UNASSIGNED: Alcohol flushing syndrome (AFS) is experienced by up to 46% of East Asians. This study aimed to review the risk of cancers in AFS patients, elucidate an exposure-response relationship, and understand risk associated with alcohol intake and cancer.
UNASSIGNED: An electronic database search of PubMed, Embase, Scopus and Cochrane Library was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines. Observational studies on AFS\' effects and all cancers risk were included. Studies including patients with existing malignancy were excluded. Dichotomous variables were pooled using the Mantel-Haenszel method with a random effects model. Sensitivity and subgroup analyses were performed. PROSPERO (CRD42023392916) protocol was followed.
UNASSIGNED: A total of 18 articles were included in the final analysis with a total of 387,521 participants. AFS was associated with an increased risk of all cancers (odds ratio [OR] 1.19, 95% confidence interval [CI] 1.06-1.34), esophageal squamous cell carcinoma (OR 1.47, 95% CI 1.05-2.05) and gastric adenocarci-noma (OR 1.40, 95% CI 1.14-1.72). Men with AFS exhibited an increased risk of all cancers (OR 1.34, 95% CI 1.13-1.59). However, this was not observed in women. All cancers risk was associated with AFS in those who consumed drink (i.e. consumed alcohol) more than 200 g of pure ethanol/week (OR 1.68, 95% CI 1.20-2.37) but not those who consumed less than 200 g of pure ethanol/week (OR 1.27, 95% CI 0.90-1.79) or non-drinkers (OR 0.99, 95% CI 0.67-1.47).
UNASSIGNED: AFS is associated with an increased risk of all cancers, particularly esophageal squamous cell carcinoma and gastric adenocarcinoma.

The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca2+-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K142A- and K142D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV-Vis absorption and emission spectroscopies support that K142D-PqqT binds tightly (Kd = 0.6 ± 0.2 μM) to La3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K142A-variants or upon addition of Ca2+ to PQQ ⸦ K142D-PqqT. Addition of benzyl alcohol to La3+-bound PQQ ⸦ K142D-PqqT (but not Ca2+-bound PQQ ⸦ K142D-PqqT, or La3+-bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.

The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.

OBJECTIVE: This study aims to explore ADH4 expression in hepatocellular carcinoma (HCC), its prognostic impact, and its immune correlation to provide novel insights into HCC prognostication and treatment.
METHODS: HCC prognostic marker genes were rigorously selected using GEO database, Lasso regression, GEPIA, Kaplan-Meier and pROC analyses. The expression of interested markers (ADH4, DNASE1L3, RDH16, LCAT, HGFAC) in HCC and adjacent tissues was assessed by Immunohistochemistry (IHC). We observed that ADH4 exhibited low expression levels in liver cancer tissues and high expression levels in normal liver tissues. However, the remaining four genes did not manifest any statistically significant differences between hepatocellular carcinoma (HCC) tissue and adjacent non-cancerous tissue. Consequently, ADH4 became the primary focus of our research. ADH4 expression was validated by signed-rank tests and unpaired Wilcoxon rank sum tests across pan-cancer and HCC datasets. Clinical significance and associations with clinicopathological variables were determined using Kaplan-Meier, logistic regression and Cox analyses on TCGA data. The ADH4-related immune responses were explored by Spearman correlation analysis using TIMER2 data. CD68, CD4, and CD19 protein levels were confirmed by IHC in HCC and non-cancerous tissues.
RESULTS: ADH4 showed significant downregulation in various cancers, particularly in HCC. Moreover, low ADH4 expression was associated with clinicopathological variables and served as an independent prognostic marker for HCC patients. Additionally, ADH4 affects a variety of biochemical functions and may influence cancer development, prognosis, and treatment by binding to immune cells. Furthermore, at the immune level, the low expression pattern of ADH4 is TME-specific, indicating that ADH4 has the potential to be used as a target for cancer immunotherapy.
CONCLUSIONS: This study highlights the diagnostic, prognostic and immunomodulatory roles of ADH4 in HCC. ADH4 could serve as a valuable biomarker for HCC diagnosis and prognosis, as well as a potential target for immunotherapeutic interventions.

Upgrading biomass-derived bioethanol to higher-order alcohols using conventional biotechnological approaches is challenging. Herein, a novel, magnetic metal-organic-framework-based cofactor regeneration system was developed using ethanol dehydrogenase (EtDH:D46G), NADH oxidase (NOX), formolase (FLS:L482S), and nicotinamide adenine dinucleotide (NAD+) for converting rice straw-derived bioethanol to acetoin. A magnetic zeolitic imidazolate framework-8@Fe3O4/NAD+ (ZIF-8@Fe3O4/NAD+) regeneration system for cell-free cascade reactions was introduced and used to encapsulate EtDH:D46G, NOX, and FLS:L482S (ENF). ZIF-8@Fe3O4/NAD+ENF created an efficient microenvironment for three-step enzyme cascades. Under the optimized conditions, the yield of acetoin from 100 mM bioethanol using ZIF-8@Fe3O4/NAD+ENF was 90.4 %. The regeneration system showed 97.1 % thermostability at 50 °C. The free enzymes retained only 16.3 % residual conversion, compared with 91.2 % for ZIF-8@Fe3O4/NAD+ENF after ten cycles. The magnetic metal-organic-framework-based cofactor regeneration system is suitable for enzymatic cascade biotransformations and can be extended to other cascade systems for potential biotechnological applications.

Asymmetric reduction of 2-chloro-1-(6-fluorochroman-2-yl)ethan-1-one (NEB-7) into 2-chloro-1-(6-fluorochroman-2-yl)ethan-1-ol (NEB-8) is the crucial step for synthesis of liposoluble β1 receptor blocker nebivolol. Four efficient and stereoselective alcohol dehydrogenases were identified, enabling the stereoselective synthesis of all enantiomers of NEB-8 at a substrate loading of 137 g·L-1 with ee values of >99% and high space-time yields. This study provides novel biocatalysts for the efficient synthesis of nebivolol precursors and uncovers the molecular basis for enantioselectivity manipulation by parametrization of Prelog\'s rule.

Clostridium thermocellum is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native adhE gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase (adh) genes. We were able to express all of them successfully in C. thermocellum. Surprisingly, however, none of them increased ethanol production, and several actually decreased it. Our findings contribute to understanding the correlation between C. thermocellum ethanol production and Adh enzyme cofactor preferences. The identification of a set of adh genes that can be successfully expressed in this organism provides a foundation for future investigations into how the properties of Adh enzymes affect ethanol production.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP+ are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pKa value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase enzyme, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.

Alcohol dehydrogenase (ADH) is an important enzyme that catalyzes alcohol oxidation and/or aldehyde reduction. As one of NAD+-dependent ADH types, iron-containing/activated ADH (Fe-ADH) is ubiquitous in Bacteria, Archaea, and Eukaryotes, possessing a similar \"tunnel-like\" structure that is composed of a domain A in its N-terminus and a domain B in its C-terminus. A conserved \"GGGS\" sequence in the domain A of Fe-ADH associates with NAD+, and one conserved Asp residue and three conserved His residues in the domain B are its catalytic active sites by surrounding with Fe atom, suggesting that it might employ similar catalytic mechanism. Notably, all the biochemically characterized Fe-ADHs from hyperthermophiles that thrive in above 80 °C possess two unique characteristics that are absent in other Fe-ADHs: thermophilicity and thermostability, thereby demonstrating that they can oxidize alcohol and reduce aldehyde at high temperature. Considering these two unique characteristics, Fe-ADHs from hyperthermophiles are potentially industrial biocatalysts for alcohol and aldehyde biotransformation at high temperature. Herein, we reviewed structural and biochemical characteristics of Fe-ADHs from hyperthermophiles, focusing on similarity and difference between Fe-ADHs from hyperthermophiles and their homologs from non-hyperthermophiles, and between hyperthermophilic archaeal Fe-ADHs and bacterial homologs. Furthermore, we proposed future directions of Fe-ADHs from hyperthermophiles.