The binding of dihydronicotinamide--adenine dinucleotide and pyridine-3-aldehyde--adenine dinucleotide by yeast alcohol dehydrogenase.
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ABSTRACT:
SUBMITTER: Dickinson FM
PROVIDER: S-EPMC1179676 | biostudies-other | 1970 Dec
REPOSITORIES: biostudies-other
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Project description:The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.
| S-EPMC1164927 | biostudies-other
Project description:1. Inhibition by pyridine of reduction of NAD by ethanol in the presence of yeast alcohol dehydrogenase was studied at 25 degrees in 60mm-glycine buffer (K(+), pH9.3). 2. The apparent Michaelis constant for ethanol increases linearly and that for NAD increases non-linearly with pyridine concentration. 3. Rates, v, observed in the presence of pyridine are lower than the values calculated from the effect of pyridine on the two apparent Michaelis constants and are described by the expression V/v={1+5.8[pyridine]}x{1+0.016(1+124 [pyridine)]/[EtOH]}x{1+0.00019(1+3.3[pyridine]+110 [pyridine](2))/[NAD]}. 4. Mixed inhibitor studies with pyridine and N(1)-methylnicotinamide chloride in 40mm-pyrophosphate buffer (Na(+), pH8.2) indicated little interaction of pyridine with the ;pyridinium site' of the dehydrogenase (interaction constant, alpha, 2.1). 5. The possible competition of ethanol and pyridine for a zinc atom in the active centre of yeast alcohol dehydrogenase is discussed.
| S-EPMC1271227 | biostudies-other
Project description:Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%).
| S-EPMC95503 | biostudies-literature
Project description:The Complex II family of enzymes, comprising respiratory succinate dehydrogenases and fumarate reductases, catalyzes reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, soluble fumarate reductases (e.g., those from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8?-N3-histidyl covalent linkage between the protein and FAD was examined. Variants SdhA-R286A/K/Y and -H242A/Y that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in the assembly of a noncovalent FAD cofactor, which led to a significant decrease (-87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The "free" and "occupied" states of the active site were linked to the reduced and oxidized states of FAD, respectively. Our data allow for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD.
| S-EPMC4731035 | biostudies-literature
Project description: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 ADH1 B, 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.
| S-EPMC3860432 | biostudies-literature
Project description: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 NADPH(2) of high purity, the maximal specific rates were similar to those obtained with NAD and NADH(2), 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 NADH(2), 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 NADPH(2) do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH(2). 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 NADPH(2) than NAD and NADH(2). 5. Phosphate behaves as a competitive inhibitor towards NADP.
| S-EPMC1214325 | biostudies-other
Project description:BackgroundIn a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol.MethodsTriple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol.ResultsIn the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol.ConclusionThis study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol.General significanceThe present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.
| S-EPMC3669398 | biostudies-literature
Project description:Butanol production by Clostridium acetobutylicum is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91?g butanol/g ethanol for AADF716L and AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59?±?0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity.IMPORTANCE Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered Clostridium acetobutylicum strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.
| S-EPMC6343042 | biostudies-literature
Project description:1. The kinetics of oxidation of butan-1-ol and propan-2-ol by NAD+, catalysed by yeast alcohol dehydrogenase, were studied at 25 degrees C from pH 5.5 to 10, and at pH 7.05 from 14 degrees to 44 degrees C, 2. Under all conditions studied the results are consistent with a mechanism whereby some dissociation of coenzyme from the active enzyme-NAD+-alcohol ternary complexes occurs, and the mechanism is therefore not strictly compulsory order. 3. A primary 2H isotopic effect on the maximum rates of oxidation of [1-2H2]butan-1-ol and [2H7]propan-2-ol was found at 25 degrees C over the pH range 5.5-10. Further, in stopped-flow experiments at pH 7.05 and 25 degrees C, there was no transient formation of NADH in the oxidation of butan-1-ol and propan-2-ol. The principal rate-limiting step in the oxidation of dependence on pH of the maximum rates of oxidation of butan-1-ol and propan-2-ol is consisten with the possibility that histidine and cysteine residues may affect or control catalysis.
| S-EPMC1165482 | biostudies-other
Project description:We explored the interactome of flavin adenine dinucleotide (FAD) and the matrix of UV-immobilised FAD bound more than 3,000 proteins from SW-620 lysate. GO enrichment analysis showed that about 600 RNA-binding proteins were bound to FAD beads and, surprisingly, more than 40 proteins showed clear dose-dependent binding competition with nanomolar affinities indicating that their direct or indirect interaction with free FAD is both specific and strong. Among the few known consensus sequences for RNA-binding proteins, we selected the PUF motif 5'-UGUANAUA-3' to investigate whether FAD is binding the Pumilio homologs PUM1 and PUM2 in their RNA-binding pocket, as both these proteins showed dose-response behaviour in the FAD versus FAD-beads. Immobilisation of an oligomer containing the consensus sequence on NHS-activated Sepharose beads yielded PUM-beads that did enrich the Pumilio homologs. Oligomer vs FAD-beads and FAD vs PUM-beads together with the FAD vs FAD-beads dose-response competition indicated that the binding of FAD does not seem to influence the binding of the RNA. It is therefore most likely that FAD binding is allosteric for PUMs.
2022-10-06 | PXD036953 | Pride