Alzheimer's disease (AD) is the most common dementia affecting one in nine people over 65. Only a handful of small-molecule drugs and the anti-β amyloid (Aβ) antibody aducanumab are approved to treat AD. However, they only serve to reduce symptoms of advanced disease. Novel treatments administered early in disease progression before the accumulation of Aβ and tau reaches the threshold where neuroinflammation is triggered and irreversible neuronal damage occurs are more likely to provide effective therapy. There is a growing body of evidence implying that mitochondrial dysfunction occurs at an early stage of AD pathology. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) binds to Aβ potentiating toxicity. Moreover, ABAD has been shown to be overexpressed in the same areas of the brain most affected by AD. Inhibiting the Aβ-ABAD protein-protein interaction without adversely affecting normal enzyme turnover is hypothesized to be a potential treatment strategy for AD. Herein, we conduct structure-activity relationship studies across a series of functionalized allopurinol derivatives to determine their ability to inhibit Aβ-mediated reduction of estradiol production from ABAD. The lead compound resulting from these studies possesses potent activity with no toxicity up to 100 μM, and demonstrates an ability to rescue defective mitochondrial metabolism in human SH-SY5Y cells and rescue both defective mitochondrial metabolism and morphology ex vivo in primary 5XFAD AD mouse model neurons.

The identification of new and more effective treatments for alcohol abuse remains a priority. Alcohol intake activates glucocorticoids, which have a key role in alcohol's reinforcing properties. Glucocorticoid effects are modulated in part by the activity of 11β-hydroxysteroid dehydrogenases (11β-HSD) acting as pre-receptors. Here, we tested the effects on alcohol intake of the 11β-HSD inhibitor carbenoxolone (CBX, 18β-glycyrrhetinic acid 3β-O-hemisuccinate), which has been extensively used in the clinic for the treatment of gastritis and peptic ulcer and is active on both 11β-HSD1 and 11β-HSD2 isoforms. We observed that CBX reduces both baseline and excessive drinking in rats and mice. The CBX diastereomer 18α-glycyrrhetinic acid 3β-O-hemisuccinate (αCBX), which we found to be selective for 11β-HSD2, was also effective in reducing alcohol drinking in mice. Thus, 11β-HSD inhibitors may be a promising new class of candidate alcohol abuse medications, and existing 11β-HSD inhibitor drugs may be potentially re-purposed for alcohol abuse treatment.

Aldehyde-alcohol dehydrogenase (AdhE) is an enzyme responsible for converting acetyl-CoA to ethanol via acetaldehyde using NADH. AdhE is composed of two catalytic domains of aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH), and forms a spirosome architecture critical for AdhE activity. Here, we present the atomic resolution (3.43 Å) cryo-EM structure of AdhE spirosomes in an extended conformation. The cryo-EM structure shows that AdhE spirosomes undergo a structural transition from compact to extended forms, which may result from cofactor binding. This transition leads to access to a substrate channel between ALDH and ADH active sites. Furthermore, prevention of this structural transition by crosslinking hampers the activity of AdhE, suggesting that the structural transition is important for AdhE activity. This work provides a mechanistic understanding of the regulation mechanisms of AdhE activity via structural transition, and a platform to modulate AdhE activity for developing antibiotics and for facilitating biofuel production.

Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity.

The associations between genetic polymorphisms in ADH1B (rs1229984) and ALDH2 (rs671), alcohol consumption, the effect of a combination of the two polymorphisms, and breast cancer risk were studied in a population of East-Asian women. In this study, 623 breast cancer cases and 1845 controls, aged 40 or above, were included. The association between ALDH2 polymorphism and breast cancer risk was validated in 2143 breast cancer cases and 3977 controls. Alcohol consumption increased the risk of breast cancer regardless of ADH1B and ALDH2 genotypes. The rs671 polymorphism of ALDH2 was independently associated with increased breast cancer risk (OR = 1.27, 95% CI = 1.02-1.58 per increment of A). The ADH1B rs1229984 polymorphism, and combined effects of the rs671 and rs1229984 polymorphisms, did not reveal any significant association with breast cancer. Stratification by menopausal status revealed that rs671 gene polymorphisms were significantly associated with breast cancer only in postmenopausal women (OR = 1.45, 95% CI = 1.03-2.05 per increment of A). This is the first study to demonstrate an independent association between ALDH2 gene variants and breast cancer in Asian women. Further studies are warranted to further elucidate the etiology of breast cancer as it relates to alcohol consumption in Asian women.

The class IV alcohol dehydrogenase gene ADH7 encodes an enzyme that is involved in ethanol and retinol metabolism. ADH7 is expressed mainly in the upper gastrointestinal tract and not in the liver, the major site of expression of the other closely related ADHs. We identified an intergenic sequence (iA1C), located between ADH7 and ADH1C, that has enhancer-blocking activity in liver-derived HepG2 cells that do not express their endogenous ADH7. This enhancer blocking function was cell- and position-dependent, with no activity seen in CP-A esophageal cells that express ADH7 endogenously. iA1C function was not specific to the ADH enhancers; it had a similar cell-specific effect on the SV40 enhancer. The CCCTC-binding factor (CTCF), an insulator binding protein, bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of ADH enhancers and thereby contributes to the cell specificity of ADH7 expression.

Lignin is a complex phenylpropanoid polymer deposited in plant cell walls. Lignin has long been recognized as an important limiting factor for the polysaccharide-oriented biomass utilizations. To mitigate lignin-associated biomass recalcitrance, numerous mutants and transgenic plants that produce lignocellulose with reduced lignin contents and/or lignins with altered chemical structures have been produced and characterised. However, it is not fully understood how altered lignin chemistry affects the supramolecular structure of lignocellulose, and consequently, its utilization properties. Herein, we conducted comprehensive chemical and supramolecular structural analyses of lignocellulose produced by a rice cad2 mutant deficient in CINNAMYL ALCOHOL DEHYDROGENASE (CAD), which encodes a key enzyme in lignin biosynthesis. By using a solution-state two-dimensional NMR approach and complementary chemical methods, we elucidated the structural details of the altered lignins enriched with unusual hydroxycinnamaldehyde-derived substructures produced by the cad2 mutant. In parallel, polysaccharide assembly and the molecular mobility of lignocellulose were investigated by solid-state 13C MAS NMR, nuclear magnetic relaxation, X-ray diffraction, and Simon's staining analyses. Possible links between CAD-associated lignin modifications (in terms of total content and chemical structures) and changes to the lignocellulose supramolecular structure are discussed in the context of the improved biomass saccharification efficiency of the cad2 rice mutant.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step of the monolignol biosynthesis, the conversion of cinnamyl aldehydes to alcohols, using NADPH as a cofactor. Seven members of the CAD gene family were identified in the genome of Brachypodium distachyon and five of these were isolated and cloned from genomic DNA. Semi-quantitative reverse-transcription PCR revealed differential expression of the cloned genes, with BdCAD5 being expressed in all tissues and highest in root and stem while BdCAD3 was only expressed in stem and spikes. A phylogenetic analysis of CAD-like proteins placed BdCAD5 on the same branch as bona fide CAD proteins from maize (ZmCAD2), rice (OsCAD2), sorghum (SbCAD2) and Arabidopsis (AtCAD4, 5). The predicted three-dimensional structures of both BdCAD3 and BdCAD5 resemble that of AtCAD5. However, the amino-acid residues in the substrate-binding domains of BdCAD3 and BdCAD5 are distributed symmetrically and BdCAD3 is similar to that of poplar sinapyl alcohol dehydrogenase (PotSAD). BdCAD3 and BdCAD5 expressed and purified from Escherichia coli both showed a temperature optimum of about 50 °C and molar weight of 49 kDa. The optimal pH for the reduction of coniferyl aldehyde were pH 5.2 and 6.2 and the pH for the oxidation of coniferyl alcohol were pH 8 and 9.5, for BdCAD3 and BdCAD5 respectively. Kinetic parameters for conversion of coniferyl aldehyde and coniferyl alcohol showed that BdCAD5 was clearly the most efficient enzyme of the two. These data suggest that BdCAD5 is the main CAD enzyme for lignin biosynthesis and that BdCAD3 has a different role in Brachypodium. All CAD enzymes are cytosolic except for BdCAD4, which has a putative chloroplast signal peptide adding to the diversity of CAD functions.

Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single-cell genome data, we identified the alcohol dehydrogenase (ADH) gene, adh/a1a, from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents. The polyextremophilic ADH/A1a exhibits a broad substrate scope including aliphatic and aromatic alcohols and is able to reduce cinnamyl-methyl-ketone and raspberry ketone in the reverse reaction, making it a possible candidate for the production of chiral compounds. Here, we report the affiliation of ADH/A1a to a rare enzyme family of microbial cinnamyl alcohol dehydrogenases and explain unique structural features for halo- and thermoadaptation.

Synechocystis salt-responsive gene 1 (sysr1) was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH) superfamily] by examining the salt tolerance of sysr1-overexpressing (sysr1-OX) tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The sysr1-OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions. Additionally, the expression levels of several stress-responsive genes were upregulated. Moreover, airborne signals from salt-stressed sysr1-OX plants triggered salinity tolerance in neighboring wild-type (WT) plants. Therefore, Sysr1 enhanced the interconversion of aldehydes to alcohols, and this occurrence might affect the quality of green leaf volatiles (GLVs) in sysr1-OX plants. Actually, the Z-3-hexenol level was approximately twofold higher in sysr1-OX plants than in WT plants within 1-2 h of wounding. Furthermore, analyses of WT plants treated with vaporized GLVs indicated that Z-3-hexenol was a stronger inducer of stress-related gene expression and salt tolerance than E-2-hexenal. The results of the study suggested that increased C6 alcohol (Z-3-hexenol) induced the expression of resistance genes, thereby enhancing salt tolerance of transgenic plants. Our results revealed a role for ADH in salinity stress responses, and the results provided a genetic engineering strategy that could improve the salt tolerance of crops.

In metabolic engineering and synthetic biology fields, there have been efforts to produce variable bioalcohol fuels, such as isobutanol and 2-phenylethanol, in order to meet industrial demands. YjgB is an aldehyde dehydrogenase from Escherichia coli that shows nicotinamide adenine dinucleotide phosphate (NADP)-dependent broad selectivity for aldehyde derivatives with an aromatic ring or small aliphatic chain. This could contribute to the design of industrial synthetic pathways. We determined the crystal structures of YjgB for both its apo-form and NADP-complexed form at resolutions of 1.55 and 2.00 Å, respectively, in order to understand the mechanism of broad substrate selectivity. The hydrophobic pocket of the active site and the nicotinamide ring of NADP(H) are both involved in conferring its broad specificity toward aldehyde substrates. In addition, based on docking-simulation data, we inferred that π-π stacking between substrates and aromatic side chains might play a crucial role in recognizing substrates. Our structural analysis of YjgB might provide insights into establishing frameworks to understand its broad substrate specificity and develop engineered enzymes for industrial biofuel synthesis.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogasterAdh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Di(2-ethylhexyl) phthalate (DEHP) is an industrial additive that is widely used as a plasticizer. Due to its widespread use, DEHP is often found in freshwater ecosystems and many freshwater species have been exposed to various levels of DEHP in natural aquatic systems. Alcohol dehydrogenase (ADH) is a metabolizing enzyme produced in response to exposure to DEHP. To evaluate the effects of DEHP exposure on the ADH metabolizing process of Chironomus, the full-length cDNA of ADH from Chironomus riparius was determined through molecular cloning and rapid amplification of cDNA ends (RACE). The expression of ADH was then analyzed during different life-cycle developmental stages and under various DEHP concentrations. In addition, a comparative and phylogenetic study among different orders of insects and vertebrates was conducted through analysis of sequence databases. The complete cDNA sequence of the ADH gene was 1134 bp in length. The amino acid sequence of C. riparius ADH was found to have a low degree of homology (around 70%) with other insects available in the databases. ADH mRNA was highly expressed during various developmental stages. ADH gene expression by C. riparius increased significantly after short-term exposure (24 h) to DEHP, regardless of the exposure concentration. ADH gene expression also increased in C. riparius following exposure to DEHP for 7 days. These results suggest that DEHP affects the metabolism associated with ADH in Chironomus species.

Candida albicans proteins located on the cell wall and in the cytoplasm have gained great attention because they are not only involved in cellular metabolism and the maintenance of integrity but also interact with host immune systems. Previous research has reported that enolase from C. albicans exhibits high immunogenicity and effectively protects mice against disseminated candidiasis. In this study, alcohol dehydrogenase (ADH) of C. albicans was cloned and purified for the first time, and this study focused on evaluating its effects on the differentiation of the human monocytic cell line THP-1. The morphological features of THP-1 cells exposed to ADH were similar to those of phorbol-12-myristate acetate-differentiated (PMA-differentiated) macrophages. Functionally, ADH enhanced the adhesion, phagocytosis, and killing capacities of THP-1 cells. A flow cytometric assay demonstrated that ADH-induced THP-1 cells significantly increased CD86 and CD11b expression. The production of IL-1β, IL-6, and TNF-α by cells increased in the presence of ADH. As expected, after pretreatment with a MEK inhibitor (U0126), ADH-induced THP-1 cells exhibited unaltered morphological features, eliminated ERK1/2 phosphorylation, prevented CD86/CD11b upregulation and inhibited pro-inflammatory cytokine increase. Collectively, these results suggest that ADH enables THP-1 cells to differentiate into macrophages via the ERK pathway, and it may play an important role in the immune response against fungal invasion.

In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease Vmax and Km for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in Vmax is likely due to excluded volume effects, which are partially counteracted by viscosity hindering release of the NAD+ product. Macromolecular crowding is further complicated by the presence of a depletion layer in solutions of dextran larger than YADH, which diminishes the hindrance from viscosity. The disparate effects from 25 g/L dextran or glucose compared to 25 g/L Ficoll or sucrose reveals that soft interactions must also be considered. Data from binary mixtures of glucose, dextran, and Ficoll support this "tuning" of opposing factors. While macromolecular crowding was originally proposed to influence proteins mainly through excluded volume effects, this work compliments the growing body of evidence revealing that other factors, such as preferential hydration, chemical interactions, and the presence of a depletion layer also contribute to the overall effect of crowding.

In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production).Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants.

In a population-based study of 613 cases and 1082 controls, alcohol dehydrogenase 1B (ADH1B) genotype was not an independent risk factor for breast cancer, although the possibility was raised that it modifies risk associated with high levels of alcohol consumption (OR 1.1, 95% confidence interval (CI) 0.8-1.6 for ADH1B*1/*1 genotype vs 0.2, 95% CI 0.1-1.0 for ADH1B*2 carriers).

Multiple genes have been implicated in Parkinson's disease (PD), including causal gene variants and risk variants typically identified using genome-wide association studies. Variants in the alcohol dehydrogenase genes ADH1C and ADH1B are among the genes that have been associated with PD, suggesting that this family of genes may be important in PD. As part of the International Parkinson's Disease Genomics Consortium's efforts to scrutinize previously reported risk factors for PD, we explored genetic variation in the alcohol dehydrogenase genes ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6, and ADH7 using imputed genome-wide association study data from 15,097 cases and 17,337 healthy controls. Rare-variant association tests and single-variant score tests did not show any statistically significant association of alcohol dehydrogenase genetic variation with the risk for PD.

The bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Å resolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering.