An integrated cofactor and co-substrate recycling pathway for the biosynthesis of 1,5-pentanediol.

BACKGROUND: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO. RESULTS: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM. CONCLUSION: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO.

Insight into the structural characteristics of self-assembled liposome with epigallocatechin gallate/alcohol dehydrogenase.

In this study, the encapsulation and structural characteristics of the self-assembled liposome formed by epigallocatechin gallate (EGCG) and alcohol dehydrogenase (ADH) were studied. According to the results, EGCG significantly increased the catalytic activity of ADH with a 33.33 % activation rate and the liposomes were able to entrap EGCG-ADH with an effectiveness of 88.94 %. The self-assembled monolayers had nanometer-sized particles, and the excellent self-assembled system was demonstrated by the low PDI value and high surface absolute potential. The scanning electron microscope showed that the self-assembled liposome was honeycomb, groove-shaped, and rough. The spectroscopic results showed that EGCG-ADH complex was formed through hydrogen bond, which changed the secondary structure of the liposome, and verified EGCG-ADH liposome system was successfully prepared. In vitro digestion experiments showed that the gastrointestinal tolerance and antioxidant activity of EGCG-ADH liposomes were significantly higher than those of free EGCG-ADH.

Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Investigating human-derived lactic acid bacteria for alcohol resistance.

BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.

MASLD is related to impaired alcohol dehydrogenase (ADH) activity and elevated blood ethanol levels: Role of TNFα and JNK.

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-1ß 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.

Theoretical Analysis of Selectivity Differences in Ketoreductases toward Aldehyde and Ketone Carbonyl Groups.

Lactobacillus kefir alcohol dehydrogenase (LkADH) and ketoreductase from Chryseobacterium sp. CA49 (ChKRED12) exhibit different chemoselectivity and stereoselectivity toward a substrate with both keto and aldehyde carbonyl groups. LkADH selectively reduces the keto carbonyl group while retaining the aldehyde carbonyl group, producing optically pure R-alcohols. In contrast, ChKRED12 selectively reduces the aldehyde group and exhibits low reactivity toward ketone carbonyls. This study investigated the structural basis for these differences and the role of specific residues in the active site. Molecular dynamics (MD) simulations and quantum chemical calculations were used to investigate the interactions between the substrate and the enzymes and the essential cause of this phenomenon. The present study has revealed that LkADH and ChKRED12 exhibit significant differences in the structure of their respective active pockets, which is a crucial determinant of their distinct chemoselectivity toward the same substrate. Moreover, residues N89, N113, and E144 within LkADH as well as Q151 and D190 within ChKRED12 have been identified as key contributors to substrate stabilization within the active pocket through electrostatic interactions and van der Waals forces, followed by hydride transfer utilizing the coenzyme NADPH. Furthermore, the enantioselectivity mechanism of LkADH has been elucidated using quantum chemical methods. Overall, these findings not only provide fundamental insights into the underlying reasons for the observed differences in selectivity but also offer a detailed mechanistic understanding of the catalytic reaction.

Identification of a Novel Peptide with Alcohol Dehydrogenase Activating Ability from Ethanol-Induced Lactococcus lactis: A Combined In Silico Prediction and In Vivo Validation.

Alcohol dehydrogenase (ADH) is a crucial rate-limiting enzyme in alcohol metabolism. Our previous research found that ethanol-induced intracellular extracts of Lactococcus lactis (L. lactis) could enhance alcohol metabolism in mice, but the responsible compounds remain unidentified. The study aimed to screen potential ADH-activating peptides from ethanol-induced L. lactis using virtual screening and molecular docking calculation. Among them, the pentapeptide FAPEG might bind to ADH through hydrophobic interaction and hydrogen bonds, then enhancing ADH activity. Spectroscopy analysis further investigated the peptide-enzyme interaction between FAPEG and ADH, including changes in the amino acid residue microenvironment and secondary structural alterations. Furthermore, FAPEG could protect against alcoholic liver injury (ALI) in mice by reducing blood alcohol concentration, enhancing the activity of antioxidant and alcohol metabolism enzymes, and attenuating alcohol-induced hepatotoxicity, which was related to the activation of the Nrf2/keap1/HO-1 signaling pathway. The study provided preliminary evidence that the generation of ADH-activating peptides in ethanol-induced L. lactis has the potential in preventing ALI in mice using in silico prediction and in vivo validation approaches.

Alcohol dehydrogenase system acts as the sole pathway for methanol oxidation in Desulfofundulus kuznetsovii strain TPOSR.

Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17T. Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17T, lacks several genes from the methyl transferase operon found in strain 17T. The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol.

Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids.

Odd-numbered fatty acids (FAs) have been widely used in nutrition, agriculture, and chemical industries. Recently, some studies showed that they could be produced from bacteria or yeast, but the products are almost exclusively odd-numbered long-chain FAs. Here we report the design and construction of two biosynthetic pathways in Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids (OMFAs) via ricinoleic acid and 10-hydroxystearic acid, respectively. The production of OMFAs was enabled by introducing a hydroxy fatty acid cleavage pathway, including an alcohol dehydrogenase from Micrococcus luteus, a Baeyer-Villiger monooxygenase from Pseudomonas putida, and a lipase from Pseudomonas fluorescens. These OMFA biosynthetic pathways were optimized by eliminating the rate-limiting step, generating heptanoic acid, 11-hydroxyundec-9-enoic acid, nonanoic acid, and 9-hydroxynonanoic acid at 7.83 mg/L, 9.68 mg/L, 9.43 mg/L and 13.48 mg/L, respectively. This work demonstrates the biological production of OMFAs in a sustainable manner in S. cerevisiae.

Elevated levels of alcohol dehydrogenase aggravate ethanol-evoked cardiac remodeling and contractile anomalies through FKBP5-yap-mediated regulation of ferroptosis and ER stress.

Alcohol intake provokes severe organ injuries including alcoholic cardiomyopathy with hallmarks of cardiac remodeling and contractile defects. This study examined the toxicity of facilitated ethanol metabolism in alcoholism-evoked changes in myocardial morphology and contractile function, insulin signaling and various cell death domains using cardiac-selective overexpression of alcohol dehydrogenase (ADH). WT and ADH mice were offered an alcohol liquid diet for 12 weeks prior to assessment of cardiac geometry, function, ER stress, apoptosis and ferroptosis. Alcohol intake provoked pronounced glucose intolerance, cardiac remodeling and contractile anomalies with apoptosis, ER stress, and ferroptosis, the effects were accentuated by ADH with the exception of global glucose intolerance. Hearts from alcohol ingesting mice displayed dampened insulin-stimulated phosphorylation of insulin receptor (tyr1146) and IRS-1 (tyrosine) along with elevated IRS-1 serine phosphorylation, the effect was augmented by ADH. Alcohol challenge dampened phosphorylation of Akt and GSK-3ß, and increased phosphorylation of c-Jun and JNK, the effects were accentuated by ADH. Alcohol challenge promoted ER stress, FK506 binding protein 5 (FKBP5), YAP, apoptosis and ferroptosis, the effects were exaggerated by ADH. Using a short-term ethanol challenge model (3 g/kg, i.p., twice in three days), we found that inhibition of FKBP5-YAP signaling or facilitated ethanol detoxification by Alda-1 alleviated ethanol cardiotoxicity. In vitro study revealed that the ethanol metabolite acetaldehyde evoked cardiac contractile anomalies, lipid peroxidation, and apoptosis, the effects of which were mitigated by Alda-1, inhibition of ER stress, FKBP5 and YAP. These data suggest that facilitated ethanol metabolism via ADH exacerbates alcohol-evoked myocardial remodeling, functional defects, and insulin insensitivity possibly through a FKBP5-YAP-associated regulation of ER stress and ferroptosis.

Intermediate product control in cascade reaction for one-pot production of ε-caprolactone by Escherichia coli.

ε-Caprolactone is an important non-toxic compound for polymer synthesis like polycaprolactone which has been widely used in drug delivery and degradable plastics. To meet the demand for a green economy, a bi-enzymatic cascade, consisting of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO), was designed and introduced into Escherichia coli to synthesize ε-caprolactone from cyclohexanol with a self-sufficient NADPH-cofactor regeneration system. To further improve the catalytic efficiency, a carbonyl group-dependent colorimetric method using inexpensive 2,4-dinitrophenylhydrazine (DNPH) was developed for assay of cyclohexanone, an intermediate production of cascade reaction. It can be used to screen mutant strains with high catalytic efficiency from high-throughput library by detecting the absorbance value in microtiter plates (MTP) instead of gas chromatography (GC) analysis. Moreover, an RBS combinatorial library was constructed for balancing the expression of ADH and CHMO from two independent transcriptional units. After the high-throughput screening based on intermediate product control, an optimal variant with higher substrate tolerance and long-term stability was obtained from RBS combinatorial library. Through a fed-batch process, ε-caprolactone production reached 148.2 mM after 70 h of reaction under the optimized conditions, which was the highest yield achieved to date.

Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity.

Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.

Oral Administration of Alcohol-Tolerant Lactic Acid Bacteria Alleviates Blood Alcohol Concentration and Ethanol-Induced Liver Damage in Rodents.

Excessive alcohol consumption can have serious negative consequences on health, including addiction, liver damage, and other long-term effects. The causes of hangovers include dehydration, alcohol and alcohol metabolite toxicity, and nutrient deficiency due to absorption disorders. Additionally, alcohol consumption can slow reaction times, making it more difficult to rapidly respond to situations that require quick thinking. Exposure to a large amount of ethanol can also negatively affect a person's righting reflex and balance. In this study, we evaluated the potential of lactic acid bacteria (LAB) to alleviate alcohol-induced effects and behavioral responses. Two LAB strains isolated from kimchi, Levilactobacillus brevis WiKim0168 and Leuconostoc mesenteroides WiKim0172, were selected for their ethanol tolerance and potential to alleviate hangover symptoms. Enzyme activity assays for alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were then conducted to evaluate the role of these bacteria in alcohol metabolism. Through in vitro and in vivo studies, these strains were assessed for their ability to reduce blood alcohol concentrations and protect against alcohol-induced liver damage. The results indicated that these LAB strains possess significant ethanol tolerance and elevate ADH and ALDH activities. LAB administration remarkably reduced blood alcohol levels in rats after excessive alcohol consumption. Moreover, the LAB strains showed hepatoprotective effects and enhanced behavioral outcomes, highlighting their potential as probiotics for counteracting the adverse effects of alcohol consumption. These findings support the development of functional foods incorporating LAB strains that can mediate behavioral improvements following alcohol intake.

Insight into the binding characteristics of epigallocatechin-3-O-gallate and alcohol dehydrogenase: Based on the spectroscopic and molecular docking analysis.

Alcohol dehydrogenase (ADH) is one of the pivotal enzymes for alcohol metabolism, which plays an important role in many physiological processes. In this study, the activation effects of epigallocatechin-3-O-gallate (EGCG) on ADH and the characteristics of the interaction were investigated via biochemical method, spectroscopy methods, and molecular docking. The results demonstrated that EGCG significantly increased the catalytic activity of ADH with a 33.33% activation rate and that EGCG blending slightly altered the microenvironment surrounding ADH aromatic amino acids, with an increase in the quantity of ß-sheet and a decrease in the α-helix. Through the thermal stability analysis, it is further shown that the interaction of the two affects the intra-molecular hydrogen bond formation of the protein, and the conformation is partially extended. Besides, a total of 8 residues in ADH participated in the docking with EGCG, among which Asp-227, Lys-231, Glu-234, Gly-365 and Glu-366 participated in the formation of hydrogen bonds. At the same time, EGCG and amino group of Lys-231 form a noncovalent bond through cation-π interaction. In particular, hydrogen bonding was beneficial to keep the stability of EGCG-ADH, which was the primary driver of ADH activity activation. The results supply a new way for EGCG to activate ADH and a theoretical basis for the development of anti-alcoholism products.

Computational analysis of human medium-chain dehydrogenases/reductases revealing substrate- and coenzyme-binding characteristics.

The medium-chain dehydrogenase/reductase (MDR) superfamily has more than 600,000 members in UniProt as of March 2023. As the family has been growing, the proportion of functionally characterized proteins has been falling behind. The aim of this project was to investigate the binding pockets of nine different MDR protein families based on sequence conservation patterns and three-dimensional structures of members within the respective families. A search and analysis methodology was developed. Using this, a total of 2000 eukaryotic MDR sequences belonging to nine different families were identified. The pairwise sequence identities within each of the families were 80-90 % for the mammalian sequences, like the levels observed for alcohol dehydrogenase, another MDR family. Twenty conserved residues were identified in the coenzyme part of the binding site by matching structural and conservation data of all nine protein families. The conserved residues in the substrate part of the binding pocket varied between the nine MDR families, implying divergent functions for the different families. Studying each family separately made it possible to identify multiple conserved residues that are expected to be important for substrate binding or catalysis of the enzymatic reaction. By combining structural data with the conservation of the amino acid residues in each protein, important residues in the binding pocket were identified for each of the nine MDRs. The obtained results add new positions of interest for the binding and activity of the enzyme family as well as fit well to earlier published data. Three distinct types of binding pockets were identified, containing no, one, or two tyrosine residues.

LncRNA LINC01339 Hinders the Development of Wilms' Tumor via MiR-135b-3p/ADH1C Axis.

Wilms' tumor is a malignant renal cancer that arises within the pediatric urinary system. This study intended to investigate how a novel long non-coding RNA LINC01339 functions in the pathogenesis of Wilms' tumor. An elevated miR-135b-3p expression as well as reduced levels of LINC01339 and ADH1C were observed in Wilms' tumor. LINC01339 mediated ADH1C expression by directly binding to miR-135b-3p. The enforced LINC01339 or ADH1C markedly hindered cell growth and migration in Wilms' tumor. The LINC01339 overexpression also repressed the growth of Wilms' tumors in vivo, whereas miR-135b-3p overexpression exerted the opposite effects on Wilms' tumor cells in vitro. Additionally, upregulating miR-135b-3p reversed LINC01339's effects on the cellular processes of Wilms' tumor cells, whereas ADH1C overexpression offset the cancer-promoting influence of miR-135b-3p upregulation on Wilms' tumor progression. Therefore, LINC01339 prevents Wilms' tumor progression by modulating the miR-135b-3p/ADH1C axis. Our findings substantiate that the LINC01339/miR-135 b-3p/ADH1C regulatory axis has potential to be a target for the treatment of Wilms' tumor.

Effects of Hovenia dulcis fruit and peduncle extract on alcohol metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried fruit and peduncle of Hovenia dulcis Thunberg (Rhamnaceae) (HD) has been used as a folk medicine to treat liver disease, detoxify alcoholism, and prevent and cure hangovers. AIM OF THE STUDY: We investigated the pharmacology of HD on the kinetics of EtOH and on the enzymes related to alcohol metabolism to seek the scientific evidence of HD to prevent hangover, the effectiveness as a folk medicine. MATERIALS AND METHODS: EtOH was orally administered 30 min after oral administration of HD boiling water extract in rats. Then, the profiles of blood EtOH concentrations were measured. Mice were reared with food containing powdered HD for 7 days, and the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in liver were measured. Hepa1c1c7 cells were cultured with the medium containing HD extract, and the activities of ADH and ALDH were measured. RESULTS: HD extract reduced the blood EtOH concentrations in rats and induced the activities of ADH and ALDH and mRNA and protein expressions of ADH1B, ALDH1A1, and ALDH2 in the liver of mice and Hepa1c1c7 cells. Dihydromyricetin, one of the ingredients of HD, significantly induced the activities of ADH and ALDH in Hepa1c1c7 cells, however, the fractions containing hydrophilic organic compounds with small molecular weight contributed the most of the activities of HD extract. CONCLUSIONS: We clarified the experimental pharmacological evidences of HD as a folk medicine to detoxify alcoholism and prevent hangovers.

Microbial alcohol dehydrogenases: recent developments and applications in asymmetric synthesis.

Alcohol dehydrogenases are a well-known group of enzymes in the class of oxidoreductases that use electron transfer cofactors such as NAD(P)+/NAD(P)H for oxidation or reduction reactions of alcohols or carbonyl compounds respectively. These enzymes are utilized mainly as purified enzymes and offer some advantages in terms of green chemistry. They are environmentally friendly and a sustainable alternative to traditional chemical synthesis of bulk and fine chemicals. Industry has implemented several whole-cell biocatalytic processes to synthesize pharmaceutically active ingredients by exploring the high selectivity of enzymes. Unlike the whole cell system where cofactor regeneration is well conserved within the cellular environment, purified enzymes require additional cofactors or a cofactor recycling system in the reaction, even though cleaner reactions can be carried out with fewer downstream work-up problems. The challenge of producing purified enzymes in large quantities has been solved in large part by the use of recombinant enzymes. Most importantly, recombinant enzymes find applications in many cascade biotransformations to produce several important chiral precursors. Inevitably, several dehydrogenases were engineered as mere recombinant enzymes could not meet the industrial requirements for substrate and stereoselectivity. In recent years, a significant number of engineered alcohol dehydrogenases have been employed in asymmetric synthesis in industry. In a parallel development, several enzymatic and non-enzymatic methods have been established for regenerating expensive cofactors (NAD+/NADP+) to make the overall enzymatic process more efficient and economically viable. In this review article, recent developments and applications of microbial alcohol dehydrogenases are summarized by emphasizing notable examples.

Solvent isotope and mutagenesis studies on the proton relay system in yeast alcohol dehydrogenase 1.

Alcohol dehydrogenase catalyzes the reversible transfer of a hydride directly from an alcohol to the nicotinamide ring of NAD+ to form an aldehyde and NADH, and the proton from the alcohol probably is transferred through a hydrogen-bonded system to the imidazole of His-48. Studies of the pH dependencies, and solvent and substrate isotope effects on the wild-type and the enzyme with His-48 substituted with Gln-48 were used to demonstrate a role for the proton relay system. The H48Q substitution increases affinities for NAD+ and NADH by ∼2-fold, suggesting that the overall protein structure is maintained. In contrast, catalytic efficiencies (V/Km) on ethanol and acetaldehyde and affinity for 2,2,2-trifluoroethanol are decreased by about 10-fold. The pH dependencies for catalytic efficiencies on ethanol and acetaldehyde (log V/Km versus pH), show pK values of about 7.5 for wild-type enzyme, but ethanol oxidation by H48Q ADH is essentially linear over the pH range from 5.5 to 9.2 with a slope of 0.47. Steady-state kinetics and substrate isotope effects suggest that the kinetic mechanism of H48Q ADH has become partly random for oxidation of ethanol. Both wild-type and H48Q ADHs have pH-independent isotope effects for oxidation (V1/Kb) of 1-butanol/1-butanol-d9 of 4, suggesting that hydride transfer is a major rate-limiting step. The pH dependence for butanol oxidation by wild type ADH shows a wavy profile over the pH range from pH 6 to 10, with a ∼2.3-fold larger V1/Kb in D2O than in H2O, an "inverse" isotope effect. The substrate isotope effect of 4 is not altered by the solvent isotope effect, suggesting concerted proton/hydride transfer. The solvent isotope effect can be explained by a ground state with a water bound to the catalytic zinc in the enzyme-NAD+ complex, and a transition state that resembles a complex with NADH and aldehyde. In contrast, the H48Q enzyme has a diminished inverse solvent isotope effect of ∼1.3 and an essentially linear pH dependence with a slope of log V1/Kb against pH of 0.49 for oxidation of 1-butanol, which together are consistent with a transition state where hydroxide ion directly accepts a proton from the 2'-hydroxyl group of the nicotinamide ribose in the proton relay system in the enzyme-NAD+-alcohol complex. The results support a catalytic role for His-48 in the proton relay system.

Involvement of Type 10 17ß-Hydroxysteroid Dehydrogenase in the Pathogenesis of Infantile Neurodegeneration and Alzheimer's Disease.

Type 10 17ß-hydroxysteroid dehydrogenase (17ß-HSD10) is the HSD17B10 gene product playing an appreciable role in cognitive functions. It is the main hub of exercise-upregulated mitochondrial proteins and is involved in a variety of metabolic pathways including neurosteroid metabolism to regulate allopregnanolone homeostasis. Deacetylation of 17ß-HSD10 by sirtuins helps regulate its catalytic activities. 17ß-HSD10 may also play a critical role in the control of mitochondrial structure, morphology and dynamics by acting as a member of the Parkin/PINK1 pathway, and by binding to cyclophilin D to open mitochondrial permeability pore. 17ß-HSD10 also serves as a component of RNase P necessary for mitochondrial tRNA maturation. This dehydrogenase can bind with the Aß peptide thereby enhancing neurotoxicity to brain cells. Even in the absence of Aß, its quantitative and qualitative variations can result in neurodegeneration. Since elevated levels of 17ß-HSD10 were found in brain cells of Alzheimer's disease (AD) patients and mouse AD models, it is considered to be a key factor in AD pathogenesis. Since data underlying Aß-binding-alcohol dehydrogenase (ABAD) were not secured from reported experiments, ABAD appears to be a fabricated alternative term for the HSD17B10 gene product. Results of this study would encourage researchers to solve the question why elevated levels of 17ß-HSD10 are present in brains of AD patients and mouse AD models. Searching specific inhibitors of 17ß-HSD10 may find candidates to reduce senile neurodegeneration and open new approaches for the treatment of AD.