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Biochemistry Mar 2021Structures of yeast alcohol dehydrogenase determined by X-ray crystallography show that the subunits have two different conformational states in each of the two dimers...
Structures of yeast alcohol dehydrogenase determined by X-ray crystallography show that the subunits have two different conformational states in each of the two dimers that form the tetramer. Apoenzyme and holoenzyme complexes relevant to the catalytic mechanism were described, but the asymmetry led to questions about the cooperativity of the subunits in catalysis. This study used cryo-electron microscopy (cryo-EM) to provide structures for the apoenzyme, two different binary complexes with NADH, and a ternary complex with NAD and 2,2,2-trifluoroethanol. All four subunits in each of these complexes are identical, as the tetramers have 2 symmetry, suggesting that there is no preexisting asymmetry and that the subunits can be independently active. The apoenzyme and one enzyme-NADH complex have "open" conformations and the inverted coordination of the catalytic zinc with Cys-43, His-66, Glu-67, and Cys-153, whereas another enzyme-NADH complex and the ternary complex have closed conformations with the classical coordination of the zinc with Cys-43, His-66, Cys-153, and a water or the oxygen of trifluoroethanol. The conformational change involves interactions of Arg-340 with the pyrophosphate group of the coenzyme and Glu-67. The cryo-EM and X-ray crystallography studies provide structures relevant for the catalytic mechanism.
Topics: Alcohol Dehydrogenase; Binding Sites; Catalysis; Cryoelectron Microscopy; Crystallography, X-Ray; Models, Molecular; Oxidation-Reduction; Protein Binding; Protein Conformation; Saccharomyces cerevisiae; Substrate Specificity
PubMed: 33620215
DOI: 10.1021/acs.biochem.0c00921 -
Physical Chemistry Chemical Physics :... Nov 2023Alcohol dehydrogenases (ADH) are a family of enzymes that catalyse the interconversion between ketones/aldehydes and alcohols in the presence of NADPH cofactor. It is...
Alcohol dehydrogenases (ADH) are a family of enzymes that catalyse the interconversion between ketones/aldehydes and alcohols in the presence of NADPH cofactor. It is challenging to desymmetrise the substituted cyclopentane-1,3-dione by engineering an ADH, while the reaction mechanism of the metal independent ADH remains elusive. Here we measured the conversion of a model substrate 2-benzyl-2-methylcyclopentane-1,3-dione by ADH and found it predominately gave the (2,3) product. Binding mode analysis of the substrate in ADH from molecular dynamics simulations disclosed the origin of the enantioselectivity of the enzyme; the opening and closing of the loop 191-205 above the substrate are responsible for shaping the binding pocket to orientate the substrate, so as to give different stereoisomer products. Using QM/MM calculations, we elucidated the reaction mechanism of ADH. Furthermore, we demonstrated the reaction profile corresponding to the production of different stereoisomers, which is in accordance with our experimental observations. This research here will shed a light on the rational engineering of ADH to achieve stereodivergent stereoisomer products.
Topics: Alcohol Dehydrogenase; Alcohols; Aldehydes; Catalysis; Ketones; Substrate Specificity
PubMed: 37955422
DOI: 10.1039/d3cp04019d -
Enzyme and Microbial Technology Jun 2022Ethyl 3-hydroxy-3-phenylpropionate (EHPP), (R)-EHPP or (S)-EHPP, is an important chiral intermediate for pharmaceuticals. Its synthesis from ethyl benzoyl acetate (EBA)...
Ethyl 3-hydroxy-3-phenylpropionate (EHPP), (R)-EHPP or (S)-EHPP, is an important chiral intermediate for pharmaceuticals. Its synthesis from ethyl benzoyl acetate (EBA) by alcohol dehydrogenase is regarded as a green method. However, scarcely any alcohol dehydrogenase has been reported competent in asymmetric synthesis of chiral EHPP at high EBA loading. Present study developed two robust and efficient bio-catalysts Mu-S2 and Mu-R4 for preparation of (S)-EHPP and (R)-EHPP respectively by rational design of alcohol dehydrogenase PcSDR from Pedobacter chitinilyticus based on molecular dynamics (MD) simulation analysis. BtGDH, a glucose dehydrogenase from Bacillus toyonensis catalyzing the oxidation of glucose for cofactor regeneration, was co-expressed with the screened mutants to form enzyme systems Mu-S2-BtGDH and Mu-R4-BtGDH. After reaction condition optimization, Mu-S2-BtGDH and Mu-R4-BtGDH were efficient in the synthesis of (S)-EHPP (94% conv. and 99% e.e.) and (R)-EHPP (99% conv. and 98% e.e.) respectively in 100 mL scale under 500 mM of EBA loading in 10 h following a substrate continuous feeding mode. After purifying, the isolated yield for each EHPP enantiomer is > 93%. This work not only provides potential biocatalysts for the industrial production of (R)-EHPP and (S)-EHPP, but also enriches the constructure-function relationship of alcohol dehydrogenases.
Topics: Alcohol Dehydrogenase; Catalysis; Phenylpropionates; Stereoisomerism
PubMed: 35364555
DOI: 10.1016/j.enzmictec.2022.110033 -
Archives of Biochemistry and Biophysics Apr 2021Enzymes typically have high specificity for their substrates, but the structures of substrates and products differ, and multiple modes of binding are observed. In this...
Enzymes typically have high specificity for their substrates, but the structures of substrates and products differ, and multiple modes of binding are observed. In this study, high resolution X-ray crystallography of complexes with NADH and alcohols show alternative modes of binding in the active site. Enzyme crystallized with the good substrates NAD and 4-methylbenzyl alcohol was found to be an abortive complex of NADH with 4-methylbenzyl alcohol rotated to a "non-productive" mode as compared to the structures that resemble reactive Michaelis complexes with NAD and 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol. The NADH is formed by reduction of the NAD with the alcohol during the crystallization. The same structure was also formed by directly crystallizing the enzyme with NADH and 4-methylbenzyl alcohol. Crystals prepared with NAD and 4-bromobenzyl alcohol also form the abortive complex with NADH. Surprisingly, crystals prepared with NAD and the strong inhibitor 1H,1H-heptafluorobutanol also had NADH, and the alcohol was bound in two different conformations that illustrate binding flexibility. Oxidation of 2-methyl-2,4-pentanediol during the crystallization apparently led to reduction of the NAD. Kinetic studies show that high concentrations of alcohols can bind to the enzyme-NADH complex and activate or inhibit the enzyme. Together with previous studies on complexes with NADH and formamide analogues of the carbonyl substrates, models for the Michaelis complexes with NAD-alcohol and NADH-aldehyde are proposed.
Topics: Alcohol Dehydrogenase; Alcohols; Animals; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Horses; Liver; Models, Chemical; NAD
PubMed: 33675814
DOI: 10.1016/j.abb.2021.108825 -
Biomolecules May 2023In our recent article (Smędra et al.: Oral form of auto-brewery syndrome. J Forensic Leg Med. 2022; 87: 102333), we showed that alcohol production can occur in the oral... (Review)
Review
In our recent article (Smędra et al.: Oral form of auto-brewery syndrome. J Forensic Leg Med. 2022; 87: 102333), we showed that alcohol production can occur in the oral cavity (oral auto-brewery syndrome) due to a disruption in the microbiota (dysbiosis). An intermediate step on the path leading to the formation of alcohol is acetaldehyde. Typically, acetic aldehyde is transformed into acetate particles inside the human body via acetaldehyde dehydrogenase. Unfortunately, acetaldehyde dehydrogenase activity is low in the oral cavity, and acetaldehyde remains there for a long time. Since acetaldehyde is a recognised risk factor for squamous cell carcinoma arising from the oral cavity, we decided to analyse the relationship linking the oral microbiome, alcohol, and oral cancer using the narrative review method, based on browsing articles in the PubMed database. In conclusion, enough evidence supports the speculation that oral alcohol metabolism must be assessed as an independent carcinogenic risk. We also hypothesise that dysbiosis and the production of acetaldehyde from non-alcoholic food and drinks should be treated as a new factor for the development of cancer.
Topics: Humans; Aldehyde Dehydrogenase; Dysbiosis; Mouth Neoplasms; Ethanol; Acetaldehyde; Microbiota; Alcohol Dehydrogenase
PubMed: 37238685
DOI: 10.3390/biom13050815 -
Journal of Agricultural and Food... May 2022The current chelation therapy has several drawbacks, including lack of selectivity, which could lead to trace metal depletion. Consequently, the proper function of...
The current chelation therapy has several drawbacks, including lack of selectivity, which could lead to trace metal depletion. Consequently, the proper function of metalloenzymes can be disrupted. Flavonoids possess chelating properties and hence interfere with the homeostasis of essential metals. We focused on zinc, an important trace metal required for the function of many enzymes and transcription factors. After making an initial evaluation of the Zn-chelating properties of a series of flavonoids, the effect of these compounds on various zinc-containing enzymes was also investigated. We performed enzyme inhibition assays spectrophotometrically using yeast and equine alcohol dehydrogenases and bovine glutamate dehydrogenase. Nine of the 21 flavonoids tested were capable of chelating Zn. Baicalein and 3-hydroxyflavone were the most potent Zn chelators under slightly acidic and neutral pH conditions. This chelation was also confirmed by the ability to reverse Zn-induced enzymatic inhibition of bovine glutamate dehydrogenase. Although some flavonoids were also able to inhibit zinc-containing alcohol dehydrogenases, this inhibition was likely not caused by Zn chelation. Luteolin was a relatively potent inhibitor of these enzymes regardless of the presence of Zn. Docking studies confirmed the binding of active flavonoids to equine alcohol dehydrogenase without any significant interaction with the catalytic zinc.
Topics: Alcohol Dehydrogenase; Animals; Cattle; Chelating Agents; Flavonoids; Glutamate Dehydrogenase; Horses; Metals; Zinc
PubMed: 35544338
DOI: 10.1021/acs.jafc.2c01446 -
Biological Psychiatry Apr 2020Alcohol use disorder (AUD) is defined by several symptom criteria, which can be dissected further at the genetic level. Over the past several years, our understanding of... (Review)
Review
Alcohol use disorder (AUD) is defined by several symptom criteria, which can be dissected further at the genetic level. Over the past several years, our understanding of the genetic factors influencing alcohol use and abuse has progressed tremendously; numerous loci have been implicated in different aspects of alcohol use. Previously known associations with alcohol-metabolizing enzymes (ADH1B, ALDH2) have been replicated definitively. In addition, novel associations with loci containing the genes KLB, GCKR, CRHR1, and CADM2 have been reported. Downstream analyses have leveraged these genetic findings to reveal important relationships between alcohol use behaviors and both physical and mental health. AUD and aspects of alcohol misuse have been shown to overlap strongly with psychiatric disorders, whereas aspects of alcohol consumption have shown stronger links to metabolism. These results demonstrate that the genetic architecture of alcohol consumption only partially overlaps with the genetics of clinically defined AUD. We discuss the limitations of using quantitative measures of alcohol use as proxy measures for AUD, and we outline how future studies will require careful phenotype harmonization to properly capture the genetic liability to AUD.
Topics: Alcohol Dehydrogenase; Alcohol Drinking; Alcoholism; Aldehyde Dehydrogenase, Mitochondrial; Ethanol; Humans; Phenotype; Polymorphism, Single Nucleotide
PubMed: 31733789
DOI: 10.1016/j.biopsych.2019.09.011 -
Biochemical Pharmacology Sep 2022Alcohol dehydrogenases (ADHs) play vital roles in alcohol metabolism and alcohol toxicity, yet little is known about microRNA-mediated regulation of the ADH gene...
Alcohol dehydrogenases (ADHs) play vital roles in alcohol metabolism and alcohol toxicity, yet little is known about microRNA-mediated regulation of the ADH gene cluster. Here, we showed that miR-29c activated ADH gene cluster transcription by targeting an enhancer element within the ADH6 gene. miR-29c is differentially expressed in alcoholic liver disease. Following biochemical and molecular evidence demonstrated that miR-29c increased ADH6 mRNA and protein levels without affecting the stability of the ADH6 transcript. Further evidence showed that exogenous miR-29c translocated into the nucleus and then unconventionally bound an enhancer element within the ADH6 gene. Luciferase reporter assay and chromatin immunoprecipitation data indicated that miR-29c activated the enhancer and increased the enrichment of RNA polymerase II at the promoter regions of ADH1A, ADH1B, ADH1C, ADH4, and ADH6. Finally, exogenous miR-29c transfection promoted the expression of ADH1A, ADH1B, ADH1C, and ADH4 pre-mRNA and mRNA transcripts from the ADH gene cluster. In conclusion, our data suggest that miR-29c might be a novel epigenetic regulator involved in ADH gene cluster activation.
Topics: Alcohol Dehydrogenase; Inactivation, Metabolic; MicroRNAs; Multigene Family; RNA, Messenger
PubMed: 35868429
DOI: 10.1016/j.bcp.2022.115182 -
Biochemistry Sep 2014Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1...
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.
Topics: Alcohol Dehydrogenase; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Protein Conformation; Saccharomyces cerevisiae
PubMed: 25157460
DOI: 10.1021/bi5006442 -
Poultry Science May 2022Ethanol is one of the most widely used and abused drugs. Following ethanol consumption, ethanol enters the bloodstream from the small intestine where it gets distributed...
Ethanol is one of the most widely used and abused drugs. Following ethanol consumption, ethanol enters the bloodstream from the small intestine where it gets distributed to peripheral tissues. In the bloodstream, ethanol is cleared from the system by the liver. The primary metabolism of ethanol uses alcohol dehydrogenase (ADH). In mammals, females appear to have higher ADH activity in liver samples than males. The purpose of the first experiment was to analyze sex differences in ADH levels following 12 d of ethanol administration (i.e., water or 2 g/kg) in male and female quail. Following the last daily treatment of ethanol, quail were euthanized, their livers were extracted, and ADH was analyzed in liver homogenate samples. Results showed that female quail had higher ADH levels, heavier livers, and a greater liver to body weight ratio than male quail. In a second experiment, we aimed to develop a blood ethanol concentration (BEC) profile for both male and female quail. Quail were administered 0.75 or 2 g/kg of ethanol and blood was collected at 0.5, 1, 2, 4, 6, 8, 12, 24 h after gavage administration. Blood ethanol concentration was analyzed using an Analox. We found that quail had a fairly rapid increase in BECs followed by a steady and slow disappearance of ethanol from the blood samples. Female quail had a lower peak of ethanol concentration and a smaller area under the curve (AUC) than male quail. The current research suggests that higher ADH levels in female quail may be responsible for increased metabolism of ethanol. In general, quail appear to eliminate ethanol more slowly than rodents. Thus, as a model, they may allow for a prolonged window with which to investigate the effects of ethanol.
Topics: Alcohol Dehydrogenase; Animals; Blood Alcohol Content; Chickens; Coturnix; Ethanol; Female; Liver; Male; Mammals; Sex Characteristics
PubMed: 35316649
DOI: 10.1016/j.psj.2022.101790