Bivalent cations stabilize yeast alcohol dehydrogenase I.
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ABSTRACT:
SUBMITTER: De Bolle X
PROVIDER: S-EPMC1218334 | biostudies-other | 1997 Apr
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC4165444 | biostudies-literature
Project description:The metalated ylide YNa [Y=(Ph3 PCSO2 Tol)- ] was employed as X,L-donor ligand for the preparation of a series of boron cations. Treatment of the bis-ylide functionalized borane Y2 BH with different trityl salts or B(C6 F5 )3 for hydride abstraction readily results in the formation of the bis-ylide functionalized boron cation [Y-B-Y]+ (2). The high donor capacity of the ylide ligands allowed the isolation of the cationic species and its characterization in solution as well as in solid state. DFT calculations demonstrate that the cation is efficiently stabilized through electrostatic effects as well as ?-donation from the ylide ligands, which results in its high stability. Despite the high stability of 2 [Y-B-Y]+ serves as viable source for the preparation of further borenium cations of type Y2 B+ ?LB by addition of Lewis bases such as amines and amides. Primary and secondary amines react to tris(amino)boranes via N-H activation across the B-C bond.
| S-EPMC5363341 | biostudies-literature
Project description:Daqu Alcohol dehydrogenase Raw sequence reads
| PRJNA759401 | ENA
Project description:The alcohol dehydrogenase (ADH) system plays a critical role in sugar metabolism involving in not only ethanol formation and consumption but also the general "cofactor balance" mechanism. Candida maltosa is able to ferment glucose as well as xylose to produce a significant amount of ethanol. Here we report the ADH system in C. maltosa composed of three microbial group I ADH genes (CmADH1, CmADH2A and CmADH2B), mainly focusing on its metabolic regulation and physiological function.Genetic analysis indicated that CmADH2A and CmADH2B tandemly located on the chromosome could be derived from tandem gene duplication. In vitro characterization of enzymatic properties revealed that all the three CmADHs had broad substrate specificities. Homo- and heterotetramers of CmADH1 and CmADH2A were demonstrated by zymogram analysis, and their expression profiles and physiological functions were different with respect to carbon sources and growth phases. Fermentation studies of ADH2A-deficient mutant showed that CmADH2A was directly related to NAD regeneration during xylose metabolism since CmADH2A deficiency resulted in a significant accumulation of glycerol.Our results revealed that CmADH1 was responsible for ethanol formation during glucose metabolism, whereas CmADH2A was glucose-repressed and functioned to convert the accumulated ethanol to acetaldehyde. To our knowledge, this is the first demonstration of function separation and glucose repression of ADH genes in xylose-fermenting yeasts. On the other hand, CmADH1 and CmADH2A were both involved in ethanol formation with NAD regeneration to maintain NADH/NAD ratio in favor of producing xylitol from xylose. In contrast, CmADH2B was expressed at a much lower level than the other two CmADH genes, and its function is to be further confirmed.
| S-EPMC2909261 | biostudies-literature
Project description:In the presence of Mn(II) ions, the u.v. absorption spectrum of retinyl phosphate (Ret-P) solubilized in Triton X-100 micelles, phosphatidylcholine liposomes or rat liver microsomes exhibited a shift from the maximum of 330 nm to 287 nm. The effect of Mn(II) was reversed by adding EDTA or phosphate buffer. The same spectral change was found in the presence of poly-L-lysine in place of Mn(II) ions. The e.s.r. spectrum of Mn(II) in the presence or in the absence of Ret-P clearly showed that approx. 75% of the initial concentration of Mn(II) ions is bound to Ret-P when the molar ratio of Ret-P to Mn(II) ions is 4:1; no such binding occurred in the presence of retinol or retinoic acid. The appearance of two isosbestic points at 303 and 368 nm, in the presence of Mn(II) ions, suggests the existence of an equilibrium between an Mn(II)-bound monomer and an Mn(II)-bound dimer of Ret-P in Triton X-100 micelles. The same effect on the u.v.-absorption spectrum of Ret-P was also induced by Co(II), Cr(II), Zn(II) and Fe(II), but not by Mg2+ or Cu(II). The formation of the 'metachromatic complex' between Ret-P and Mn(II) or Co(II) inhibited the synthesis of retinyl phosphate mannose (Ret-P-Man) from exogenous and endogenous Ret-P and guanosine diphosphate [14C]mannose when bovine serum albumin was added after the metal ion. However, the order of addition did not influence Ret-P-Man synthesis in incubations containing MgCl2, which does not form the metachromatic complex with Ret-P. These results suggest that the bioavailability of proteins, polyamines and metal ions may control the extent to which Ret-P can be mannosylated in the intact membrane.
| S-EPMC1152308 | biostudies-other
Project description:Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut- phenotype. The Mut- phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut- strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut- increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.
| S-EPMC7972947 | biostudies-literature
Project description:The Michael hydratase - alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of ?,?-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.
| S-EPMC4052635 | biostudies-literature
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:1. The excretion of H(+) ions, with practically equivalent uptake of K(+) ions (from 0.1m-potassium chloride), occurs during the aerobic oxidation of ethanol. 2. Acetaldehyde and acetic acid formed at the same time are quantitatively equal to the amount of ethanol oxidized. 3. A slow uptake of K(+) ions occurs during the oxidation of acetaldehyde and a more rapid uptake during the oxidation of d-glyceraldehyde 3-phosphate. 4. The anaerobic reduction of methylene blue is studied, and the inhibitory effect of K(+) and other inorganic cations on the system demonstrated. 5. The cation requirement for equal inhibitory effect is parallel with the reciprocals of the transport affinities for the ;physiological K-carrier' (as taken from Conway & Duggan, 1958). 6. The cation inhibition of methylene blue reduction is reversed by treatment of the yeast with Teepol or by freezing-and-thawing. 7. Azide is shown to inhibit the reduction of methylene blue with intact cells. The inhibition is partially reversed by Teepol treatment and completely by freezing-and-thawing.
| S-EPMC1198284 | biostudies-other
Project description:1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited in the presence of 1,10-phenanthroline. 2. A conformational change in the enzyme's structure is induced by 1,10-phenanthroline, and is abolished in the presence of NADH. 1,10-Phenanthroline binds to the enzyme competitively with respect to NADH, with a stoicheiometry of 2 mol of 1,10-phenanthroline/144000g of enzyme. 3. 1,10-Phenanthroline induces a time-dependent dissociation of Zn2+ from the enzyme, which is in correlation with its inhibitions. 4. Spectrophotometric measurement indicates that the dissociation of half (2 zinc atoms/tetramer) of the total zinc content of the enzyme correlates with the full inhibition of its activity. Measurement of the tightly bound Zn2+ by atomic absorption photometry confirms this. 5. A proposition is advanced that the tetrameric molecule of yeast alcohol dehydrogenase possesses an inherent asymmetry, with four monomeric subunits being arranged in two mutually symmetrical pairs.
| S-EPMC1172812 | biostudies-other