ADH1B, the adipocyte-enriched alcohol dehydrogenase, plays an essential, cell-autonomous role in human adipogenesis.

Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its expression in adipose tissue is reduced in obesity and increased by insulin stimulation. Interference with ADH1B expression has also been reported to impair adipocyte function. To better understand the role of ADH1B in adipocytes, we used CRISPR/Cas9 to delete ADH1B in human adipose stem cells (ASC). Cells lacking ADH1B failed to differentiate into mature adipocytes manifested by minimal triglyceride accumulation and a marked reduction in expression of established adipocyte markers. As ADH1B is capable of converting retinol to retinoic acid (RA), we conducted rescue experiments. Incubation of ADH1B-deficient preadipocytes with 9-cis-RA, but not with all-transretinol, significantly rescued their ability to accumulate lipids and express markers of adipocyte differentiation. A homozygous missense variant in ADH1B (p.Arg313Cys) was found in a patient with congenital lipodystrophy of unknown cause. This variant significantly impaired the protein's dimerization, enzymatic activity, and its ability to rescue differentiation in ADH1B-deficient ASC. The allele frequency of this variant in the Middle Eastern population suggests that it is unlikely to be a fully penetrant cause of severe lipodystrophy. In conclusion, ADH1B appears to play an unexpected, crucial and cell-autonomous role in human adipocyte differentiation by serving as a necessary source of endogenous retinoic acid.

Single-cell and genome-wide Mendelian randomization identifies causative genes for gout.

BACKGROUND: Gout is a prevalent manifestation of metabolic osteoarthritis induced by elevated blood uric acid levels. The purpose of this study was to investigate the mechanisms of gene expression regulation in gout disease and elucidate its pathogenesis. METHODS: The study integrated gout genome-wide association study (GWAS) data, single-cell transcriptomics (scRNA-seq), expression quantitative trait loci (eQTL), and methylation quantitative trait loci (mQTL) data for analysis, and utilized two-sample Mendelian randomization study to comprehend the causal relationship between proteins and gout. RESULTS: We identified 17 association signals for gout at unique genetic loci, including four genes related by protein-protein interaction network (PPI) analysis: TRIM46, THBS3, MTX1, and KRTCAP2. Additionally, we discerned 22 methylation sites in relation to gout. The study also found that genes such as TRIM46, MAP3K11, KRTCAP2, and TM7SF2 could potentially elevate the risk of gout. Through a Mendelian randomization (MR) analysis, we identified three proteins causally associated with gout: ADH1B, BMP1, and HIST1H3A. CONCLUSION: According to our findings, gout is linked with the expression and function of particular genes and proteins. These genes and proteins have the potential to function as novel diagnostic and therapeutic targets for gout. These discoveries shed new light on the pathological mechanisms of gout and clear the way for future research on this condition.

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.

Modeling alcohol-associated liver disease in humans using adipose stromal or stem cell-derived organoids.

Alcohol-associated liver disease (ALD) is a prevalent liver disease, yet research is hampered by the lack of suitable and reliable human ALD models. Herein, we generated human adipose stromal/stem cell (hASC)-derived hepatocellular organoids (hAHOs) and hASC-derived liver organoids (hALOs) in a three-dimensional system using hASC-derived hepatocyte-like cells and endodermal progenitor cells, respectively. The hAHOs were composed of major hepatocytes and cholangiocytes. The hALOs contained hepatocytes and nonparenchymal cells and possessed a more mature liver function than hAHOs. Upon ethanol treatment, both steatosis and inflammation were present in hAHOs and hALOs. The incubation of hALOs with ethanol resulted in increases in the levels of oxidative stress, the endoplasmic reticulum protein thioredoxin domain-containing protein 5 (TXNDC5), the alcohol-metabolizing enzymes ADH1B and ALDH1B1, and extracellular matrix accumulation, similar to those of liver tissues from patients with ALD. These results present a useful approach for understanding the pathogenesis of ALD in humans, thus facilitating the discovery of effective treatments.

Effectiveness of genetic feedback on alcohol metabolism to reduce alcohol consumption in young adults: an open-label randomized controlled trial.

BACKGROUND: It is unclear whether brief interventions using the combined classification of alcohol-metabolizing enzymes aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) together with behavioral changes in alcohol use can reduce excessive alcohol consumption. This study aimed to examine the effects of a brief intervention based on the screening of ALDH2 and ADH1B gene polymorphisms on alcohol consumption in Japanese young adults. METHODS: In this open-label randomized controlled trial, we enrolled adults aged 20-30 years who had excessive drinking behavior (average amount of alcohol consumed: men, ≥ 4 drinks/per day and women, ≥ 2 drinks/per day; 1 drink = 10 g of pure alcohol equivalent). Participants were randomized into intervention or control group using a simple random number table. The intervention group underwent saliva-based genotyping of alcohol-metabolizing enzymes (ALDH2 and ADH1B), which were classified into five types. A 30-min in-person or online educational counseling was conducted approximately 1 month later based on genotyping test results and their own drinking records. The control group received traditional alcohol education. Average daily alcohol consumption was calculated based on the drinking diary, which was recorded at baseline and at 3 and 6 months of follow-up. The primary endpoint was average daily alcohol consumption, and the secondary endpoints were the alcohol-use disorder identification test for consumption (AUDIT-C) score and behavioral modification stages assessed using a transtheoretical model. RESULTS: Participants were allocated to the intervention (n = 100) and control (n = 96) groups using simple randomization. Overall, 28 (29.2%) participants in the control group and 21 (21.0%) in the intervention group did not complete the follow-up. Average alcohol consumption decreased significantly from baseline to 3 and 6 months in the intervention group but not in the control group. The reduction from baseline alcohol consumption values and AUDIT-C score at 3 months were greater in the intervention group than in the control group (p < 0.001). In addition, the behavioral modification stages were significantly changed by the intervention (p < 0.001). CONCLUSIONS: Genetic testing for alcohol-metabolizing enzymes and health guidance on type-specific excessive drinking may be useful for reducing sustained average alcohol consumption associated with behavioral modification. TRIAL REGISTRATION: R000050379, UMIN000044148, Registered on June 1, 2021.

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.

Classification and functional origins of stereocomplementary alcohol dehydrogenases for asymmetric synthesis of chiral secondary alcohols: A review.

Alcohol dehydrogenases (ADHs) mediated biocatalytic asymmetric reduction of ketones have been widely applied in the synthesis of optically active secondary alcohols with highly reactive hydroxyl groups ligated to the stereogenic carbon and divided into (R)- and (S)-configurations. Stereocomplementary ADHs could be applied in the synthesis of both enantiomers and are increasingly accepted as the "first of choice" in green chemistry due to the high atomic economy, low environmental factor, 100 % theoretical yield, and high environmentally friendliness. Due to the equal importance of complementary alcohols, development of stereocomplementary ADHs draws increasing attention. This review is committed to summarize recent advance in discovery of naturally evolved and tailor-made stereocomplementary ADHs, unveil the molecular mechanism of stereoselective catalysis in views of classification and functional basis, and provide guidance for further engineering the stereoselectivity of ADHs for the industrial biosynthesis of chiral secondary alcohol of industrial relevance.

Elevated ADH5 expression suggested better prognosis in kidney renal clear cell carcinoma (KIRC) and related to immunity through single-cell and bulk RNA-sequencing.

BACKGROUND: Despite the rapid advances in modern medical technology, kidney renal clear cell carcinoma (KIRC) remains a challenging clinical problem in urology. Researchers urgently search for useful markers to break through the therapeutic conundrum due to its high lethality. Therefore, the study explores the value of ADH5 on overall survival (OS) and the immunology of KIRC. METHODS: The gene expression matrix and clinical information on ADH5 in the TCGA database were validated using external databases and qRT-PCR. To confirm the correlation between ADH5 and KIRC prognosis, univariate/multivariate Cox regression analysis was used. We also explored the signaling pathways associated with ADH5 in KIRC and investigated its association with immunity. RESULTS: The mRNA and protein levels showed an apparent downregulation of ADH5 in KIRC. Correlation analysis revealed that ADH5 was directly related to histological grade, clinical stage, and TMN stage (p < 0.05). Univariate and multivariate Cox regression analysis identified ADH5 as an independent factor affecting the prognosis of KIRC. Enrichment analysis looked into five ADH5-related signaling pathways. The results showed no correlation between ADH5 and TMB, TNB, and MSI. From an immunological perspective, ADH5 was found to be associated with the tumor microenvironment, immune cell infiltration, and immune checkpoints. Lower ADH5 expression was associated with greater responsiveness to immunotherapy. Single-cell sequencing revealed that ADH5 is highly expressed in immune cells. CONCLUSION: ADH5 could be a promising prognostic biomarker and a potential therapeutic target for KIRC. Besides, it was found that KIRC patients with low ADH5 expression were more sensitive to immunotherapy.

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.

A phenome-wide association and Mendelian randomisation study of alcohol use variants in a diverse cohort comprising over 3 million individuals.

BACKGROUND: Alcohol consumption is associated with numerous negative social and health outcomes. These associations may be direct consequences of drinking, or they may reflect common genetic factors that influence both alcohol consumption and other outcomes. METHODS: We performed exploratory phenome-wide association studies (PheWAS) of three of the best studied protective single nucleotide polymorphisms (SNPs) in genes encoding ethanol metabolising enzymes (ADH1B: rs1229984-T, rs2066702-A; ADH1C: rs698-T) using up to 1109 health outcomes across 28 phenotypic categories (e.g., substance-use, mental health, sleep, immune, cardiovascular, metabolic) from a diverse 23andMe cohort, including European (N ≤ 2,619,939), Latin American (N ≤ 446,646) and African American (N ≤ 146,776) populations to uncover new and perhaps unexpected associations. These SNPs have been consistently implicated by both candidate gene studies and genome-wide association studies of alcohol-related behaviours but have not been investigated in detail for other relevant phenotypes in a hypothesis-free approach in such a large cohort of multiple ancestries. To provide insight into potential causal effects of alcohol consumption on the outcomes significant in the PheWAS, we performed univariable two-sample and one-sample Mendelian randomisation (MR) analyses. FINDINGS: The minor allele rs1229984-T, which is protective against alcohol behaviours, showed the highest number of PheWAS associations across the three cohorts (N = 232, European; N = 29, Latin American; N = 7, African American). rs1229984-T influenced multiple domains of health. We replicated associations with alcohol-related behaviours, mental and sleep conditions, and cardio-metabolic health. We also found associations with understudied traits related to neurological (migraines, epilepsy), immune (allergies), musculoskeletal (fibromyalgia), and reproductive health (preeclampsia). MR analyses identified evidence of causal effects of alcohol consumption on liability for 35 of these outcomes in the European cohort. INTERPRETATION: Our work demonstrates that polymorphisms in genes encoding alcohol metabolising enzymes affect multiple domains of health beyond alcohol-related behaviours. Understanding the underlying mechanisms of these effects could have implications for treatments and preventative medicine. FUNDING: MVJ, NCK, SBB, SSR and AAP were supported by T32IR5226 and 28IR-0070. SSR was also supported by NIDA DP1DA054394. NCK and RBC were also supported by R25MH081482. ASH was supported by funds from NIAAA K01AA030083. JLMO was supported by VA 1IK2CX002095. JLMO and JJMM were also supported by NIDA R21DA050160. JJMM was also supported by the Kavli Postdoctoral Award for Academic Diversity. EGA was supported by K01MH121659 from the NIMH/NIH, the Caroline Wiess Law Fund for Research in Molecular Medicine and the ARCO Foundation Young Teacher-Investigator Fund at Baylor College of Medicine. MSA was supported by the Instituto de Salud Carlos III and co-funded by the European Union Found: Fondo Social Europeo Plus (FSE+) (P19/01224, PI22/00464 and CP22/00128).

Structural elucidation, binding sites exploration and biological activities of bound phenolics from Radix Puerariae Thomsonii.

Radix puerariae thomsonii (RPT) contains many phenolics and exhibits various health benefits. Although the free phenolics in RPT have been identified, the composition and content of bound phenolics, which account for approximately 20% of the total phenolic content, remain unknown. In this study, 12 compounds were isolated and identified from RPT-bound phenolic extracts, of which 2 were novel and 6 were reported first in RPT. ORAC and PSC antioxidant activities of 12 compounds, as well as their effects on alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), α-glucosidase, and α-amylase were evaluated. Genistein exhibited the highest ORAC activity, while daidzin demonstrated superior PSC activity. Five compounds, including two new compounds, exhibited the ability to activate both ADH and ALDH. All the compounds except 4-hydroxyphenylacetic acid methyl ester and 2,4,4'-trihydroxydeoxybenzoin demonstrated inhibitory effects on α-glucosidase and α-amylase. Alkaline hydrolysis and stepwise enzymatic hydrolysis revealed that bound phenolics in RPT mainly exist within starch.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

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.

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.

Identification of Corn Peptides with Alcohol Dehydrogenase Activating Activity Absorbed by Caco-2 Cell Monolayers.

Alcohol dehydrogenase (ADH) plays a pivotal role in constraining alcohol metabolism. Assessing the ADH-activating activity in vitro can provide insight into the capacity to accelerate ethanol metabolism in vivo. In this study, ADH-activating peptides were prepared from corn protein meal (CGM) using enzymatic hydrolysis, and these peptides were subsequently identified following simulated gastrointestinal digestion and their absorption through the Caco-2 cell monolayer membrane. The current investigation revealed that corn protein hydrolysate hydrolyzed using alcalase exhibited the highest ADH activation capability, maintaining an ADH activation rate of 52.93 ± 2.07% following simulated gastrointestinal digestion in vitro. After absorption through the Caco-2 cell monolayer membrane, ADH-activating peptides were identified. Among them, SSNCQPF, TGCPVLQ, and QPQQPW were validated to possess strong ADH activation activity, with EC50 values of 1.35 ± 0.22 mM, 2.26 ± 0.16 mM, and 2.73 ± 0.13 mM, respectively. Molecular Docking revealed that the activation of ADH occurred via the formation of a stable complex between the peptide and the active center of ADH by hydrogen bonds and hydrophobic interactions. The results of this study also suggest that corn protein hydrolysate could be a novel functional dietary element that helps protects the liver from damage caused by alcohol and aids in alcohol metabolism.

Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-Enzymatic Synthesis of Rivastigmine.

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.

[Metagenomics unveils the differences in the functions of microbial community of medium-temperature Daqu before and after maturation].

Daqu is the saccharifying, fermenting, and aroma-producing agent used in Baijiu brewing, and its maturation is crucial for obtaining high-quality Daqu. Previous studies have explored the microbial community composition and diversity before and after maturation. However, little is known about the changes in the functions of microbial community. In this study, based on the analyses of enzyme activities and volatile compounds of medium-temperature Daqu before and after maturation, metagenomics was used to analyze the differences in the composition of microbial community and the potential functions, with the aim to explore the microorganisms involved in changes in enzyme activities and important volatiles. The results showed that the moisture (P≤0.05), starch content, liquefying activity, saccharifying activity (P≤0.05), and fermentative activity decreased, while the acidity and esterifying activity (P≤0.05) increased after Daqu maturation. In the meantime, the composition of volatile compounds changed significantly (P=0.001), with significant decreases in the contents of aromatic alcohols and esters as well as significant increases in the contents of pyrazines, ketones, and higher fatty alcohols. The relative abundances of Mucorales (34.8%-23.0%) and Eurotiales (34.3%-20.1%) decreased in matured Daqu, and functional predictions showed these changes decreased the gene abundances of α-amylase, α-glucosidase, alcohol dehydrogenase, and alcohol dehydrogenase (NADP+) (P > 0.05), resulting in lower levels of liquefying activity (P > 0.05), saccharifying activity (P≤0.05), fermentative activity (P > 0.05), as well as aromatic alcohols such as phenylethyl alcohol (P≤0.05). In addition, higher relative abundances of Saccharomycetales (2.9%-16.6%), Lactobacillales (14.9%-23.6%), and Bacillales (0.8%-3.8%) were observed after maturation, and they were conducive to improving the gene abundances of alcohol O-acetyltransferase, carboxylesterase, acetolactate decarboxylase, (R)-acetoin dehydrogenase, and (S)-acetoin dehydrogenase (P≤0.05), resulting in significantly higher levels of esterifying activity and pyrazines (P≤0.05). The microorganisms involved in the changes in enzyme activities and important volatiles before and after Daqu maturation were studied at the gene level in this work, which may facilitate further rational regulation for Daqu production.

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.

Aß-binding with alcohol dehydrogenase drives Alzheimer's disease pathogenesis: A review.

Although Alzheimer's disease (AD) characterized with senile plaques and neurofibrillary tangles has been found for over 100 years, its molecular mechanisms are ambiguous. More worsely, the developed medicines targeting amyloid-beta (Aß) and/or tau hyperphosphorylation did not approach the clinical expectations in patients with moderate or severe AD until now. This review unveils the role of a vicious cycle between Aß-derived formaldehyde (FA) and FA-induced Aß aggregation in the onset course of AD. Document evidence has shown that Aß can bind with alcohol dehydrogenase (ADH) to form the complex of Aß/ADH (ABAD) and result in the generation of reactive oxygen species (ROS) and aldehydes including malondialdehyde, hydroxynonenal and FA; in turn, ROS-derived H2O2 and FA promotes Aß self-aggregation; subsequently, this vicious cycle accelerates neuron death and AD occurrence. Especially, FA can directly induce neuron death by stimulating ROS generation and tau hyper hyperphosphorylation, and impair memory by inhibiting NMDA-receptor. Recently, some new therapeutical methods including inhibition of ABAD activity by small molecules/synthetic polypeptides, degradation of FA by phototherapy or FA scavengers, have been developed and achieved positive effects in AD transgenic models. Thus, breaking the vicious loop may be promising interventions for halting AD progression.