliton. The latter tends to infinity as V → V0 + 0 and V → ∞. The presence of dissipation leads to a finite soliton path length. An equilibrium velocity of soliton in an external electric field is calculated. It is shown that there is a maximum intensity of an electric field at which a steady motion of a soliton is possible. The soliton mobility is calculated for the stable or ohmic brunch.

  13. NDP kinase reactivity towards 3TC nucleotides.

    PubMed

    Kreimeyer, A; Schneider, B; Sarfati, R; Faraj, A; Sommadossi, J P; Veron, M; Deville-Bonne, D

    2001-05-01

    Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.

  14. Nucleotide sequence of mouse satellite DNA.

    PubMed Central

    Hörz, W; Altenburger, W

    1981-01-01

    The nucleotide sequence of uncloned mouse satellite DNA has been determined by analyzing Sau96I restriction fragments that correspond to the repeat unit of the satellite DNA. An unambiguous sequence of 234 bp has been obtained. The sequence of the first 250 bases from dimeric satellite fragments present in Sau96I limit digests corresponds almost exactly to two tandemly arranged monomer sequences including a complete Sau96I site in the center. This is in agreement with the hypothesis that a low level of divergence which cannot be detected in sequence analyses of uncloned DNA is responsible for the appearance of dimeric fragments. Most of the sequence of the 5% fraction of Sau96 monomers that are susceptible to TaqI has also been determined and has been found to agree completely with the prototype sequence. The monomer sequence is internally repetitious being composed of eight diverged subrepeats. The divergence pattern has interesting implications for theories on the evolution of mouse satellite DNA. PMID:6261227

  15. Adenine nucleotides of the stria vascularis.

    PubMed

    Thalmann, I; Marcus, N Y; Thalmann, R

    1979-01-01

    The levels of the adenine nucleotides ATP, ADP, and AMP in the stria vascularis were measured under normal conditions, and following various durations of ischemia. The concentrations of these compounds were used for the calculation of the adenylate energy charge, the energy status and the phosphorylation state of the stria. Following 10 min of ischemia the adenylate energy charge had decreased three fold, the energy status seven fold and the phosphorylation state 14 fold. To study the potential for recovery of strial function following various brief and prolonged ischemic intervals, a method for the perfusion of the ear via the anterior inferior cerebellar artery was developed. For various reasons it was found advantageous to use "artifical blood" as perfusate, relying upon fluorocarbons as oxygen carriers. The endolymphatic potential was used as electrical indicator of strial function. Recovery of the endolymphatic potential following brief periods of ischemia was paralleled by a corresponding increase of the ATP levels and a drastic decrease of the AMP levels of the stria vascularis. Preliminary results on the effects of substrate-free perfusion are presented.

  16. Functional compartmentation of the nucleotide pool

    SciTech Connect

    Volkin, E.

    1981-01-01

    Various lines of evidence show that the total cellular ribonucleoside triphosphate pool serves as the percursor pool for RNA synthesis. Other reports strongly support the thesis that the ribonucleotide pool is compartmentalized. In particular, the suggestion has been put forward that, in some cell lines, nucleosides such as uridine are rapidly channeled into the putative functional pool. The present experiments were designed to obtain a more direct answer to the question of nucleotide pool compartmentation. From these data, one can assess the feasibility of using specific activities of the total pool nucleoside triphosphates for calculating rates of RNA synthesis. Two cell lines were used in this investigation. A rat transformed tracheal cell line, cloned from a keratinizing squamous cell carcinoma, was grown as a stratified epithelium, and Novikoff hepatoma cell line, grown in suspension culture. Tritiated nucleoside and /sup 32/PO/sub 4/ were added to cells in exponential growth. Under the conditions used, between 85 to 100% of the RNA is hydrolyzed to 5'-mononucleotides. Furthermore, the enzyme was shown to be almost free of 5'-nucleotidase and totally devoid of deaminase and phosphoryl-transferring activities. (ERB)

  17. Phanerochaete chrysosporium Cellobiohydrolase and Cellobiose Dehydrogenase Transcripts in Wood

    PubMed Central

    Vallim, Marcelo A.; Janse, Bernard J. H.; Gaskell, Jill; Pizzirani-Kleiner, Aline A.; Cullen, Daniel

    1998-01-01

    The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation. PMID:9572973

  18. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hydroxybutyric dehydrogenase test system. 862.1380 Section 862.1380 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test...

  19. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  20. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Isocitric dehydrogenase test system. 862.1420 Section 862.1420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test...

  1. NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

    PubMed Central

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Ma, Yanhe

    2015-01-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD+ as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn174 was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases. PMID:26150461

  2. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene.

    PubMed

    Sadeghi, H Mir Mohammad; Ahmadi, R; Aghaabdollahian, S; Mofid, M R; Ghaemi, Y; Abedi, D

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities.

  3. Genetics Home Reference: 2-methylbutyryl-CoA dehydrogenase deficiency

    MedlinePlus

    ... down proteins from food into smaller parts called amino acids. Amino acids can be further processed to provide energy for ... methylbutyryl-CoA dehydrogenase deficiency cannot process a particular amino acid called isoleucine. Most cases of 2-methylbutyryl-CoA ...

  4. Distribution of the Pyruvate Dehydrogenase Complex in Developing Soybean Cotyledons

    USDA-ARS?s Scientific Manuscript database

    The somewhat surprising report that storage proteins and oil are non-uniformly distributed in the cotyledons of developing soybeans prompted us to determine the spatial distribution of the mitochondrial and plastidial forms of the pyruvate dehydrogenase complex (PDC). It has been proposed that pla...

  5. Red Algal Bromophenols as Glucose 6-Phosphate Dehydrogenase Inhibitors

    PubMed Central

    Mikami, Daisuke; Kurihara, Hideyuki; Kim, Sang Moo; Takahashi, Koretaro

    2013-01-01

    Five bromophenols isolated from three Rhodomelaceae algae (Laurencia nipponica, Polysiphonia morrowii, Odonthalia corymbifera) showed inhibitory effects against glucose 6-phosphate dehydrogenase (G6PD). Among them, the symmetric bromophenol dimer (5) showed the highest inhibitory activity against G6PD. PMID:24152564

  6. Molecular properties of succinate dehydrogenase isolated from Micrococcus luteus (lysodeikticus).

    PubMed Central

    Crowe, B A; Owen, P

    1983-01-01

    Succinate dehydrogenase (EC 1.3.99.1) of Micrococcus luteus was selectively precipitated from Triton X-100-solubilized membranes by using specific antiserum. The precipitated enzyme contained equimolar amounts of four polypeptides with apparent molecular weights of 72,000, 30,000, 17,000, and 15,000. The 72,000 polypeptide possessed a covalently bound flavin prosthetic group and appeared to be strongly antigenic as judged by immunoprinting experiments. Low-temperature absorption spectroscopy revealed the presence of cytochrome b556 in the antigen complex. By analogy with succinate dehydrogenase purified from other sources, the 72,000 and 30,000 polypeptides were considered to represent subunits of the succinate dehydrogenase enzyme, whereas one (or both) of the low-molecular-weight polypeptides was attributed to the apoprotein of the b-type cytochrome. A succinate dehydrogenase antigen cross-reacting with the M. luteus enzyme complex could be demonstrated in membranes of Micrococcus roseus, Micrococcus flavus, and Sarcina lutea, but not in the membranes isolated from a wide variety of other gram-positive and gram-negative bacteria. Images PMID:6402500

  7. Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase.

    PubMed Central

    Neuberger, M S; Patterson, R A; Hartley, B S

    1979-01-01

    An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization. Images Fig. 1. Fig. 2. PMID:393250

  8. Genetics Home Reference: 3-hydroxyacyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... step that metabolizes groups of fats called medium-chain fatty acids and short-chain fatty acids. Mutations in the HADH gene lead ... a shortage of 3-hydroxyacyl-CoA dehydrogenase. Medium-chain and short-chain fatty acids cannot be metabolized ...

  9. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sorbitol dehydrogenase test system. 862.1670 Section 862.1670 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES.... Measurements obtained by this device are used in the diagnosis and treatment of liver disorders such as...

  10. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false 6-Phosphogluconate dehydrogenase test system. 862.1565 Section 862.1565 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias. (b...

  11. Efficiency of superoxide anions in the inactivation of selected dehydrogenases

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

    2010-09-01

    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 rad 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.

  12. Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate ▿ †

    PubMed Central

    Rocha, Joana; Popescu, Alma O.; Borges, Patrícia; Mil-Homens, Dalila; Moreira, Leonilde M.; Sá-Correia, Isabel; Fialho, Arsénio M.; Frazão, Carlos

    2011-01-01

    Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate. PMID:21602353

  13. Virtual and experimental high-throughput screening (HTS) in search of novel inosine 5'-monophosphate dehydrogenase II (IMPDH II) inhibitors

    NASA Astrophysics Data System (ADS)

    Dunkern, Torsten; Prabhu, Arati; Kharkar, Prashant S.; Goebel, Heike; Rolser, Edith; Burckhard-Boer, Waltraud; Arumugam, Premkumar; Makhija, Mahindra T.

    2012-11-01

    IMPDH (Inosine 5'-monophosphate dehydrogenase) catalyzes a rate-limiting step in the de novo biosynthesis of guanine nucleotides. IMPDH inhibition in sensitive cell types (e.g., lymphocytes) blocks proliferation (by blocking RNA and DNA synthesis as a result of decreased cellular levels of guanine nucleotides). This makes it an interesting target for cancer and autoimmune disorders. Currently available IMPDH inhibitors such as mycophenolic acid (MPA, uncompetitive inhibitor) and nucleoside analogs (e.g., ribavirin, competitive inhibitor after intracellular activation by phosphorylation) have unfavorable tolerability profiles which limit their use. Hence, the quest for novel IMPDH inhibitors continues. In the present study, a ligand-based virtual screening using IMPDH inhibitor pharmacophore models was performed on in-house compound collection. A total of 50,000 virtual hits were selected for primary screen using in vitro IMPDH II inhibition up to 10 μM. The list of 2,500 hits (with >70 % inhibition) was further subjected to hit confirmation for the determination of IC50 values. The hits obtained were further clustered using maximum common substructure based formalism resulting in 90 classes and 7 singletons. A thorough inspection of these yielded 7 interesting classes in terms of mini-SAR with IC50 values ranging from 0.163 μM to little over 25 μM. The average ligand efficiency was found to be 0.3 for the best class. The classes thus discovered represent structurally novel chemotypes which can be taken up for further development.

  14. Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes.

    PubMed

    Nakamura, T; Yokota, S; Muramoto, Y; Tsutsui, K; Oguri, Y; Fukui, K; Takabe, T

    1997-05-01

    Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the plant biosynthetic pathway that leads to glycinebetaine. Rice plants (Oryza sativa L.), albeit considered a typical non-glycinebetaine accumulating species, have been found to express this enzyme at low levels. This observation evokes an interest in phylogenic evolution of the enzyme in the plant kingdom. It is reported here that rice plants possess the ability to take up exogenously added betaine aldehyde through the roots and convert it to glycinebetaine, resulting in an enhanced salt-tolerance of the plants. A gene encoding a putative BADH from the rice genome was also cloned and sequenced. The gene was found to contain 14 introns, and the overall nucleotide sequence of the coding region is c. 78% identical to that of the barley BADH cDNA. Cloning of a partial BADH cDNA from rice was accomplished by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence of the cloned fragment was found to be identical to the corresponding exon regions of the rice genomic BADH gene. The deduced amino acid sequences of rice and barley BADH both contain a C-terminal tripeptide SKL, a signal known to target preproteins to microbodies. This localization was confirmed by an immuno-gold labeling study of transgenic tobacco harboring barley cDNA, which showed BADH protein inside peroxisomes. Northern blot analysis revealed that the level of BADH mRNA is salt-inducible.

  15. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

  16. Inosine 5'-Monophosphate Dehydrogenase (IMPDH) as a Potential Target for the Development of a New Generation of Antiprotozoan Agents.

    PubMed

    Fotie, Jean

    2016-06-19

    Inosine-5'-monophosphate dehydrogenase (IMPDH) is a metabolic enzyme that catalyzes the critical step in guanine nucleotide biosynthesis, and thus is at the center of cell growth and proliferation. However, although this enzyme has been exploited as potential target for the development of immunosuppressive, anticancer, and antiviral agents, the functional importance of IMPDH as a promising antiprotozoan drug target is still in its infancy mainly because of the availability of alternative nucleotides metabolic pathways in many of these parasites. This situation suggests that the inhibition of IMPDH might have little to no effect on the survival of protozoan parasites. As a result, no IMPDH inhibitor is currently commercially available or has advanced to clinical trials as a potential antiprotozoan drug. Nevertheless, recent advances toward the development of selective inhibitors of the IMPDH enzyme from Crystosporidium parvum as potential drug candidates against cryptosporidiosis should revive further investigations of this drug target in other protozoa parasites. The current review examines the chemical structures and biological activities of reported protozoan's IMPDH inhibitors. SciFinder was used to broadly pinpoint reports published on the topic in the chemical literature, with no specific time frame. Opportunities and challenges towards the development of inhibitors of IMPDH enzymes from protozoa parasites as potential chemotherapies toward the respective diseases they cause are also discussed.

  17. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme glucose-6...

  18. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme glucose-6...

  19. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme glucose-6...

  20. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme glucose-6...

  1. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme glucose-6...

  2. Spatial variability of the dehydrogenase activity in forest soils

    NASA Astrophysics Data System (ADS)

    Błońska, Ewa; Lasota, Jarosław

    2014-05-01

    The aim of this study was to assess the spatial variability of the dehydrogenase activity (DH) in forest soils using geostatistics. We have studied variability soil dehydrogenase and their relationship with variability of some physic-chemical properties. Two study areas (A and B) were set up in southern Poland in the Zlotoryja Forest District. Study areas were covered by different types of vegetation (A- broadleaf forest with beech, ash and sycamore), B- coniferous forest with Norway spruce). The soils were classified as Dystric Cambisols (WRB 2006). The samples for laboratory testing were collected from 49 places on each areas. 15 cm of surface horizon of soil were taken (with previously removed litter). Dehydrogenase activity was marked with Lenhard's method according to the Casida procedure. Soil pH, nitrogen (N) and soil organic carbon (C) content (by LECO CNS 2000 carbon analyzer) was marked. C/N ratio was calculated. Particle size composition was determined using laser diffraction. Statistical analysis were performed using STATISTICA 10 software. Geostatistical analysis and mapping were done by application of GS 9+ (Gamma Design) and Surfer 11 (Golden Software). The activity of DH ranged between 5,02 and 71,20 mg TPP• kg-1 •24 h-1 on the A area and between 0,94 and 16,47 mg TPP• kg-1 •24 h-1. Differences in spatial variability of the analised features were noted. The variability of dehydrogenase activity on the A study area was described by an exponential model, whereas on the B study area the spatial correlation has not been noted. The relationship of dehydrogenase activity with the remaining parameters of soil was noted only in the case of A study area. The variability of organic carbon content on the A and B study areas were described by an exponential model. The variability of nitrogen content on both areas were described by an spherical model.

  3. Glutamate dehydrogenases: the why and how of coenzyme specificity.

    PubMed

    Engel, Paul C

    2014-01-01

    NAD(+) and NADP(+), chemically similar and with almost identical standard oxidation-reduction potentials, nevertheless have distinct roles, NAD(+) serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD(+)-dependent for glutamate oxidation, NADP(+)-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD(+) reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD(+) but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP(+) reduction by NADH, maintaining the coenzyme pools at different oxidation-reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD(+)-dependent, NADP(+)-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD(+) or for NADP(+) has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2'- and 3'-hydroxyls, dictating NAD(+) specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD(+) only, NADP(+) only, or in higher animals both.

  4. Pho5p and Newly Identified Nucleotide Pyrophosphatases/ Phosphodiesterases Regulate Extracellular Nucleotide Phosphate Metabolism in Saccharomyces cerevisiae

    PubMed Central

    Kennedy, Eileen J.; Pillus, Lorraine; Ghosh, Gourisankar

    2005-01-01

    Extracellular nucleotides play many biological roles, including intercellular communication and modulation of nucleotide receptor signaling, and are dependent on the phosphorylation state of the nucleotide. Regulation of nucleotide phosphorylation is necessary, and a specialized class of enzymes, nucleotide pyrophosphatases/phosphodiesterases (E-NPPs), has been identified in mammals to perform this function. Although the E-NPP class is conserved among complex eukaryotes, this system has not yet been identified in Saccharomyces cerevisiae. Using genetic and biochemical experiments, we show that two orthologs of the E-NPP family, referred to as Npp1p and Npp2p, exist in budding yeast and can perform nucleotide phosphate hydrolysis. This activity is enhanced during phosphate starvation, where hydrolyzed phosphates can be imported from extracellular sources and utilized to overcome phosphate starvation through the activity of the Pho5p acid phosphatase. The added compensatory effect by Pho5p is also a newly established role for Pho5p. This study demonstrates that extracellular nucleotide phosphate metabolism appears to be controlled by at least two independent regulatory mechanisms, uniting phosphate starvation with extracellular nucleotide regulation. PMID:16278456

  5. Catalytic Mechanism of Short Ethoxy Chain Nonylphenol Dehydrogenase Belonging to a Polyethylene Glycol Dehydrogenase Group in the GMC Oxidoreductase Family

    PubMed Central

    Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

    2013-01-01

    Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor. PMID:23306149

  6. NADH dehydrogenase-like behavior of nitrogen-doped graphene and its application in NAD(+)-dependent dehydrogenase biosensing.

    PubMed

    Gai, Pan-Pan; Zhao, Cui-E; Wang, Ying; Abdel-Halim, E S; Zhang, Jian-Rong; Zhu, Jun-Jie

    2014-12-15

    A novel electrochemical biosensing platform for nicotinamide adenine dinucleotide (NAD(+))-dependent dehydrogenase catalysis was designed using the nitrogen-doped graphene (NG), which had properties similar to NADH dehydrogenase (CoI). NG mimicked flavin mononucleotide (FMN) in CoI and efficiently catalyzed NADH oxidation. NG also acted as an electron transport "bridge" from NADH to the electrode due to its excellent conductivity. In comparison with a bare gold electrode, an 800 mV decrease in the overpotential for NADH oxidation and CoI-like behavior were observed at NG-modified electrode, which is the largest decrease in overpotential for NADH oxidation reported to date. The catalytic rate constant (k) for the CoI-like behavior of NG was estimated to be 2.3×10(5) M(-1) s(-1), which is much higher than that of other previously reported FMN analogs. The Michaelis-Menten constant (Km) of NG was 26 μM, which is comparable to the Km of CoI (10 μM). Electrodes modified with NG and NG/gold nanoparticals/formate dehydrogenase (NG/AuNPs/FDH) showed excellent analytical performance for the detection of NADH and formate. This electrode fabrication strategy could be used to create a universal biosensing platform for developing NAD(+)-dependent dehydrogenase biosensors and biofuel cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity.

    PubMed

    Robertson, D; Smith, C; Bolwell, G P

    1996-01-01

    UDP-glucose dehydrogenase is responsible for channelling UDP-glucose into the pool of UDP-sugars utilized in the synthesis of wall matrix polysaccharides and glycoproteins. It has been purified to homogeneity from suspension-cultured cells of French bean by a combination of hydrophobic-interaction chromatography, gel filtration and dye-ligand chromatography. The enzyme had a subunit of Mr 40,000. Km values were measured for UDP-glucose as 5.5 +/- 1.4 mM and for NAD+ as 20 +/- 3 microM. It was subject to inhibition by UDP-xylose. UDP-glucose dehydrogenase activity co-purified with alcohol dehydrogenase activity from suspension-cultured cells, elicitor-treated cells and elongating hypocotyls, even when many additional chromatographic steps were employed subsequently. The protein from each source was resolved into virtually identical patterns of isoforms on two-dimensional isoelectric focusing/PAGE. However, a combination of peptide mapping and sequence analysis, gel analysis using activity staining and kinetic analysis suggests that both activities are a function of the same protein. An antibody was raised and used to immunolocalize UDP-glucose dehydrogenase to developing xylem and phloem of French bean hypocotyl. Together with data published previously, these results are consistent with an important role in the regulation of carbon flux into wall matrix polysaccharides.

  8. Short Chain Dehydrogenase/Reductase Rdhe2 Is a Novel Retinol Dehydrogenase Essential for Frog Embryonic Development*

    PubMed Central

    Belyaeva, Olga V.; Lee, Seung-Ah; Adams, Mark K.; Chang, Chenbei; Kedishvili, Natalia Y.

    2012-01-01

    The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates. PMID:22291023

  9. Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

    PubMed

    Rozeboom, Henriëtte J; Yu, Shukun; Mikkelsen, Rene; Nikolaev, Igor; Mulder, Harm J; Dijkstra, Bauke W

    2015-12-01

    The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer. © 2015 The Protein Society.

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

    PubMed

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

    2012-03-01

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

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

    SciTech Connect

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

    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.

  12. Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes.

    PubMed Central

    Orchinik, M; Murray, T F; Franklin, P H; Moore, F L

    1992-01-01

    The recently characterized corticosteroid receptor on amphibian neuronal membranes appears to mediate rapid, stress-induced changes in male reproductive behaviors. Because the transduction mechanisms associated with this receptor are unknown, we performed radioligand binding studies to determine whether this steroid receptor is negatively modulated by guanyl nucleotides. The binding of [3H]corticosterone to neuronal membranes was inhibited by nonhydrolyzable guanyl nucleotides in both equilibrium saturation binding and titration studies. The addition of guanyl nucleotide plus unlabeled corticosterone induced a rapid phase of [3H]corticosterone dissociation from membranes that was not induced by addition of unlabeled ligand alone. Furthermore, the equilibrium binding of [3H]corticosterone and the sensitivity of the receptor to modulation by guanyl nucleotides were both enhanced by Mg2+. These results are consistent with the formation of a ternary complex of steroid, receptor, and guanine nucleotide-binding protein that is subject to regulation by guanyl nucleotides. Therefore, rapid signal transduction through corticosteroid receptors on neuronal membranes appears to be mediated by guanine nucleotide-binding proteins. PMID:1570300

  13. Identifying 2'-O-methylationation sites by integrating nucleotide chemical properties and nucleotide compositions.

    PubMed

    Chen, Wei; Feng, Pengmian; Tang, Hua; Ding, Hui; Lin, Hao

    2016-06-01

    2'-O-methylationation is an important post-transcriptional modification and plays important roles in many biological processes. Although experimental technologies have been proposed to detect 2'-O-methylationation sites, they are cost-ineffective. As complements to experimental techniques, computational methods will facilitate the identification of 2'-O-methylationation sites. In the present study, we proposed a support vector machine-based method to identify 2'-O-methylationation sites. In this method, RNA sequences were formulated by nucleotide chemical properties and nucleotide compositions. In the jackknife cross-validation test, the proposed method obtained an accuracy of 95.58% for identifying 2'-O-methylationation sites in the human genome. Moreover, the model was also validated by identifying 2'-O-methylation sites in the Mus musculus and Saccharomyces cerevisiae genomes, and the obtained accuracies are also satisfactory. These results indicate that the proposed method will become a useful tool for the research on 2'-O-methylation.

  14. Nucleic acid analysis using terminal-phosphate-labeled nucleotides

    DOEpatents

    Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

    2008-04-22

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  15. Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

    PubMed

    Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

    2014-07-01

    Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

    PubMed Central

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha

    2012-01-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)+ to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+ (S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, where Km,NAD is the Km for NAD+ and Km,NADP is the Km for NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+ as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156. PMID:22344653

  17. Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase.

    PubMed

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha; Kang, Yun Chan; Lee, Jung-Kul

    2012-05-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

  18. Nucleotide sequence of the pyruvate decarboxylase gene from Zymomonas mobilis.

    PubMed

    Neale, A D; Scopes, R K; Wettenhall, R E; Hoogenraad, N J

    1987-02-25

    Pyruvate decarboxylase (EC 4.1.1.1), the penultimate enzyme in the alcoholic fermentation pathway of Zymomonas mobilis, converts pyruvate to acetaldehyde and carbon dioxide. The complete nucleotide sequence of the structural gene encoding pyruvate decarboxylase from Zymomonas mobilis has been determined. The coding region is 1704 nucleotides long and encodes a polypeptide of 567 amino acids with a calculated subunit mass of 60,790 daltons. The amino acid sequence was confirmed by comparison with the amino acid sequence of a selection of tryptic fragments of the enzyme. The amino acid composition obtained from the nucleotide sequence is in good agreement with that obtained experimentally.

  19. Nucleotide Accumulation Induced in Staphylococcus aureus by Glycine

    PubMed Central

    Strominger, Jack L.; Birge, Claire H.

    1965-01-01

    Strominger, Jack L. (Washington University School of Medicine, St. Louis, Mo.), and Claire H. Birge. Nucleotide accumulation induced in Staphylococcus aureus by glycine. J. Bacteriol. 89:1124–1127. 1965.—High concentrations of glycine induce accumulation of four uridine nucleotides in Staphylococcus aureus. Investigations of their structure suggest that these compounds are uridine diphosphate (UDP)-acetylmuramic acid, UDP-acetylmuramyl-gly-d-glu-l-lys, UDP-acetylmuramyl-l-ala-d-glu-l-lys and UDP-acetylmuramyl-gly-d-glu-l-lys-d-ala-d-ala. The mechanism by which glycine may induce uridine nucleotide accumulation and protoplast formation is discussed. Images PMID:14276106

  20. Dependence of the Excitability of Pituitary Cells on Cyclic Nucleotides

    PubMed Central

    Stojilkovic, Stanko S.; Kretschmannova, Karla; Tomic, Melanija; Stratakis, Constantine A.

    2012-01-01

    Cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate are intracellular (second) messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. These enzymes are involved in a broad array of signal transduction pathways mediated by the cyclic nucleotide monophosphates and their kinases, which control multiple aspects of cell function through the phosphorylation of protein substrates. Here, we review the findings and working hypotheses on the role of the cyclic nucleotides and their kinases in the control of electrical activity of the endocrine pituitary cells and the plasma membrane channels involved in this process. PMID:22564128

  1. Single-nucleotide polymorphism discovery by targeted DNA photocleavage.

    PubMed

    Hart, Jonathan R; Johnson, Martin D; Barton, Jacqueline K

    2004-09-28

    Single-nucleotide polymorphisms are the largest source of genetic variation in humans. We report a method for the discovery of single-nucleotide polymorphisms within genomic DNA. Pooled genomic samples are amplified, denatured, and annealed to generate mismatches at polymorphic DNA sites. Upon photoactivation, these DNA mismatches are then cleaved site-specifically by using a small molecular probe, a bulky metallointercalator, Rhchrysi or Rhphzi. Fluorescent labeling of the cleaved products and separation by capillary electrophoresis permits rapid identification with single-base resolution of the single-nucleotide polymorphism site. This method is remarkably sensitive and minor allele frequencies as low as 5% can be readily detected.

  2. Building phylogenetic trees by using gene Nucleotide Genomic Signals.

    PubMed

    Cristea, Paul Dan

    2012-01-01

    Nucleotide genomic signal (NuGS) methodology allows a molecular level approach to determine distances between homologous genes or between conserved equivalent non-coding genome regions in various species or individuals of the same species. Therefore, distances between the genes of species or individuals can be computed and phylogenetic trees can be built. The paper illustrates the use of the nucleotide imbalance (N) and nucleotide pair imbalance (P) signals to determine the distances between the genes of several Hominidae. The results are in accordance with those of other genetic or phylogenetic approaches to establish distances between Hominidae species.

  3. Time-resolved FRET for single-nucleotide polymorphism genotyping

    NASA Astrophysics Data System (ADS)

    Andreoni, Alessandra; Nardo, Luca; Bondani, Maria

    2009-05-01

    By tens-of-picosecond resolved fluorescence detection (TCSPC, time-correlated single-photon counting) we study Förster resonance energy transfer between a donor and a black-hole-quencher acceptor bound at the 5'- and 3'-positions of a synthetic DNA oligonucleotide. This dual labelled oligonucleotide is annealed with either the complementary sequence or with sequences that mimic single-nucleotide polymorphic gene sequences: they differ in one nucleotide at positions near either the ends or the center of the oligonucleotide. We find donor fluorescence decay times whose values are definitely distinct and discuss the feasibility of single nucleotide polymorphism genotyping by this method.

  4. Diquat-induced cellular pyridine nucleotide redox changes and alteration of metabolic enzyme activities in colonic carcinoma cells.

    PubMed

    Circu, Magdalena L; Maloney, Ronald E; Aw, Tak Yee

    2017-02-25

    Previously we have shown that the redox cycler menadione (MQ) induced cellular pyridine nucleotide redox imbalance that was linked to a decrease in aerobic glycolysis and perturbation of the mitochondrial respiratory activity due to the redox cycling of the compound; these processes were potentiated by low glucose. In this study, we investigated how colonic epithelial cells maintained pyridine nucleotide (NAD(+)/NADH and NADP(+)/NADPH) redox homeostasis upon acute metabolic variation and exposure to the redox cycling diquat (DQ). Our results show that DQ challenge disrupted cellular NADH/NAD(+) redox status and enhanced cellular NADPH generation. Notably, DQ-induced NADH decrease was associated with enhanced lactate production, a process that was potentiated by glucose availability, but not by the mitochondrial substrates, succinate or malate/glutamate. In addition, DQ increased glucose 6-phoshate dehydrogenase (G6PDH) activity consistent with glucose diversion towards pentose phosphate pathway. As a consequence, steady-state NADPH levels were maintained during MQ challenge at normal glucose. In contrast and despite increased G6PDH and malic enzyme (ME) activities, DQ induced cellular NADPH-to-NADP(+) shift at low glucose, a situation that was reversed by mitochondrial substrates. Collectively, these results are consistent with increased aerobic glycolysis by DQ and specific metabolic changes leading to enhanced NADPH generation upon oxidative challenge.

  5. Nucleotide Sequence of the Akv env Gene

    PubMed Central

    Lenz, Jack; Crowther, Robert; Straceski, Anthony; Haseltine, William

    1982-01-01

    The sequence of 2,191 nucleotides encoding the env gene of murine retrovirus Akv was determined by using a molecular clone of the Akv provirus. Deduction of the encoded amino acid sequence showed that a single open reading frame encodes a 638-amino acid precursor to gp70 and p15E. In addition, there is a typical leader sequence preceding the amino terminus of gp70. The locations of potential glycosylation sites and other structural features indicate that the entire gp70 molecule and most of p15E are located on the outer side of the membrane. Internal cleavage of the env precursor to generate gp70 and p15E occurs immediately adjacent to several basic amino acids at the carboxyl terminus of gp70. This cleavage generates a region of 42 uncharged, relatively hydrophobic amino acids at the amino terminus of p15E, which is located in a position analogous to the hydrophobic membrane fusion sequence of influenza virus hemagglutinin. The mature polypeptides are predicted to associate with the membrane via a region of 30 uncharged, mostly hydrophobic amino acids located near the carboxyl terminus of p15E. Distal to this membrane association region is a sequence of 35 amino acids at the carboxyl terminus of the env precursor, which is predicted to be located on the inner side of the membrane. By analogy to Moloney murine leukemia virus, a proteolytic cleavage in this region removes the terminal 19 amino acids, thus generating the carboxyl terminus of p15E. This leaves 15 amino acids at the carboxyl terminus of p15E on the inner side of the membrane in a position to interact with virion cores during budding. The precise location and order of the large RNase T1-resistant oligonucleotides in the env region were determined and compared with those from several leukemogenic viruses of AKR origin. This permitted a determination of how the differences in the leukemogenic viruses affect the primary structure of the env gene products. PMID:6283170

  6. Expression of lactate dehydrogenase C correlates with poor prognosis in renal cell carcinoma.

    PubMed

    Hua, Yibo; Liang, Chao; Zhu, Jundong; Miao, Chenkui; Yu, Yajie; Xu, Aimin; Zhang, Jianzhong; Li, Pu; Li, Shuang; Bao, Meiling; Yang, Jie; Qin, Chao; Wang, Zengjun

    2017-03-01

    Lactate dehydrogenase C is an isoenzyme of lactate dehydrogenase and a member of the cancer-testis antigens family. In this study, we aimed to investigate the expression and functional role of lactate dehydrogenase C and its basic mechanisms in renal cell carcinoma. First, a total of 133 cases of renal cell carcinoma samples were analysed in a tissue microarray, and Kaplan-Meier survival curve analyses were performed to investigate the correlation between lactate dehydrogenase C expression and renal cell carcinoma progression. Lactate dehydrogenase C protein levels and messenger RNA levels were significantly upregulated in renal cell carcinoma tissues, and the patients with positive lactate dehydrogenase C expression had a shorter progression-free survival, indicating the oncogenic role of lactate dehydrogenase C in renal cell carcinoma. In addition, further cytological experiments demonstrated that lactate dehydrogenase C could prompt renal cell carcinoma cells to produce lactate, and increase metastatic and invasive potential of renal cell carcinoma cells. Furthermore, lactate dehydrogenase C could induce the epithelial-mesenchymal transition process and matrix metalloproteinase-9 expression. In summary, these findings showed lactate dehydrogenase C was associated with poor prognosis in renal cell carcinoma and played a pivotal role in the migration and invasion of renal cell carcinoma cells. Lactate dehydrogenase C may act as a novel biomarker for renal cell carcinoma progression and a potential therapeutic target for the treatment of renal cell carcinoma.

  7. [Effect of hypobaric hypoxia on the dehydrogenase activities of respiration and photosynthetic metabolism in barley seedlings].

    PubMed

    Voytsekovskaya, S A; Astafurova, T P; Verkhoturova, G S; Postovalova, V M

    2015-01-01

    Hypobaric hypoxia effects on enzymes of photosynthesis and respiration metabolism were explored in 8-day old seedlings of barley Hordeum vulgare L. in the dark or light. 16-hour exposure in rarified atmosphere that causes reductions of partial pressure of air gases and, consequently, hypobaric hypoxia (P(air) = 8.3 κPa, pO2 = 1.7 κPa, pCO2 = 0.003 κPa) up-regulated the activities of piruvate kinase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase and NADP x N-glyceraldehyde phosphate dehydrogenase both in the dark and light. NAD- and NAD-N-malate dehydrogenase activities were down-regulated. Levels of NAD- and NAD x H- malate dehydrogenases were decreased. Activation of the NADP-malic enzyme activity, invariably high activity of NADP-isocitrate dehydrogenase and growth of NADP x N- glyceraldehyde phosphate dehydrogenase are considered as a mechanism of barley seedlings adaptation to hypobaric hypoxia.

  8. Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases.

    PubMed Central

    Sanderson, S J; Khan, S S; McCartney, R G; Miller, C; Lindsay, J G

    1996-01-01

    Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1 (substrate-specific 2-oxoacid decarboxylase) and E3 (dihydrolipoamide dehydrogenase) components and isolated E2/X (where E2 is dihydrolipoamide acyltransferase) core assembly. Selective cleavage of the protein X component of the purified E2/X core with the proteinase arg C decreases the activity of the reconstituted complex to residual levels (i.e. 8-12%); however, significant recovery of reconstitution is achieved on addition of a large excess (i.e. 50-fold) of parent E3. N-terminal sequence analysis of the truncated 35,000-M(r) protein X fragment locates the site of cleavage by arg C at the extreme N-terminal boundary of a putative E3-binding domain and corresponds to the release of a 15,000-M(r) N-terminal fragment comprising both the lipoyl and linker sequences. In native PDC this region of protein X is shown to be partly protected from proteolytic attack by the presence of E3. Recovery of complex activity in the presence of excess E3 after arg C treatment is thought to result from low-affinity interactions with the partly disrupted subunit-binding domain on X and/or the intact analogous subunit binding domain on E2. Contrasting recoveries for arg C-modified E2/X/E1 core, and untreated E2/E1 core of the 2-oxoglutarate dehydrogenase complex, reconstituted with excess bovine heart E3, pig heart E3 or yeast E3 point to subtle differences in subunit interactions with heterologous E3s and offer an explanation for the inability of previous investigators to achieve restoration of PDC function after selective proteolysis of the protein X component. PMID:8870656

  9. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

    PubMed

    Kondo, Jiro; Westhof, Eric

    2011-10-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

  10. The TyrA family of aromatic-pathway dehydrogenases in phylogenetic context

    PubMed Central

    Song, Jian; Bonner, Carol A; Wolinsky, Murray; Jensen, Roy A

    2005-01-01

    Background The TyrA protein family includes members that catalyze two dehydrogenase reactions in distinct pathways leading to L-tyrosine and a third reaction that is not part of tyrosine biosynthesis. Family members share a catalytic core region of about 30 kDa, where inhibitors operate competitively by acting as substrate mimics. This protein family typifies many that are challenging for bioinformatic analysis because of relatively modest sequence conservation and small size. Results Phylogenetic relationships of TyrA domains were evaluated in the context of combinatorial patterns of specificity for the two substrates, as well as the presence or absence of a variety of fusions. An interactive tool is provided for prediction of substrate specificity. Interactive alignments for a suite of catalytic-core TyrA domains of differing specificity are also provided to facilitate phylogenetic analysis. tyrA membership in apparent operons (or supraoperons) was examined, and patterns of conserved synteny in relationship to organismal positions on the 16S rRNA tree were ascertained for members of the domain Bacteria. A number of aromatic-pathway genes (hisHb, aroF, aroQ) have fused with tyrA, and it must be more than coincidental that the free-standing counterparts of all of the latter fused genes exhibit a distinct trace of syntenic association. Conclusion We propose that the ancestral TyrA dehydrogenase had broad specificity for both the cyclohexadienyl and pyridine nucleotide substrates. Indeed, TyrA proteins of this type persist today, but it is also common to find instances of narrowed substrate specificities, as well as of acquisition via gene fusion of additional catalytic domains or regulatory domains. In some clades a qualitative change associated with either narrowed substrate specificity or gene fusion has produced an evolutionary "jump" in the vertical genealogy of TyrA homologs. The evolutionary history of gene organizations that include tyrA can be deduced in

  11. Purification and preliminary characterization of alcohol dehydrogenase from Aspergillus nidulans.

    PubMed Central

    Creaser, E H; Porter, R L; Britt, K A; Pateman, J A; Doy, C H

    1985-01-01

    Aspergillus alcohol dehydrogenase is produced in response to growth in the presence of a wide variety of inducers, of which the most effective are short-chain alcohols and ketones, e.g. butan-2-one and propan-2-ol. The enzyme can be readily extracted from fresh or freeze-dried cells and purified to homogeneity on Blue Sepharose in a single step by using specific elution with NAD+ and pyrazole. The pure enzyme has Mr 290 000 by electrophoresis or gel filtration; it is a homopolymer with subunit Mr 37 500 by electrophoresis in sodium dodecyl sulphate; its amino acid composition corresponds to Mr 37 900, and the native enzyme contains one zinc atom per subunit. The enzyme is NAD-specific and has a wide substrate activity in the forward and reverse reactions; its activity profile is not identical with those of other alcohol dehydrogenases. PMID:3156582

  12. A specific radiochemical assay for pyrroline-5-carboxylate dehydrogenase.

    PubMed

    Small, C; Jones, M E

    1987-03-01

    Previous studies of pyrroline-5-carboxylate dehydrogenase have been conducted using a spectrophotometric method to monitor substrate-dependent NAD(P)H production. For the assay of the mammalian enzyme, the spectrophotometric assay was found to be unacceptable for kinetic studies as the production of NAD(P)H was nonlinear with time and protein concentration. An assay which measures radiolabeled glutamate production by this enzyme in the presence of NAD+ from radiolabeled pyrroline-5-carboxylate has been developed. Separation of substrate from product is achieved by column chromatography using Dowex 50 cation-exchange resin. The product isolated by this procedure was identified as glutamate. This new assay is linear with time and protein concentration and gives reproducible results. The assay is not influenced by competing enzyme activities, such as glutamate dehydrogenase, in a liver homogenate so that quantitative conversion of pyrroline-5-carboxylate to glutamate is observed.

  13. Regional development of glutamate dehydrogenase in the rat brain.

    PubMed

    Leong, S F; Clark, J B

    1984-07-01

    The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (greater than 2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons greater than midbrain = hypothalamus greater than cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90-day-old adult value, but not in other regions. The enzyme-specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90-day-old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.

  14. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    NASA Astrophysics Data System (ADS)

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-07-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

  15. NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts.

    PubMed Central

    Nosek, J; Fukuhara, H

    1994-01-01

    The genes encoding the NADH dehydrogenase subunits of respiratory complex I have not been identified so far in the mitochondrial DNA (mtDNA) of yeasts. In the linear mtDNA of Candida parapsilosis, we found six new open reading frames whose sequences were unambiguously homologous to those of the genes known to code for NADH dehydrogenase subunit proteins of different organisms, i.e., ND1, ND2, ND3, ND4L, ND5, and ND6. The gene for ND4 also appears to be present, as judged from hybridization experiments with a Podospora gene probe. Specific transcripts from these open reading frames (ND genes) could be detected in the mitochondria. Hybridization experiments using C. parapsilosis genes as probes suggested that ND genes are present in the mtDNAs of a wide range of yeast species including Candida catenulata, Pichia guilliermondii, Clavispora lusitaniae, Debaryomyces hansenii, Hansenula polymorpha, and others. Images PMID:7521869

  16. Pressure regulation of malic dehydrogenase in reversed micelles.

    PubMed

    Klyachko, N L; Levashov, P A; Levashov, A V; Balny, C

    1999-01-27

    Malic dehydrogenase (MDH) studied in water and reversed micelles upon pressure application revealed a difference in catalysis. Whereas MDH in water appeared to be not sensitive to the pressure increasing, the catalytic activity of MDH in reversed micelles showed bell-shaped dependencies both on pressure and surfactant hydration degree, w0. The catalytic activity of MDH was found to be maximal under moderate pressure equal to 300-500 bar and at w0 approximately 14 with the difference between lowest and highest levels of the catalytic activity amounted to about 10 times. The work presented demonstrates for the first time the co-operative effect of reversed micelles and pressure application to malic dehydrogenase leading to the enzyme regulation that cannot be realized in aqueous solution. Copyright 1999 Academic Press.

  17. Alcohol Dehydrogenase and Ethanol in the Stems of Trees 1

    PubMed Central

    Kimmerer, Thomas W.; Stringer, Mary A.

    1988-01-01

    Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out. PMID:16666209

  18. Enantioselective oxidation of aldehydes catalyzed by alcohol dehydrogenase.

    PubMed

    Könst, Paul; Merkens, Hedda; Kara, Selin; Kochius, Svenja; Vogel, Andreas; Zuhse, Ralf; Holtmann, Dirk; Arends, Isabel W C E; Hollmann, Frank

    2012-09-24

    Teaching old dogs new tricks: Alcohol dehydrogenases (ADHs) may be established redox biocatalysts but they still are good for a few surprises. ADHs can be used to oxidize aldehydes, and this was demonstrated by the oxidative dynamic kinetic resolution of profens. In the presence of a suitable cofactor regeneration system, this reaction can occur with high selectivity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

    PubMed

    Kisiela, Michael; Skarka, Adam; Ebert, Bettina; Maser, Edmund

    2012-03-01

    Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including

  20. Parasite Lactate Dehydrogenase for Diagnosis of Plasmodium Falciparum. Phase II.

    DTIC Science & Technology

    1997-04-01

    Diagnosis of Plasmodium Falciparum PRINCIPAL INVESTIGATOR: Robert C. Piper, Ph.D. CONTRACTING ORGANIZATION: Flow, Incorporated Portland, Oregon 97201...Phase 11 (24 Mar 95 - 23 Mar 97) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Parasite Lactate Dehydrogenase for Diagnosis of Plasmodium Falciparum DAMD...that infected patients become ill. Four species of Plasmodium infect humans. P. falciparum accounts for -85 % of the world’s malaria. P. falciparum is

  1. Inhibition of membrane-bound succinate dehydrogenase by fluorescamine.

    PubMed

    Jay, D; Jay, E G; Garcia, C

    1993-12-01

    Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

  2. Reappraisal of the Regulation of Lactococcal l-Lactate Dehydrogenase

    PubMed Central

    van Niel, Ed W. J.; Palmfeldt, Johan; Martin, Rani; Paese, Marco; Hahn-Hägerdal, Bärbel

    2004-01-01

    Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed. PMID:15006814

  3. Reducing nontemplated 3' nucleotide addition to polynucleotide transcripts

    DOEpatents

    Kao, C. Cheng

    2000-01-01

    Non-template 3' nucleotide addition to a transcript is reduced by transcribing a transcript from a template comprising an ultimate and/or penultimate 5' ribose having a C'2 substituent such as methoxy, which reduces non-template 3' nucleotide addition to the transcript. The methods are shown to be applicable to a wide variety of polymerases, including Taq, T7 RNA polymerase, etc.

  4. Pressure response of (31)P chemical shifts of adenine nucleotides.

    PubMed

    Karl, Matthias; Spoerner, Michael; Pham, Thuy-Vy; Narayanan, Sunilkumar Puthenpurackal; Kremer, Werner; Kalbitzer, Hans Robert

    2017-03-30

    High pressure NMR spectroscopy is a powerful method for identifying rare conformational states of proteins from the pressure response of their chemical shifts. Many proteins have bound adenine nucleotides at their active centers, in most cases in a complex with Mg(2+)-ions. The (31)P NMR signals of phosphate groups of the nucleotides can be used as probes for conformational transitions in the proteins themselves. For distinguishing protein specific pressure effects from trivial pressure responses not due to the protein interaction, data of the pressure response of the free nucleotides must be available. Therefore, the pressure response of (31)P chemical shifts of the adenine nucleotides AMP, ADP, and ATP and their Mg(2+)-complexes has been determined at pH values several units distant from the respective pK-values. It is clearly non-linear for most of the resonances. A negative first order pressure coefficient B1 was determined for all (31)P resonances except Mg(2+)·AMP indicating an upfield shift of the resonances with pressure. The smallest and largest negative values are obtained for the α-phosphate group of ADP and β-phosphate group of Mg(2+)·ATP with -0.32 and -4.59ppm/GPa, respectively. With exception of the α-phosphate group of Mg(2+)·AMP the second order pressure coefficients are positive leading to a saturation like behaviour. The pressure response of the adenine nucleotides is similar but not identical to that observed earlier for guanine nucleotides. The obtained data show that the chemical shift pressure response of the different phosphate groups is rather different dependent on the position of phosphate group in the nucleotide and the nucleotide used. Copyright © 2016. Published by Elsevier B.V.

  5. Microsporidia: Why Make Nucleotides if You Can Steal Them?

    PubMed Central

    Dean, Paul; Hirt, Robert P.

    2016-01-01

    Microsporidia are strict obligate intracellular parasites that infect a wide range of eukaryotes including humans and economically important fish and insects. Surviving and flourishing inside another eukaryotic cell is a very specialised lifestyle that requires evolutionary innovation. Genome sequence analyses show that microsporidia have lost most of the genes needed for making primary metabolites, such as amino acids and nucleotides, and also that they have only a limited capacity for making adenosine triphosphate (ATP). Since microsporidia cannot grow and replicate without the enormous amounts of energy and nucleotide building blocks needed for protein, DNA, and RNA biosynthesis, they must have evolved ways of stealing these substrates from the infected host cell. Providing they can do this, genome analyses suggest that microsporidia have the enzyme repertoire needed to use and regenerate the imported nucleotides efficiently. Recent functional studies suggest that a critical innovation for adapting to intracellular life was the acquisition by lateral gene transfer of nucleotide transport (NTT) proteins that are now present in multiple copies in all microsporidian genomes. These proteins are expressed on the parasite surface and allow microsporidia to steal ATP and other purine nucleotides for energy and biosynthesis from their host. However, it remains unclear how other essential metabolites, such as pyrimidine nucleotides, are acquired. Transcriptomic and experimental studies suggest that microsporidia might manipulate host cell metabolism and cell biological processes to promote nucleotide synthesis and to maximise the potential for ATP and nucleotide import. In this review, we summarise recent genomic and functional data relating to how microsporidia exploit their hosts for energy and building blocks needed for growth and nucleic acid metabolism and we identify some remaining outstanding questions. PMID:27855212

  6. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    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.

  7. Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species.

    PubMed

    Starkov, Anatoly A; Fiskum, Gary; Chinopoulos, Christos; Lorenzo, Beverly J; Browne, Susan E; Patel, Mulchand S; Beal, M Flint

    2004-09-08

    Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

  8. Characterization of two β-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

    PubMed

    Takahashi, Kento; Nakanishi, Fumika; Tomita, Takeo; Akiyama, Nagisa; Lassak, Kerstin; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2016-11-01

    Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg(2+), thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

  9. Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase.

    PubMed

    Keung, W M; Vallee, B L

    1993-02-15

    Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde with a Ki of 40 nM, and uncompetitively with respect to the coenzyme NAD+. The human cytosolic aldehyde dehydrogenase isozyme (ALDH-II) is nearly 3 orders of magnitude less sensitive to daidzin inhibition. Daidzin does not inhibit human class I, II, or III alcohol dehydrogenases, nor does it have any significant effect on biological systems that are known to be affected by other isoflavones. Among more than 40 structurally related compounds surveyed, 12 inhibit ALDH-I, but only prunetin and 5-hydroxydaidzin (genistin) combine high selectivity and potency, although they are 7- to 15-fold less potent than daidzin. Structure-function relationships have established a basis for the design and synthesis of additional ALDH inhibitors that could both be yet more potent and specific.

  10. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.

    PubMed

    Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen; Zhang, Xian-Man; Braddock, Demetrios T; Albright, Ronald A; Prigaro, Brett J; Wood, John L; Bhanot, Sanjay; MacDonald, Michael J; Jurczak, Michael J; Camporez, Joao-Paulo; Lee, Hui-Young; Cline, Gary W; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2014-06-26

    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 oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

  11. Purification and characterization of dimeric dihydrodiol dehydrogenase from dog liver.

    PubMed

    Sato, K; Nakanishi, M; Deyashiki, Y; Hara, A; Matsuura, K; Ohya, I

    1994-09-01

    High NADP(+)-linked dihydrodiol dehydrogenase activity was detected in dog liver cytosol, from which a dimeric enzyme composed of M(r) 39,000 subunits was purified to homogeneity. The enzyme oxidized trans-cyclohexanediol, and trans-dihydrodiols of benzene and naphthalene, the [1R,2R]-isomers of which were selectively oxidized. In the reverse reaction in the presence of NADPH as a coenzyme, the enzyme reduced alpha-dicarbonyl compounds, such as methylglyoxal, 3-deoxyglucosone, and diacetyl, and some compounds with a carbonyl group, such as glyceraldehyde, lactaldehyde, and acetoin. 4-Hydroxyphenylketones and ascorbates inhibited the enzyme. The results of steady-state kinetic analyses indicated that the reaction proceeds through an ordered bi bi mechanism with the coenzyme binding to the free enzyme, and suggested that the inhibitors bind to the enzyme-NADP+ binary complex. The dimeric enzyme was detected in liver and kidney of dog, and was immunochemically similar to the dimeric enzymes from monkey kidney, rabbit lens, and pig liver. The sequences (total 127 amino acid residues) of eight peptides derived on enzymatic digestion of the dog liver enzyme did not show significant similarity with the primary structures of members of the aldo-keto reductase and short chain dehydrogenase superfamilies, which include monomeric dihydrodiol dehydrogenases and carbonyl reductase, respectively.

  12. Functional Analysis of a Mosquito Short Chain Dehydrogenase Cluster

    PubMed Central

    Mayoral, Jaime G.; Leonard, Kate T.; Defelipe, Lucas A.; Turjansksi, Adrian G.; Nouzova, Marcela; Noriegal, Fernando G.

    2013-01-01

    The short chain dehydrogenases (SDR) constitute one the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP+-dependent and they have S-enantioselectivity and preference for secondary alcohols with 8–15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity towards the (10S)-isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes. PMID:23238893

  13. Inhibition of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide.

    PubMed

    Rosas-Rodríguez, Jesús A; Figueroa-Soto, Ciria G; Valenzuela-Soto, Elisa M

    2010-01-01

    Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.

  14. An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

    PubMed

    Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2015-05-01

    An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity.

  15. Nucleotide diversity analysis highlights functionally important genomic regions

    PubMed Central

    Tatarinova, Tatiana V.; Chekalin, Evgeny; Nikolsky, Yuri; Bruskin, Sergey; Chebotarov, Dmitry; McNally, Kenneth L.; Alexandrov, Nickolai

    2016-01-01

    We analyzed functionality and relative distribution of genetic variants across the complete Oryza sativa genome, using the 40 million single nucleotide polymorphisms (SNPs) dataset from the 3,000 Rice Genomes Project (http://snp-seek.irri.org), the largest and highest density SNP collection for any higher plant. We have shown that the DNA-binding transcription factors (TFs) are the most conserved group of genes, whereas kinases and membrane-localized transporters are the most variable ones. TFs may be conserved because they belong to some of the most connected regulatory hubs that modulate transcription of vast downstream gene networks, whereas signaling kinases and transporters need to adapt rapidly to changing environmental conditions. In general, the observed profound patterns of nucleotide variability reveal functionally important genomic regions. As expected, nucleotide diversity is much higher in intergenic regions than within gene bodies (regions spanning gene models), and protein-coding sequences are more conserved than untranslated gene regions. We have observed a sharp decline in nucleotide diversity that begins at about 250 nucleotides upstream of the transcription start and reaches minimal diversity exactly at the transcription start. We found the transcription termination sites to have remarkably symmetrical patterns of SNP density, implying presence of functional sites near transcription termination. Also, nucleotide diversity was significantly lower near 3′ UTRs, the area rich with regulatory regions. PMID:27774999

  16. Nucleotide diversity analysis highlights functionally important genomic regions.

    PubMed

    Tatarinova, Tatiana V; Chekalin, Evgeny; Nikolsky, Yuri; Bruskin, Sergey; Chebotarov, Dmitry; McNally, Kenneth L; Alexandrov, Nickolai

    2016-10-24

    We analyzed functionality and relative distribution of genetic variants across the complete Oryza sativa genome, using the 40 million single nucleotide polymorphisms (SNPs) dataset from the 3,000 Rice Genomes Project (http://snp-seek.irri.org), the largest and highest density SNP collection for any higher plant. We have shown that the DNA-binding transcription factors (TFs) are the most conserved group of genes, whereas kinases and membrane-localized transporters are the most variable ones. TFs may be conserved because they belong to some of the most connected regulatory hubs that modulate transcription of vast downstream gene networks, whereas signaling kinases and transporters need to adapt rapidly to changing environmental conditions. In general, the observed profound patterns of nucleotide variability reveal functionally important genomic regions. As expected, nucleotide diversity is much higher in intergenic regions than within gene bodies (regions spanning gene models), and protein-coding sequences are more conserved than untranslated gene regions. We have observed a sharp decline in nucleotide diversity that begins at about 250 nucleotides upstream of the transcription start and reaches minimal diversity exactly at the transcription start. We found the transcription termination sites to have remarkably symmetrical patterns of SNP density, implying presence of functional sites near transcription termination. Also, nucleotide diversity was significantly lower near 3' UTRs, the area rich with regulatory regions.

  17. Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation.

    PubMed

    Khalpey, Zain; Yuen, Ada H Y; Lavitrano, Marialuisa; McGregor, Christopher G A; Kalsi, Kameljit K; Yacoub, Magdi H; Smolenski, Ryszard T

    2007-10-01

    Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors.Ecto-5'-nucleotidase (E5'N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.

  18. Single nucleotide polymorphisms in nucleotide excision repair genes, cancer treatment, and head and neck cancer survival

    PubMed Central

    Wyss, Annah B.; Weissler, Mark C.; Avery, Christy L.; Herring, Amy H.; Bensen, Jeannette T.; Barnholtz-Sloan, Jill S.; Funkhouser, William K.

    2014-01-01

    Purpose Head and neck cancers (HNC) are commonly treated with radiation and platinum-based chemotherapy, which produce bulky DNA adducts to eradicate cancerous cells. Because nucleotide excision repair (NER) enzymes remove adducts, variants in NER genes may be associated with survival among HNC cases both independently and jointly with treatment. Methods Cox proportional hazards models were used to estimate race-stratified (White, African American) hazard ratios (HRs) and 95 % confidence intervals for overall (OS) and disease-specific (DS) survival based on treatment (combinations of surgery, radiation, and chemotherapy) and 84 single nucleotide polymorphisms (SNPs) in 15 NER genes among 1,227 HNC cases from the Carolina Head and Neck Cancer Epidemiology Study. Results None of the NER variants evaluated were associated with survival at a Bonferroni-corrected alpha of 0.0006. However, rs3136038 [OS HR = 0.79 (0.65, 0.97), DS HR = 0.69 (0.51, 0.93)] and rs3136130 [OS HR = 0.78 (0.64, 0.96), DS HR = 0.68 (0.50, 0.92)] of ERCC4 and rs50871 [OS HR = 0.80 (0.64, 1.00), DS HR = 0.67 (0.48, 0.92)] of ERCC2 among Whites, and rs2607755 [OS HR = 0.62 (0.45, 0.86), DS HR = 0.51 (0.30, 0.86)] of XPC among African Americans were suggestively associated with survival at an uncorrected alpha of 0.05. Three SNP-treatment joint effects showed possible departures from additivity among Whites. Conclusions Our study, a large and extensive evaluation of SNPs in NER genes and HNC survival, identified mostly null associations, though a few variants were suggestively associated with survival and potentially interacted additively with treatment. PMID:24487794

  19. Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase

    USDA-ARS?s Scientific Manuscript database

    We have developed an invitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana a2b2-hetero tetrameric pyruvate dehydrogenase (E1) plus A.thaliana E1-kinase (AtPDK). Upon addition of MgATP...

  20. Levels of Alpha-Glycerophosphate Dehydrogenase, Triosephosphate Isomerase and Lactic Acid Dehydrogenase in Muscles of the Cockroach, ’Periplaneta americana’ L.,

    DTIC Science & Technology

    The level of alpha-glycerophosphate dehydrogenase is slightly higher in leg muscle than in thoracic muscle of the American cockroach, Periplaneta ... americana . Triosephosphate isomerase in leg muscle is about twice that of thoracic muscle. There is little lactic acid dehydrogenase in both muscles. (Author)

  1. A quantitative histochemical study of lactate dehydrogenase and succinate dehydrogenase activities in the membrana granulosa of the ovulatory follicle of the rat.

    PubMed

    Zoller, L C; Enelow, R

    1983-11-01

    Using a microdensitometer, lactate dehydrogenase and succinate dehydrogenase activities were measured in the membrana granulosa of the rat ovulatory follicle. Ovaries were removed on each day of the oestrous cycle; oestrus, dioestrus-1, dioestrus-2, and proestrus; and enzyme activities measured in the membrana granulosa as a whole and in four regions within it: peripheral (PR), antral (AR), cumulus oophorus (CO) and corona radiata (CR). Throughout the cycle, lactate dehydrogenase activity was greatest in PR. On oestrus, lactate dehydrogenase activity was progressively less in AR, CO and CR. On dioestrus-1, activity was identical in AR and CO and less in CR. On dioestrus-2, activity was greater in AR than in CO or CR. By proestrus, activity was equal in AR, CO and CR. In the membrana granulosa as a whole, and in each region, lactate dehydrogenase activity declined as ovulation approached. In contrast, succinate dehydrogenase activity in the membrana granulosa as a whole and in PR was constant throughout the cycle. Activity fluctuated in the other regions. Succinate dehydrogenase activity on oestrus was greatest in PR, less in AR and CO and least in CR. On the remaining days, succinate dehydrogenase activity was greatest in PR and less but equal in the remainder of the membrana granulosa.

  2. X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons

    SciTech Connect

    VandeBerg, J.L.; Aivaliotis, M.J.; Samollow, P.B. )

    1992-12-01

    Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model. 61 refs., 1 fig., 4 tabs.

  3. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations.

    PubMed Central

    Fang, Jie; Hsu, Betty Y L; MacMullen, Courtney M; Poncz, Mortimer; Smith, Thomas J; Stanley, Charles A

    2002-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cells (ED(50), 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide

  4. Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity.

    PubMed

    Bernocchi, G; Barni, S

    1983-12-01

    Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.

  5. Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component of human. cap alpha. -ketoacid dehydrogenase complexes

    SciTech Connect

    Pons, G.; Raefsky-Estrin, C.; Carothers, D.J.; Pepin, R.A.; Javed, A.A.; Jesse, B.W.; Ganapathi, M.K.; Samols, D.; Patel, M.S.

    1988-03-01

    cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.

  6. Structural Insights into the Drosophila melanogaster Retinol Dehydrogenase, a Member of the Short-Chain Dehydrogenase/Reductase Family

    PubMed Central

    Hofmann, Lukas; Tsybovsky, Yaroslav; Alexander, Nathan S.; Babino, Darwin; Leung, Nicole Y.; Montell, Craig; Banerjee, Surajit; von Lintig, Johannes; Palczewski, Krzysztof

    2016-01-01

    The 11-cis-retinylidene chromophore of visual pigments isomerizes upon interaction with a photon, initiating a downstream cascade of signaling events that ultimately lead to visual perception. 11-cis-Retinylidene is regenerated through enzymatic transformations collectively called the visual cycle. The first and rate-limiting enzymatic reaction within this cycle, i.e., the reduction of all-trans-retinal to all-trans-retinol, is catalyzed by retinol dehydrogenases. Here, we determined the structure of Drosophila melanogaster photoreceptor retinol dehydrogenase (PDH) isoform C that belongs to the short-chain dehydrogenase/reductase (SDR) family. This is the first reported structure of a SDR that possesses this biologically important activity. Two crystal structures of the same enzyme grown under different conditions revealed a novel conformational change of the NAD+ cofactor, likely representing a change during catalysis. Amide hydrogen–deuterium exchange of PDH demonstrated changes in the structure of the enzyme upon dinucleotide binding. In D. melanogaster, loss of PDH activity leads to photoreceptor degeneration that can be partially rescued by transgenic expression of human RDH12. Based on the structure of PDH, we analyzed mutations causing Leber congenital amaurosis 13 in a homology model of human RDH12 to obtain insights into the molecular basis of RDH12 disease-causing mutations. PMID:27809489

  7. Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

    PubMed

    Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.

  8. Evidence for distinct dehydrogenase and isomerase sites within a single 3. beta. -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

    SciTech Connect

    Luu-The, V.; Takahashi, Masakazu; de Launoit, Y.; Dumont, M.; Lachance, Y.; Labrie, F. )

    1991-09-10

    Complementary DNA encoding human 3{beta}-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3-{beta}-HSD) has been expressed in transfected GH{sub 4}C{sub 1} with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of ({sup 3}H)-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3{beta}-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5{alpha}-androstane-17{beta}-carboxamide) and its analogues of 5-androstenedione to 4-androstenedione with an approximately 1,000-fold higher K{sub i} value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3-{beta}-HSD protein. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

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

    PubMed

    Zhang, Mian; Liu, Can; Hu, Meng-yue; Zhang, Ji; Xu, Ping; Li, Feng; Zhong, Ze-yu; Liu, Li; Liu, Xiao-dong

    2015-04-01

    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. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  10. The amino acid sequence of ribitol dehydrogenase-F, a mutant enzyme with improved xylitol dehydrogenase activity.

    PubMed

    Homsi-Brandeburgo, M I; Toyama, M H; Marangoni, S; Ward, R J; Giglio, J R; Hartley, B S

    1999-05-01

    A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 --> Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 --> V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 --> P196 mutation that would disrupt a surface alpha-helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.

  11. High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles

    PubMed Central

    Figueroa-Teran, Rubi; Pak, Heidi; Blomquist, Gary J.; Tittiger, Claus

    2016-01-01

    Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (−)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (−)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (−)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (−)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition. PMID:26953347

  12. High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles.

    PubMed

    Figueroa-Teran, Rubi; Pak, Heidi; Blomquist, Gary J; Tittiger, Claus

    2016-09-01

    Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  13. Mutating three residues in the bovine rod cyclic nucleotide-activated channel can switch a nucleotide from inactive to active.

    PubMed Central

    Scott, S P; Cummings, J; Joe, J C; Tanaka, J C

    2000-01-01

    Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N(1)-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the alpha subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N(1)-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod alpha subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N(1)-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences. PMID:10777730

  14. The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

    PubMed Central

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Tang, Yufan; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2016-01-01

    Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and –insensitive groups, and the functions of CmADHs were discussed. PMID:27242871

  15. Cytokinin Metabolism of Pathogenic Fungus Leptosphaeria maculans Involves Isopentenyltransferase, Adenosine Kinase and Cytokinin Oxidase/Dehydrogenase

    PubMed Central

    Trdá, Lucie; Barešová, Monika; Šašek, Vladimír; Nováková, Miroslava; Zahajská, Lenka; Dobrev, Petre I.; Motyka, Václav; Burketová, Lenka

    2017-01-01

    Among phytohormones, cytokinins (CKs) play an important role in controlling crucial aspects of plant development. Not only plants but also diverse microorganisms are able to produce phytohormones, including CKs, though knowledge concerning their biosynthesis and metabolism is still limited. In this work we demonstrate that the fungus Leptosphaeria maculans, a hemi-biotrophic pathogen of oilseed rape (Brassica napus), causing one of the most damaging diseases of this crop, is able to modify the CK profile in infected B. napus tissues, as well as produce a wide range of CKs in vitro, with the cis-zeatin derivatives predominating. The endogenous CK spectrum of L. maculans in vitro consists mainly of free CK bases, as opposed to plants, where other CK forms are mostly more abundant. Using functional genomics, enzymatic and feeding assays with CK bases supplied to culture media, we show that L. maculans contains a functional: (i) isopentenyltransferase (IPT) involved in cZ production; (ii) adenosine kinase (AK) involved in phosphorylation of CK ribosides to nucleotides; and (iii) CK-degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Our data further indicate the presence of cis–trans isomerase, zeatin O-glucosyltransferase(s) and N6-(Δ2-isopentenyl)adenine hydroxylating enzyme. Besides, we report on a crucial role of LmAK for L. maculans fitness and virulence. Altogether, in this study we characterize in detail the CK metabolism of the filamentous fungi L. maculans and report its two novel components, the CKX and CK-related AK activities, according to our knowledge for the first time in the fungal kingdom. Based on these findings, we propose a model illustrating CK metabolism pathways in L. maculans. PMID:28785249

  16. Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

    PubMed Central

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Chin, James En Wai; Liu, Xiaoping; Striepen, Boris; Makowska-Grzyska, Magdalena; Kim, Youngchang; Joachimiak, Andrzej; Hedstrom, Lizbeth; Cuny, Gregory D.

    2013-01-01

    Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition and gastroenteritis as well as posing a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5′-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and > 500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD+. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An x-ray crystal structure of a representative E•IMP•inhibitor complex is also presented. Overall, the secondary amine derivative 15a (Q67) demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines. PMID:23668331

  17. [Medium-chain acyl-CoA dehydrogenase deficiency: contribution of molecular biology].

    PubMed

    Cartier, N; Lepetit, N; Rocchiccioli, F; Bougnères, P F

    1994-03-01

    Medium-chain acyl-CoA dehydrogenase deficiency is the most frequent cause of defective congenital fatty acid oxidation. Its molecular characterization is now possible. Case n. 1. A girl, 15 month-old, was admitted because she suffered from fever and vomiting, requiring the administration of aspirin. One day later, she showed signs of drowsiness and hypotonia; her blood glucose concentration was 0.3 g/l. She was given intravenous glucose and this episode rapidly passed. Case n. 2. A boy, brother of the preceding patient, was routinely investigated; he was never symptomatic. Case n. 3. A boy, sibling of the two preceding children, was admitted at the age of 18 months because he had gone into a coma during a febrile episode. His blood glucose concentration was 0.15 g/l. This episode was rapidly resolved by a glucose infusion. His fasting blood concentrations of glucose, non esterified fatty acids. beta-hydroxybutyrate, lactate and pyruvate were normal as were his blood carnitine and ammonia, but he showed elevated urinary excretion of dicarboxylic acids. Genomic DNA was extracted from peripheral leukocytes of the three sibs and their parents. The A-->G mutation at nucleotide 985 of the MCAD gene was detected by amplification and creation of a restriction site (ACRS). The implicated segment of this gene was amplified by PCR. ACRS showed that the symptomatic children were homozygous for the A-->G mutation, whereas their parents were heterozygous. The third asymptomatic child did not carry the mutation. Molecular biology techniques are appropriate for diagnosing this potentially lethal disease and their use for screening is important for disease prevention.

  18. Genetic Profiles of Korean Patients With Glucose-6-Phosphate Dehydrogenase Deficiency

    PubMed Central

    Lee, Jaewoong; Choi, Hayoung; Kim, Jiyeon; Kwon, Ahlm; Jang, Woori; Chae, Hyojin; Kim, Myungshin; Kim, Yonggoo; Lee, Jae Wook; Chung, Nack-Gyun

    2017-01-01

    Background We describe the genetic profiles of Korean patients with glucose-6-phosphate dehydrogenase (G6PD) deficiencies and the effects of G6PD mutations on protein stability and enzyme activity on the basis of in silico analysis. Methods In parallel with a genetic analysis, the pathogenicity of G6PD mutations detected in Korean patients was predicted in silico. The simulated effects of G6PD mutations were compared to the WHO classes based on G6PD enzyme activity. Four previously reported mutations and three newly diagnosed patients with missense mutations were estimated. Results One novel mutation (p.Cys385Gly, labeled G6PD Kangnam) and two known mutations [p.Ile220Met (G6PD São Paulo) and p.Glu416Lys (G6PD Tokyo)] were identified in this study. G6PD mutations identified in Koreans were also found in Brazil (G6PD São Paulo), Poland (G6PD Seoul), United States of America (G6PD Riley), Mexico (G6PD Guadalajara), and Japan (G6PD Tokyo). Several mutations occurred at the same nucleotide, but resulted in different amino acid residue changes in different ethnic populations (p.Ile380 variant, G6PD Calvo Mackenna; p.Cys385 variants, Tomah, Madrid, Lynwood; p.Arg387 variant, Beverly Hills; p.Pro396 variant, Bari; and p.Pro396Ala in India). On the basis of the in silico analysis, Class I or II mutations were predicted to be highly deleterious, and the effects of one Class IV mutation were equivocal. Conclusions The genetic profiles of Korean individuals with G6PD mutations indicated that the same mutations may have arisen by independent mutational events, and were not derived from shared ancestral mutations. The in silico analysis provided insight into the role of G6PD mutations in enzyme function and stability. PMID:28028996

  19. The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebrocortical mitochondria.

    PubMed

    Lai, J C; Sheu, K F

    1987-08-01

    The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxybutyrate on the flux of PDHC (as determined by [1-14C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-14C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-14C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861-1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.

  20. Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia.

    PubMed

    Trung Cao, Thanh; Tsai, Ming-An; Yang, Chung-Da; Wang, Pei-Chyi; Kuo, Tsun-Yung; Gabriel Chen, Hsu-Chung; Chen, Shih-Chu

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), derived from the outer-membrane protein (OMP) fraction, has been used as a potential candidate for vaccine development. The gene-encoding 37 kDa GAPDH outer membrane protein (OMP) from Edwardsiella ictaluri was amplified using polymerase chain reaction (PCR) and was cloned and expressed in Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and nucleotide and amino acid sequencing were used to analyze the expressed antigenic protein and gene encoding this protein. Comparative DNA and protein sequence analysis of GAPDH from E. ictaluri GAPDHs from several Gram-negative bacterial species within the Enterobacteriaceae family revealed that the GAPDHs within this group are highly conserved and share a sequence similarity of 75-100% with E. ictaluri GDPDH. Rabbit antiserum raised against the E. ictaluri recombinant GAPDH (rGAPDH) protein recognized purified GADPH, indicating that it has a strong immunogenicity. Tilapia fish were intraperitoneally immunized with formalin-killed E. ictaluri whole cells, and rGAPDH (30 μg fish(-1)) from E. ictaluri, both of which were emulsified in ISA 763A adjuvant. At 3 months after immunization, fish were challenged with the E. tarda strain to assess vaccine efficacy; the relative percent survival (RPS) values were found to exceed 71.4%. The specific mean antibody titer log2 level of groups vaccinated with rGAPDH at 3 months was significantly higher than that of non-vaccinated fish (control group). Therefore, this recombinant protein can be considered a multi-purpose candidate vaccine against several pathogenic bacteria.