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Molecules (Basel, Switzerland) Jul 2023A biomimetic mineralization method was used in the facile and rapid preparation of nanoflowers for immobilizing alcohol dehydrogenase (ADH). The method mainly uses ADH...
A biomimetic mineralization method was used in the facile and rapid preparation of nanoflowers for immobilizing alcohol dehydrogenase (ADH). The method mainly uses ADH as an organic component and zinc phosphate as an inorganic component to prepare flower-like ADH/Zn(PO) organic-inorganic hybrid nanoflowers (HNFs) with the high specific surface area through a self-assembly process. The synthesis conditions of the ADH HNFs were optimized and its morphology was characterized. Under the optimum enzymatic reaction conditions, the Michaelis-Menten constant () of ADH HNFs (β-NAD as substrate) was measured to be 3.54 mM, and the half-maximal inhibitory concentration (IC) of the positive control ranitidine (0.2-0.8 mM) was determined to be 0.49 mM. Subsequently, the inhibitory activity of natural medicine Pursh and nine small-molecule compounds on ADH was evaluated using ADH HNFs. The inhibition percentage of the aqueous extract of is 57.9%. The vanillic acid, protocatechuic acid, gallic acid, and naringenin have obvious inhibitory effects on ADH, and their percentages of inhibition are 55.1%, 68.3%, 61.9%, and 75.5%, respectively. Moreover, molecular docking analysis was applied to explore the binding modes and sites of the four most active small-molecule compounds to ADH. The results of this study can broaden the application of immobilized enzymes through biomimetic mineralization, and provide a reference for the discovery of ADH inhibitors from natural products.
Topics: Alcohol Dehydrogenase; Nanostructures; Biomimetics; Molecular Docking Simulation
PubMed: 37513303
DOI: 10.3390/molecules28145429 -
Applied Microbiology and Biotechnology Aug 2021(S)-4-Chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol are key pharmaceutical intermediates for the synthesis of bepotastine and cloperastine, respectively....
(S)-4-Chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol are key pharmaceutical intermediates for the synthesis of bepotastine and cloperastine, respectively. However, the biocatalytic approach to prepare these bulky diaryl ketones remains challenging because of the low activity of naturally occurring alcohol dehydrogenases (ADH). In the present study, ADH seq5, which has an adequate binding pocket volume and accepts bulky diaryl ketones, was further engineered with a binding pocket of increased hydrophobicity. Based on molecular simulation and binding free energy analyses, a small mutation library was constructed, and mutant seq5-D150I with a threefold increase in k and a low K was obtained successfully. The comparison of kinetic parameters, binding free energy, docking conformation, and critical catalytic distances calculated by molecular dynamic simulations revealed the source of increased activity. To develop a practical approach with seq5-D150I, reaction conditions including pH, temperature, buffer, and metal ions were optimised and applied to synthesise (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol with high enantiomeric excess. The space-time yields for (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol increased dramatically to as high as 263.4 g∙L day and 150 g∙L day, respectively, which, to our knowledge, is the highest reported yield to date. These results show that the biocatalytic approach with seq5-D150I may be practical for future industrial applications.Key points An alcohol dehydrogenase was engineered based on binding free energy analysis. The mutant seq5-D150I obtained a threefold increase in k and a low K. Two important pharmaceutical intermediates were obtained with high space-time yield.
Topics: Alcohol Dehydrogenase; Biocatalysis; Hydrophobic and Hydrophilic Interactions; Kinetics; Piperidines; Pyridines; Stereoisomerism
PubMed: 34342711
DOI: 10.1007/s00253-021-11413-9 -
Clinical Laboratory Apr 2018Autoimmune hepatitis (AIH) is a progressive inflammatory hepatopathy and an important cause of end-stage liver. The liver cells' destruction is reflected by increased...
BACKGROUND
Autoimmune hepatitis (AIH) is a progressive inflammatory hepatopathy and an important cause of end-stage liver. The liver cells' destruction is reflected by increased activity of different enzymes in the serum. These enzymes include alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which play a significant role in the metabolism of many biological substances and exist mainly in the liver. In this study we investigated the activity of alcohol dehydrogenase and its isoenzymes and the total activity of ALDH in the sera of patients with autoimmune hepatitis.
METHODS
Serum samples were taken for routine biochemical investigation from 32 patients with autoimmune hepatitis and from 40 healthy subjects. Class I and II of ADH and ALDH activity was measured by the spectrofluorometric method. For measurement of class III ADH and total ADH activity we employed the photometric methods.
RESULTS
The activity of the class I ADH isoenzyme was significantly higher in the sera of patients with autoimmune hepatitis. The median activity of this isoenzyme in the patients group was approximately 63% (3.94 mU/L) higher than the control level (1.46 mU/L). For this reason, the total ADH activity was also significantly increased. The activities of other ADH isoenzymes and ALDH tested were unchanged.
CONCLUSIONS
The activity of total ADH and class I isoenzymes in the sera of patients with autoimmune hepatitis is increased, and it seems to be caused by the release of alcohol dehydrogenase from damaged liver cells.
Topics: Adult; Aged; Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Female; Hepatitis, Autoimmune; Humans; Isoenzymes; Liver; Male; Middle Aged; Oxidation-Reduction
PubMed: 29739065
DOI: 10.7754/Clin.Lab.2017.170925 -
Plant Molecular Biology Mar 2021The study shows the biochemical and enzymatic divergence between the two aldehyde-alcohol dehydrogenases of the alga Polytomella sp., shedding light on novel aspects of...
The study shows the biochemical and enzymatic divergence between the two aldehyde-alcohol dehydrogenases of the alga Polytomella sp., shedding light on novel aspects of the enzyme evolution amid unicellular eukaryotes. Aldehyde-alcohol dehydrogenases (ADHEs) are large metalloenzymes that typically perform the two-step reduction of acetyl-CoA into ethanol. These enzymes consist of an N-terminal acetylating aldehyde dehydrogenase domain (ALDH) and a C-terminal alcohol dehydrogenase (ADH) domain. ADHEs are present in various bacterial phyla as well as in some unicellular eukaryotes. Here we focus on ADHEs in microalgae, a diverse and polyphyletic group of plastid-bearing unicellular eukaryotes. Genome survey shows the uneven distribution of the ADHE gene among free-living algae, and the presence of two distinct genes in various species. We show that the non-photosynthetic Chlorophyte alga Polytomella sp. SAG 198.80 harbors two genes for ADHE-like enzymes with divergent C-terminal ADH domains. Immunoblots indicate that both ADHEs accumulate in Polytomella cells growing aerobically on acetate or ethanol. ADHE1 of ~ 105-kDa is found in particulate fractions, whereas ADHE2 of ~ 95-kDa is mostly soluble. The study of the recombinant enzymes revealed that ADHE1 has both the ALDH and ADH activities, while ADHE2 has only the ALDH activity. Phylogeny shows that the divergence occurred close to the root of the Polytomella genus within a clade formed by the majority of the Chlorophyte ADHE sequences, next to the cyanobacterial clade. The potential diversification of function in Polytomella spp. unveiled here likely took place after the loss of photosynthesis. Overall, our study provides a glimpse at the complex evolutionary history of the ADHE in microalgae which includes (i) acquisition via different gene donors, (ii) gene duplication and (iii) independent evolution of one of the two enzymatic domains.
Topics: Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Algal Proteins; Amino Acid Sequence; Chlorophyta; Genetic Variation; Mass Spectrometry; Microalgae; Phylogeny; Proteomics; Sequence Analysis, DNA; Sequence Homology, Amino Acid
PubMed: 33415608
DOI: 10.1007/s11103-020-01105-9 -
Angewandte Chemie (International Ed. in... Sep 2018Alcohol dehydrogenases can act as powerful catalysts in the preparation of optically pure γ-hydroxy-δ-lactones by means of an enantioconvergent dynamic redox...
Alcohol dehydrogenases can act as powerful catalysts in the preparation of optically pure γ-hydroxy-δ-lactones by means of an enantioconvergent dynamic redox isomerization of readily available Achmatowicz-type pyranones. Imitating the traditionally metal-mediated "borrowing hydrogen" approach to shuffle hydrides across molecular architectures and interconvert functional groups, this chemoinspired and purely biocatalytic interpretation effectively expands the enzymatic toolbox and provides new opportunities in the assembly of multienzyme cascades and tailor-made cellular factories.
Topics: Alcohol Dehydrogenase; Biocatalysis; Escherichia coli; Hydrogen; Isomerism; Lactones; Oxidation-Reduction; Oxidoreductases; Recombinant Proteins; Stereoisomerism
PubMed: 29984878
DOI: 10.1002/anie.201804911 -
Biotechnology and Applied Biochemistry Apr 2023There are three prominent alcohol dehydrogenases superfamilies: short-chain, medium-chain, and iron-containing alcohol dehydrogenases (FeADHs). Many members are valuable... (Review)
Review
There are three prominent alcohol dehydrogenases superfamilies: short-chain, medium-chain, and iron-containing alcohol dehydrogenases (FeADHs). Many members are valuable catalysts for producing industrially relevant products such as active pharmaceutical intermediates, chiral synthons, biopolymers, biofuels, and secondary metabolites. However, FeADHs are the least explored enzymes among the superfamilies for commercial tenacities. They portray a conserved structure having a "tunnel-like" cofactor and substrate binding site with particular functions, despite representing high sequence diversity. Interestingly, phylogenetic analysis demarcates enzymes catalyzing distinct native substrates where closely related clades convert similar molecules. Further, homologs from various mesophilic and thermophilic microbes have been explored for designing a solvent and temperature-resistant enzyme for industrial purposes. The review explores different iron-containing alcohol dehydrogenases potential engineering of the enzymes and substrates helpful in manufacturing commercial products.
Topics: Alcohol Dehydrogenase; Phylogeny; Amino Acid Sequence; Iron; Binding Sites
PubMed: 35751426
DOI: 10.1002/bab.2376 -
Redox Biology May 2024Elevated fasting ethanol levels in peripheral blood frequently found in metabolic dysfunction-associated steatohepatitis (MASLD) patients even in the absence of alcohol...
Elevated fasting ethanol levels in peripheral blood frequently found in metabolic dysfunction-associated steatohepatitis (MASLD) patients even in the absence of alcohol consumption are discussed to contribute to disease development. To test the hypothesis that besides an enhanced gastrointestinal synthesis a diminished alcohol elimination through alcohol dehydrogenase (ADH) may also be critical herein, we determined fasting ethanol levels and ADH activity in livers and blood of MASLD patients and in wild-type ± anti-TNFα antibody (infliximab) treated and TNFα mice fed a MASLD-inducing diet. Blood ethanol levels were significantly higher in patients and wild-type mice with MASLD while relative ADH activity in blood and liver tissue was significantly lower compared to controls. Both alterations were significantly attenuated in MASLD diet-fed TNFα mice and wild-type mice treated with infliximab. Moreover, alcohol elimination was significantly impaired in mice with MASLD. In in vitro models, TNFα but not IL-1β or IL-6 significantly decreased ADH activity. Our data suggest that elevated ethanol levels in MASLD patients are related to TNFα-dependent impairments of ADH activity.
Topics: Mice; Humans; Animals; Alcohol Dehydrogenase; Tumor Necrosis Factor-alpha; Infliximab; Ethanol; Fatty Liver; Alcohol Drinking
PubMed: 38493749
DOI: 10.1016/j.redox.2024.103121 -
Molecules (Basel, Switzerland) Feb 2020Diazo compounds are versatile reagents in chemical synthesis and biology due to the tunable reactivity of the diazo functionality and its compatibility with living...
Diazo compounds are versatile reagents in chemical synthesis and biology due to the tunable reactivity of the diazo functionality and its compatibility with living systems. Much effort has been made in recent years to explore their accessibility and synthetic potential; however, their preparation through stereoselective enzymatic asymmetric synthesis has been scarcely reported in the literature. Alcohol dehydrogenases (ADHs, also called ketoreductases, KREDs) are powerful redox enzymes able to reduce carbonyl compounds in a highly stereoselective manner. Herein, we have developed the synthesis and subsequent bioreduction of nine α-diazo-β-keto esters to give optically active α-diazo-β-hydroxy esters with potential applications as chiral building blocks in chemical synthesis. Therefore, the syntheses of prochiral α-diazo-β-keto esters bearing different substitution patterns at the adjacent position of the ketone group (NCH, ClCH, BrCH, CHOCH, NCSCH, CH, and Ph) and in the alkoxy portion of the ester functionality (Me, Et, and Bn), were carried out through the diazo transfer reaction to the corresponding β-keto esters in good to excellent yields (81-96%). After performing the chemical reduction of α-diazo-β-keto esters with sodium borohydride and developing robust analytical conditions to monitor the biotransformations, their bioreductions were exhaustively studied using in-house made overexpressed and commercially available KREDs. Remarkably, the corresponding α-diazo-β-hydroxy esters were obtained in moderate to excellent conversions (60 to >99%) and high selectivities (85 to >99% ) after 24 h at 30 °C. The best biotransformations in terms of conversion and enantiomeric excess were successfully scaled up to give the expected chiral alcohols with almost the same activity and selectivity values observed in the enzyme screening experiments.
Topics: Alcohol Dehydrogenase; Bacterial Proteins; Catalysis; Escherichia coli; Esters; Rhodococcus
PubMed: 32093093
DOI: 10.3390/molecules25040931 -
MBio Jan 2019Butanol production by is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report...
Butanol production by 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 AAD and AAD, 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. 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 strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.
Topics: Acetone; Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Butanols; Catalytic Domain; Clostridium acetobutylicum; Ethanol; Fermentation; Metabolic Engineering; Molecular Dynamics Simulation; Mutagenesis, Site-Directed
PubMed: 30670620
DOI: 10.1128/mBio.02683-18 -
Biosensors & Bioelectronics Oct 2015In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes...
In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes for biofuel cell applications. The bioanode architectures were designed with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles, along with different immobilization techniques. Three different polymeric matrices were tested (tetrabutyl ammonium bromide (TBAB)-modified Nafion; octyl-modified linear polyethyleneimine (C8-LPEI); and cellulose) in the DET studies. The modified Nafion membrane provided the best electrical communication between enzymes and the electrode surface, with catalytic currents as high as 16.8 ± 2.1 µA cm(-2). Then, a series of ferrocene redox polymers were evaluated for MET. The redox polymer 1,1'-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI) provided the best electrochemical response. Using this polymer, the electrochemical assays conducted in the presence of MWCNTs and MWCNTs-Au indicated a Jmax of 781 ± 59 µA cm(-2) and 925 ± 68 µA cm(-2), respectively. Overall, from the results obtained here, DET using the PQQ-dependent ADH and AldDH still lacks high current density, while the bioanodes that operate via MET employing ferrocene-modified LPEI redox polymers show efficient energy conversion capability in ethanol/air biofuel cells.
Topics: Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Bioelectric Energy Sources; Electrodes; Electron Transport; Enzymes, Immobilized; Ethanol; Ferrous Compounds; Fluorocarbon Polymers; Gluconobacter; Models, Molecular; Nanotubes, Carbon; Oxidation-Reduction; PQQ Cofactor; Polyethyleneimine
PubMed: 25988787
DOI: 10.1016/j.bios.2015.05.011