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.

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher T 50 15 $$ {\mathrm{T}}_{50}^{15} $$ , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

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.

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.

Interaction of genetic variation at ADH1B and MLXIPL with alcohol consumption for elevated serum urate level and gout among people of European ethnicity.

BACKGROUND: Alcohol consumption is a risk factor for hyperuricaemia and gout. Multiple single-nucleotide polymorphisms (SNPs) have been identified as associated with both alcohol consumption and serum urate or gout in separate genome-wide association studies (GWAS). This study aimed to identify and characterise interactions between these shared signals of genetic association and alcohol consumption for serum urate level, hyperuricaemia, and gout. METHODS: This research was conducted using the UK Biobank resource. The association of alcohol consumption with serum urate and gout was tested among 458,405 European participants. Candidate SNPs were identified by comparing serum urate, gout, and alcohol consumption GWAS for shared signals of association. Multivariable-adjusted linear and logistic regression analyses were conducted with the inclusion of interaction terms to identify SNP-alcohol consumption interactions for association with serum urate level, hyperuricaemia, and gout. The nature of these interactions was characterised using genotype-stratified association analyses. RESULTS: Alcohol consumption was associated with elevated serum urate and gout. For serum urate level, non-additive interactions were identified between alcohol consumption and rs1229984 at the ADH1B locus (P = 3.0 × 10-44) and rs6460047 at the MLXIPL locus (P = 1.4 × 10-4). ADH1B also demonstrated interaction with alcohol consumption for hyperuricaemia (P = 7.9 × 10-13) and gout (P = 8.2 × 10-9). Beer intake had the most significant interaction with ADH1B for association with serum urate and gout among men, while wine intake had the most significant interaction among women. In the genotype-stratified association analyses, ADH1B and MLXIPL were associated with serum urate level and ADH1B was associated with hyperuricaemia and gout among consumers of alcohol but not non-consumers. CONCLUSIONS: In this large study of European participants, novel interactions with alcohol consumption were identified at ADH1B and MLXIPL for association with serum urate level and at ADH1B for association with hyperuricaemia and gout. The association of ADH1B with serum urate and gout may occur through the modulation of alcohol metabolism rate among consumers of alcohol.

Association between alcohol dehydrogenase polymorphisms (rs1229984, rs1573496, rs1154460, and rs284787) and susceptibility to head and neck cancers: A systematic review and meta-analysis.

OBJECTIVE: Head and neck cancer (HNC) is a prevalent and complex group of malignancies with increasing incidence globally. Alcohol dehydrogenases (ADHs) play a crucial role in alcohol metabolism, and their polymorphisms have been linked to HNC risk. This systematic review and meta-analysis aims to evaluate the association between ADH polymorphisms and susceptibility to HNCs, incorporating additional analyses and adding more studies to increase power and accuracy of the results. DESIGN: Subgroup analysis, meta-regression analysis, and sensitivity analyses were conducted to explore potential differences within the data and assess the stability of pooled odds ratios (ORs). To mitigate the risk of false conclusions from meta-analyses, a trial sequential analysis was performed. RESULTS: For ADH1B rs1229984, the pooled OR (95 % confidence interval (CI)) was 0.73 (0.65, 0.82), 0.42 (0.35, 0.50), 0.57 (0.44, 0.73), 0.56 (0.50, 0.62), and 0.80 (0.73, 0.88), as well as for ADH7 rs1573496, the pooled OR was 0.72 (0.62, 0.85), 0.36 (0.17, 0.74), 0.76 (0.64, 0.91), 0.80 (0.71, 0.91), and 0.38 (0.18, 0.78) with a p < 0.05 in all allelic, homozygous, heterozygous, recessive, and dominant models, respectively. However, no significant association was found between the ADH7 rs1154460 and rs284787 polymorphisms and the risk of HNC with pooled ORs of 1.11 (p = 0.19) and 1.09 (p = 0.24) for the recessive model, respectively. The ethnicities, tumor subsites, control sources, sample sizes, quality scores, and Hardy-Weinberg equilibrium statuses were confounding factors. CONCLUSION: The ADH1B rs1229984 and ADH7 rs1573496 polymorphisms are significantly associated with a reduced risk of HNC.

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.

ALDH2 dysfunction and alcohol cooperate in cancer stem cell enrichment.

The alcohol metabolite acetaldehyde is a potent human carcinogen linked to esophageal squamous cell carcinoma (ESCC) initiation and development. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulation causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K dominant negative mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent among East Asians. Heterozygous ALDH2*2 increases the risk for the development of ESCC and other alcohol-related cancers. Despite its prevalence and link to malignant transformation, how ALDH2 dysfunction influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three-dimensional organoids and xenograft models. We find that alcohol exposure and ALDH2*2 cooperate to increase putative ESCC cancer stem cells with high CD44 expression (CD44H cells) linked to tumor initiation, repopulation and therapy resistance. Concurrently, ALHD2*2 augmented alcohol-induced reactive oxygen species and DNA damage to promote apoptosis in the non-CD44H cell population. Pharmacological activation of ALDH2 by Alda-1 inhibits this phenotype, suggesting that acetaldehyde is the primary driver of these changes. Additionally, we find that Aldh2 dysfunction affects the response to cisplatin, a chemotherapeutic commonly used for the treatment of ESCC. Aldh2 dysfunction facilitated enrichment of CD44H cells following cisplatin-induced oxidative stress and cell death in murine organoids, highlighting a potential mechanism driving cisplatin resistance. Together, these data provide evidence that ALDH2 dysfunction accelerates ESCC pathogenesis through enrichment of CD44H cells in response to genotoxic stressors such as environmental carcinogens and chemotherapeutic agents.

Construction of prophage-free and highly-transformable Limosilactobacillus reuteri strains and their use for production of 1,3-propanediol.

The lactic acid bacterium Limosilactobacillus reuteri (formerly Lactobacillus reuteri) is a desirable host for the production of 1,3-propanediol (1,3-PDO) from glycerol when 1,3-PDO is used in the food or cosmetic industry. However, the production is hindered by strain instability, causing cell lysis, and difficult gene manipulation. This study reveals that the stability of L. reuteri DSM 20016 and its 1,3-PDO production, especially in the alcohol dehydrogenases (ADHs)-deletion mutants, are greatly enhanced after the deletion of two prophages (Φ3 and Φ4) present in the L. reuteri's chromosome. The resulting phage-free and ADHs-deletion mutant could produce >825 mM 1,3-PDO in 48 h without cell lysis at the theoretical maximum yield on glucose of ~2 mol/mol. Compared to the wild-type strain, the mutant exhibited a 45.2% increase in 1,3-PDO production titer and a 2.1-fold increase in yield. In addition, this study reports that the transformation efficiency of L. reuteri Δadh2Δadh6 mutant strains were greatly enhanced by >300-fold after the deletion of prophage Φ3, probably due to the removal of a restriction-modification (RM) system which resides in the phage genome. With improved stability and higher transformation efficiency, recombinant L. reuteri DSM 20016 Δadh2Δadh6ΔΦ3ΔΦ4 can be a more reliable and amenable host for industrial applications.

Genetic influences on alcohol flushing in East Asian populations.

BACKGROUND: Although it is known that variation in the aldehyde dehydrogenase 2 (ALDH2) gene family influences the East Asian alcohol flushing response, knowledge about other genetic variants that affect flushing symptoms is limited. METHODS: We performed a genome-wide association study meta-analysis and heritability analysis of alcohol flushing in 15,105 males of East Asian ancestry (Koreans and Chinese) to identify genetic associations with alcohol flushing. We also evaluated whether self-reported flushing can be used as an instrumental variable for alcohol intake. RESULTS: We identified variants in the region of ALDH2 strongly associated with alcohol flushing, replicating previous studies conducted in East Asian populations. Additionally, we identified variants in the alcohol dehydrogenase 1B (ADH1B) gene region associated with alcohol flushing. Several novel variants were identified after adjustment for the lead variants (ALDH2-rs671 and ADH1B-rs1229984), which need to be confirmed in larger studies. The estimated SNP-heritability on the liability scale was 13% (S.E. = 4%) for flushing, but the heritability estimate decreased to 6% (S.E. = 4%) when the effects of the lead variants were controlled for. Genetic instrumentation of higher alcohol intake using these variants recapitulated known associations of alcohol intake with hypertension. Using self-reported alcohol flushing as an instrument gave a similar association pattern of higher alcohol intake and cardiovascular disease-related traits (e.g. stroke). CONCLUSION: This study confirms that ALDH2-rs671 and ADH1B-rs1229984 are associated with alcohol flushing in East Asian populations. Our findings also suggest that self-reported alcohol flushing can be used as an instrumental variable in future studies of alcohol consumption.

The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum.

The Wood-Ljungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (Ald-Adh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum ∆adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the Adh-Ald route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to ∆adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these 'stored assets' for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.

An alcohol dehydrogenase 7 gene polymorphism associates with both acute and chronic pain in sickle cell disease.

Introduction: As the most distressing complication of sickle cell disease (SCD), pain is marked by considerable heterogenicity. In this study we explored the potential association of alcohol dehydrogenase 7 gene (ADH7) polymorphism rs971074 with sickle cell pain. Methods: We analyzed clinical phenotypes and the rs971074 single-nucleotide polymorphism in ADH7 by MassARRAY-iPlex analysis in a cohort of SCD patients. Results: The synonymous rs971074 was significantly associated with both acute and chronic pain in SCD. Patients with the minor T allele(s) recorded significantly more crisis episodes and severe chronic pain symptoms. Conclusion: Our study has identified the rs971074 minor T allele as a genetic biomarker potentially influencing acute and chronic pain. These findings may ultimately help inform strategies to develop precision pain therapies in SCD.

Light-Driven CO2 Reduction with a Surface-Displayed Enzyme Cascade-C3N4 Hybrid.

Efficient and cost-effective conversion of CO2 to biomass holds the potential to address the climate crisis. Light-driven CO2 conversion can be realized by combining inorganic semiconductors with enzymes or cells. However, designing enzyme cascades for converting CO2 to multicarbon compounds is challenging, and inorganic semiconductors often possess cytotoxicity. Therefore, there is a critical need for a straightforward semiconductor biohybrid system for CO2 conversion. Here, we used a visible-light-responsive and biocompatible C3N4 porous nanosheet, decorated with formate dehydrogenase, formaldehyde dehydrogenase, and alcohol dehydrogenase to establish an enzyme-photocoupled catalytic system, which showed a remarkable CO2-to-methanol conversion efficiency with an apparent quantum efficiency of 2.48% in the absence of externally added electron mediator. To further enable the in situ transformation of methanol into biomass, the enzymes were displayed on the surface of Komagataella phaffii, which was further coupled with C3N4 to create an organic semiconductor-enzyme-cell hybrid system. Methanol was produced through enzyme-photocoupled CO2 reduction, achieving a rate of 4.07 mg/(L·h), comparable with reported rates from photocatalytic systems employing mediators or photoelectrochemical cells. The produced methanol can subsequently be transported into the cell and converted into biomass. This work presents a sustainable, environmentally friendly, and cost-effective enzyme-photocoupled biocatalytic system for efficient solar-driven conversion of CO2 within a microbial cell.

Complementation of reducing power for 5-hydroxyvaleric acid and 1,5-pentanediol production via glucose dehydrogenase in Escherichia coli whole-cell system.

One of the key intermediates, 5-hydroxyvaleric acid (5-HV), is used in the synthesis of polyhydroxyalkanoate monomer, δ-valerolactone, 1,5-pentanediol (1,5-PDO), and many other substances. Due to global environmental problems, eco-friendly bio-based synthesis of various platform chemicals and key intermediates are socially required, but few previous studies on 5-HV biosynthesis have been conducted. To establish a sustainable bioprocess for 5-HV production, we introduced gabT encoding 4-aminobutyrate aminotransferase and yqhD encoding alcohol dehydrogenase to produce 5-HV from 5-aminovaleric acid (5-AVA), through glutarate semialdehyde in Escherichia coli whole-cell reaction. As, high reducing power is required to produce high concentrations of 5-HV, we newly introduced glucose dehydrogenase (GDH) for NADPH regeneration system from Bacillus subtilis 168. By applying GDH with D-glucose and optimizing the parameters, 5-HV conversion rate from 5-AVA increased from 47% (w/o GDH) to 82% when using 200 mM (23.4 g/L) of 5-AVA. Also, it reached 56% conversion in 2 h, showing 56 mM/h (6.547 g/L/h) productivity from 200 mM 5-AVA, finally reaching 350 mM (41 g/L) and 14.6 mM/h (1.708 g/L/h) productivity at 24 h when 1 M (117.15 g/L) 5-AVA was used. When the whole-cell system with GDH was expanded to produce 1,5-PDO, its production was also increased 5-fold. Considering that 5-HV and 1,5-PDO production depends heavily on the reducing power of the cells, we successfully achieved a significant increase in 5-HV and 1,5-PDO production using GDH.

Class III Alcohol Dehydrogenase Plays a Key Role in the Onset of Alcohol-Related/-Associated Liver Disease as an S-Nitrosoglutathione Reductase in Mice.

Lipid accumulation in the liver due to chronic alcohol consumption (CAC) is crucial in the development of alcohol liver disease (ALD). It is promoted by the NADH/NAD ratio increase via alcohol dehydrogenase (ADH)-dependent alcohol metabolism and lipogenesis increase via peroxisome proliferator-activated receptor γ (PPARγ) in the liver. The transcriptional activity of PPARγ on lipogenic genes is inhibited by S-nitrosylation but activated by denitrosylation via S-nitrosoglutathione reductase (GSNOR), an enzyme identical to ADH3. Besides ADH1, ADH3 also participates in alcohol metabolism. Therefore, we investigated the specific contribution of ADH3 to ALD onset. ADH3-knockout (Adh3-/-) and wild-type (WT) mice were administered a 10% ethanol solution for 12 months. Adh3-/- exhibited no significant pathological changes in the liver, whereas WT exhibited marked hepatic lipid accumulation (p < 0.005) with increased serum transaminase levels. Adh3-/- exhibited no death during CAC, whereas WT exhibited a 40% death. Liver ADH3 mRNA levels were elevated by CAC in WT (p < 0.01). The alcohol elimination rate measured after injecting 4 g/kg ethanol was not significantly different between two strains, although the rate was increased in both strains by CAC. Thus, ADH3 plays a key role in the ALD onset, likely by acting as GSNOR.

Physiological responses and Ethylene-Response AP2/ERF Factor expression in Indica rice seedlings subjected to submergence and osmotic stress.

BACKGROUND: The increased frequency of heavy rains in recent years has led to submergence stress in rice paddies, severely affecting rice production. Submergence causes not only hypoxic stress from excess water in the surrounding environment but also osmotic stress in plant cells. We assessed physiological responses and Ethylene-Response AP2/ERF Factor regulation under submergence conditions alone and with ionic or nonionic osmotic stress in submergence-sensitive IR64 and submergence-tolerant IR64-Sub1 Indica rice cultivars. RESULTS: Our results indicate that both IR64 and IR64-Sub1 exhibited shorter plant heights and root lengths under submergence with nonionic osmotic stress than normal condition and submergence alone. IR64-Sub1 seedlings exhibited a significantly lower plant height under submergence conditions alone and with ionic or nonionic osmotic stress than IR64 cultivars. IR64-Sub1 seedlings also presented lower malondialdehyde (MDA) concentration and higher survival rates than did IR64 seedlings after submergence with ionic or nonionic osmotic stress treatment. Sub1A-1 affects reactive oxygen species (ROS) accumulation and antioxidant enzyme activity in rice. The results also show that hypoxia-inducible ethylene response factors (ERF)-VII group and alcohol dehydrogenase 1 (ADH1) and lactate dehydrogenase 1 (LDH1) genes exhibited different expression levels under nonionic or ionic osmotic stress during submergence on rice. CONCLUSIONS: Together, these results demonstrate that complex regulatory mechanisms are involved in responses to the aforementioned forms of stress and offer new insights into the effects of submergence and osmotic stress on rice.

Enhancing the activity of a monomeric alcohol dehydrogenase for site-specific applications by site-directed mutagenesis.

Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric control. Thus, a toolkit of active and robust monomeric enzymes is desirable for such applications. In this study, we engineered one of the rare examples of monomeric alcohol dehydrogenases for improved catalytic characteristics by site-directed mutagenesis. The enzyme from the hyperthermophilic archaeon Thermococcus kodakarensis naturally exhibits high thermostability and a broad substrate spectrum, but only low activity at moderate temperatures. The best enzyme variants showed an ~5-fold (2-heptanol) and 9-fold (3-heptanol) higher activity while preserving enantioselectivity and good thermodynamic stability. These variants also exhibited modified kinetic characteristics regarding regioselectivity, pH dependence and activation by NaCl.

Association of ADH1B rs1229984, ADH1C rs698, and ALDH2 rs671 with Alcohol abuse and Alcoholic Cirrhosis in People Living in Northeast Vietnam.

OBJECTIVE: Alcohol abuse can cause developing cirrhosis, even liver cancer. Several single nucleotide polymorphisms (SNPs) of ADH1B, ADH1C, and ALDH2 genes have been reported to be associated with alcohol abuse and alcoholic cirrhosis (ALC). This study investigated the association between three SNPs of ADH1B rs1229984, ADH1C rs698, and ALDH2 rs671 with alcohol abuse and ALC in people living in the Northeast region of Vietnam. METHODS: 306 male participants were recruited including 206 alcoholics (106 ALC, 100 without ALC) and 100 healthy non-alcoholics. Clinical characteristics were collected by clinicians. Genotypes were identified by Sanger sequencing. Chi-Square (χ2) and Fisher-exact tests were used to assess the differences in age and clinical characteristics, Child-Pugh score, frequencies of alleles and genotypes. RESULT: Our data showed that the frequency of ALDH2*1 was significantly higher in alcoholics (88.59%) and ALC groups (93.40%) than that of healthy non-alcoholics (78.50%) with p=0.0009 and non-ALC group (83.50%) with p=0.002, respectively. We detected opposite results when examined ALDH2*2. Frequency of combined genotypes with high acetaldehyde accumulation were significantly lower in alcoholics and ALC group than those of control groups with p=0.005 and p=0.008, respectively. Meanwhile, the proportion of combined genotypes with non-acetaldehyde accumulation were significantly two times higher in the ALC group (19.98%) than those of the non-ALC group (8%) with p=0.035. These combined genotypes showed a decreasing trend in the Child-Pugh score from likely phenotype causing risk for non-acetaldehyde accumulation to high acetaldehyde accumulation. CONCLUSION: The ALDH2*1 allele was found as a risk factor for alcohol abuse and ALC, and combined genotypes of ADH1B rs1229984, ADH1C rs698, and ALDH2 rs671 with non-acetaldehyde accumulation increase ALC risk. In contrast, ALDH2*2 and the genotype combinations related to high acetaldehyde accumulation were protective factors against alcohol abuse and ALC.

Risk factors for the development of second primary esophageal squamous-cell carcinoma after endoscopic resection for esophageal squamous-cell carcinoma according to genetic polymorphisms related to alcohol and nicotine metabolism.

BACKGROUND: Multiple development of esophageal squamous-cell carcinoma is explained by field cancerization and is associated with alcohol consumption and smoking. We investigated the association between the development of second primary esophageal squamous-cell carcinoma after endoscopic resection for esophageal squamous-cell carcinoma and genetic polymorphisms related to alcohol and nicotine metabolism. METHODS: The study group comprised 56 patients with esophageal squamous-cell carcinoma after endoscopic resection. The main variables were the following: (i) cumulative incidence and total number of second primary esophageal squamous-cell carcinoma according to genetic polymorphisms in alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and cytochrome P450 2A6; and (ii) risk factors of second primary esophageal squamous-cell carcinoma identified using a multivariate Cox proportional-hazards model. The frequencies of alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and cytochrome P450 2A6 genetic polymorphisms in the buccal mucosa were analyzed. RESULTS: The median follow-up was 92.8 months (range: 2.7-134.2). Slow-metabolizing alcohol dehydrogenase 1B was associated with a higher 7-year cumulative incidence of second primary esophageal squamous-cell carcinoma (fast-metabolizing alcohol dehydrogenase 1B vs slow-metabolizing alcohol dehydrogenase 1B: 20.5% vs 71.4%, P = 0.006). Slow-metabolizing alcohol dehydrogenase 1B (relative risk [95% confidence interval]: 3.17 [1.49-6.73]), inactive aldehyde dehydrogenase 2 (2.17 [1.01-4.63]) and poorly-metabolizing cytochrome P450 2A6 (4.63 [1.74-12.33]) had a significantly higher total number of second primary esophageal squamous-cell carcinoma per 100 person-years. In the multivariate Cox proportional-hazards model, slow-metabolizing alcohol dehydrogenase 1B was a significant risk factor of the development of second primary esophageal squamous-cell carcinoma (hazard ratio 9.92, 95% confidence interval: 2.35-41.98, P = 0.0018). CONCLUSIONS: Slow-metabolizing alcohol dehydrogenase 1B may be a significant risk factor for the development of second primary esophageal squamous-cell carcinoma. In addition, inactive aldehyde dehydrogenase 2 and poorly-metabolizing cytochrome P450 2A6 may be important factors.

Infantile Neurodegeneration Results from Mutants of 17ß-Hydroxysteroid Dehydrogenase Type 10 Rather Than Aß-Binding Alcohol Dehydrogenase.

Type 10 17ß-hydroxysteroid dehydrogenase (17ß-HSD10), a homo-tetrameric multifunctional protein with 1044 residues encoded by the HSD17B10 gene, is necessary for brain cognitive function. Missense mutations result in infantile neurodegeneration, an inborn error in isoleucine metabolism. A 5-methylcytosine hotspot underlying a 388-T transition leads to the HSD10 (p.R130C) mutant to be responsible for approximately half of all cases suffering with this mitochondrial disease. Fewer females suffer with this disease due to X-inactivation. The binding capability of this dehydrogenase to Aß-peptide may play a role in Alzheimer's disease, but it appears unrelated to infantile neurodegeneration. Research on this enzyme was complicated by reports of a purported Aß-peptide-binding alcohol dehydrogenase (ABAD), formerly referred to as endoplasmic-reticulum-associated Aß-binding protein (ERAB). Reports concerning both ABAD and ERAB in the literature reflect features inconsistent with the known functions of 17ß-HSD10. It is clarified here that ERAB is reportedly a longer subunit of 17ß-HSD10 (262 residues). 17ß-HSD10 exhibits L-3-hydroxyacyl-CoA dehydrogenase activity and is thus also referred to in the literature as short-chain 3-hydorxyacyl-CoA dehydrogenase or type II 3-hydorxyacyl-CoA dehydrogenase. However, 17ß-HSD10 is not involved in ketone body metabolism, as reported in the literature for ABAD. Reports in the literature referring to ABAD (i.e., 17ß-HSD10) as a generalized alcohol dehydrogenase, relying on data underlying ABAD's activities, were found to be unreproducible. Furthermore, the rediscovery of ABAD/ERAB's mitochondrial localization did not cite any published research on 17ß-HSD10. Clarification of the purported ABAD/ERAB function derived from these reports on ABAD/ERAB may invigorate this research field and encourage new approaches to the understanding and treatment of HSD17B10-gene-related disorders. We establish here that infantile neurodegeneration is caused by mutants of 17ß-HSD10 but not ABAD, and so we conclude that ABAD represents a misnomer employed in high-impact journals.