Alcohol Dehydrogenase Research Articles

Identification of candidate biomarkers for gastric cancer by bioinformatics analysis of pooled microarray gene expression datasets in Gene Expression Omnibus.

467 Background: Gastric cancer (GC) remains one of the most prevalent malignant diseases worldwide. The high mortality rate is largely due to the limited treatment options and the challenges in diagnosing the cancer at an early stage. Understanding the genetic differences driving the proliferation of GC is crucial for advancing diagnostic methods and developing targeted treatments. This study aims to identify biomarkers involved in the pathogenesis of GC through integrated bioinformatics analysis. Methods: Extracted from the Geo Expression Omnibus, three DataSets (GSE118916, GSE79973, and GSE13861) were analyzed for differentially expressed genes (DEGs) using the R statistical language. The three data sets were then compared for common DEGs, which would serve as the focus of this study. These DEGs were functionally analyzed for pathways by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A protein–protein interaction (PPI) network was constructed to screen out candidate genes. Results: The results revealed that 123 DEGs of the three datasets containing 26 upregulated genes and 97 downregulated genes were ascertained in our study. The GO and KEGG enrichment analysis results showed that the functions of DEGs were mainly involved in the retinol metabolic process, xenobiotic metabolic process, collagen type I trimer, potassium: proton exchanging ATPase complex, benzaldehyde dehydrogenase (NAD+) activity, alcohol dehydrogenase (NADP+) activity, Collecting duct acid secretion, and Tyrosine metabolism. Through the PPI analysis network, the core genes with the highest degree of 7 nodes were selected: COL1A1, COL1A2, COL11A1, THBS2, ATP4A, COL10A1, and CXCL8. Conclusions: The 7 genes identified in this study may play a vital regulatory role in the occurrence and development of GC and may also be potential therapeutic targets. Further mechanistic studies of the pathogenesis and treatment of GC may be able to identify new targets using these shared pathways. These findings could contribute to a better understanding of the origins of gastric cancer and facilitate the development of early interventions. The bioinformatics analysis conducted in this study offers new insights and directions for further research on gastric cancer.

Identifying a robust alcohol dehydrogenase for developing one-pot chemobiocatalytic route toward 2,5-bis(hydroxymethyl)furan from glucose

Identifying a robust alcohol dehydrogenase for developing one-pot chemobiocatalytic route toward 2,5-bis(hydroxymethyl)furan from glucose

Loop engineering of enzymes to control their immobilization and ultimately fabricate more efficient heterogeneous biocatalysts.

Enzyme immobilization is indispensable for enhancing enzyme performance in various industrial applications. Typically, enzymes require specific spatial arrangements for optimal functionality, underscoring the importance of correct orientation. Despite well-known N- or C-terminus tailoring techniques, alternatives for achieving orientation control are limited. Here, we propose a novel approach that tailors the enzyme surface with engineered His-rich loops. To that aim, we first solve the X-ray crystal structure of a hexameric alcohol dehydrogenase from Thermus thermophilus HB27 (TtHBDH) (PDB: 9FBD). Guided by this 3D structure, we engineer the enzyme surface with a new loop enriched with six His residues to control enzyme orientation. Molecular dynamics simulations reveal that the engineered loop's imidazole rings have greater solvent accessibility than those in native His residues, allowing for more efficient enzyme immobilization on certain metal chelate-functionalized carriers. Using carriers functionalized with iron (III)-catechol, the apparent Vmax of the immobilized loop variant doubles the immobilized His-tagged one, and vice versa when both variants are immobilized on carriers functionalized with copper (II)-imidodiacetic acid. His-tagged and loop-engineered TtHBDH show high operational stability reaching 100% bioconversion after 10 reaction cycles, yet the loop variant is faster than the His-tagged one.

Exploration of alcohol dehydrogenase EutG from Bacillus tropicus as an eco-friendly approach for the degradation of polycyclic aromatic compounds

Polycyclic aromatic compounds (PACs) are pervasive environmental contaminants derived from diverse sources including pyrogenic (e.g., combustion processes), petrogenic (e.g., crude oil), and biological origins. They are commonly found in gasoline, coal, and crude oil, reflecting their prevalence and varied origins in natural and anthropogenic activities. The aim of this study is to use Bacillus tropicus which is a spore-forming, gram-positive and facultative anaerobic bacteria, containing a gene for PACs degradtion. In this study bacterial sample was collected from women’s vaginal discharge through streaking and spreading techniques. The DNA was extracted from bacterial culture and then the bacterium was identified through 16S rRNA which appeared to be B.tropicus. Then the computational analysis was conducted where the sequence similarity and functional analysis of alcohol dehydrogenase EutG protein from B.tropicus was analyzed through PSI-BLAT and SMART tool, respectively. The PSI-BLAST showed 100% query coverage score and 9 domains of alcohol dehydrogenase EutG protein were predicted through SMART tool. The quality of the protein was also assessed through ProQ server with a predicted LQ score of 8.091, a Maxsub score of − 0.350 and a z score of − 10.76. Then the phylogentic analysis was conducted to know the evolutionary relationship and closely related taxa. The 3D structure of the protein was predicted through SWISS MODEL and its quality was predicted through ERRAT with overall qauality factor of 98.708. The Ramachandran plot also predicted its quality and showed that 93.8% residues were in the most favored region. After this, 3D stucture of PACs were obtained from PubChem and molecular docking of the protein was performed with each of the compound. The lowest energy of − 10.3 was obtained with Indeno[1,2,3-cd] pyrene and the best docked complex was visulaized through discover studio to analyze its binding residues. Lastly, in-silico site-directed mutagenesis studies were performed which showed that the EutG gene (codes for alcoholic dehydrogenase) obtained from B. tropicus, will not get altered or have any decreasing effect on the enzyme’s stability if it goes through any mutations. This suggests that B. tropicus can act as an efficient, non-virulent, and reliable candidate for the eco-friendly and cost-effective bioremediation of PACs.

Expression evaluation of exogenous and endogenous alcohol dehydrogenase genes in transgenic Arabidopsis

A lot of endogenous genes, as well as genes from related species, are transformed back into crops for overexpression to improve their corresponding traits. However, almost all of these transgenic events remain at the testing stage. Most of the singular transgenic events of crops approved for commercial release are developed by the transformation and heterologous expression of exogenous genes from distant species. To detect the differences in expression, protein accumulation, and enzyme activity between transformed exogenous and endogenous genes, the coding sequences (CDSs) of the alcohol dehydrogenase (ADH) genes were cloned from dicotyledonous Arabidopsis, monocotyledonous maize, and prokaryotic Escherichia coli, constructed into expression vector pBI121-cMycNY, and used to transform wild-type Arabidopsis, respectively. Three homozygous T3 lines with a single integration site were screened for each of the three transformed genes by antibiotic screening, polymerase chain reaction (PCR) identification, and genomic DNA resequencing. Real-time quantitative PCR (RT-qPCR) analysis showed that the relative expression levels of the transformed exogenous ZmADH and EcADH genes were ten or tens of times higher than that of the transformed endogenous AtADH gene. After confirming the encoded proteins of these transformed genes by Western blotting, enzyme-linked immunosorbent assay (ELISA) showed that the accumulation levels of the proteins encoded by the transformed genes ZmADH and EcADH were significantly higher than that encoded by the transformed endogenous gene AtADH. Enzyme reaction assay showed that the ADH activities of the T3 lines transformed by the exogenous genes ZmADH and EcADH were also significantly higher than that transformed by the endogenous gene AtADH as well as the wild-type control. These results indicated that exogenous genes were more conducive to transgenic improvement of crops.

Molecular Understanding of Activity Changes of Alcohol Dehydrogenase in Deep Eutectic Solvents.

Deep eutectic solvents (DESs) have emerged as promising solvents for biocatalysis. While their impact on enzyme solvation and stabilization has been studied for several enzyme classes, their role in substrate binding is yet to be investigated. Herein, molecular dynamics (MD) simulations of horse-liver alcohol dehydrogenase (HLADH) are performed in choline chloride-ethylene glycol (ChCl-EG) and choline chloride-glycerol (ChCl-Gly) at varying water concentrations. In the DES solutions, the active site was significantly constricted, and its flexibility reduced when compared to the aqueous medium. Importantly, the cavity size follows a similar trend as the catalytic activity of HLADH and as such explains previously observed activity changes. To understand the impact on the binding of the substrate (cyclohexanone), an umbrella sampling (US) setup was established to calculate the free energy changes along the substrate binding tunnel of HLADH. The US combined with replica exchange and NADH in its cofactor pocket provided the best sampling of the entire active site, explaining why the cyclohexanone binding on HLADH is reduced with increasing DES content. As different components in these multicomponent mixtures influence the substrate binding, we additionally applied the US setup to study the ability of the DES components to be present inside the substrate tunnel. The presented approach may become useful to understand enzyme behaviors in DESs and to enable the design of more enzyme-compatible and tunable solvents.

The CaCAD1-CaPOA1 module positively regulates pepper resistance to cold stress by increasing lignin accumulation.

Low-temperature stress is a major environmental constraint, limiting the growth, development, and yield of peppers. Cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POA) are two key enzymes in lignin synthesis, participating in monolignol biosynthesis and monolignol polymerization, respectively. Although CAD and POA are known to play central roles in lignin biosynthesis and plant responses to abiotic stress, their functions in peppers remain poorly understood. In this study, we demonstrated the interaction between CaCAD1 and CaPOA1, which collectively positively regulated lignin biosynthesis in peppers. Additionally, CaCAD1 and CaPOA1 expression was induced by low temperatures, with expression levels gradually increasing with prolonged cold treatment. Silencing of CaCAD1 or CaPOA1 increased the sensitivity of pepper plants to low temperatures. On the other hand, overexpression of CaCAD1 and CaPOA1 in Arabidopsis enhanced its reactive oxygen species scavenging ability and improved plant tolerance to freezing conditions. In summary, the CaCAD1-CaPOA1 module was shown to play a crucial role in pepper cold tolerance, providing valuable insights and targets for future molecular breeding efforts aimed at enhancing pepper cold tolerance.

New Insights into the Pathogenesis of Alcoholic Liver Disease Based on Global Research.

Alcoholic liver disease (ALD) is the leading cause of death among alcohol-related diseases, yet its pathogenesis remains incompletely understood. This article employs data mining methods to conduct an indepth study of articles on ALD published in the past three decades, aiming to elucidate the pathogenesis of ALD. Firstly, articles related to the pathogenesis of ALD were retrieved from the Web of Science (WOS) database. CiteSpace 6.1.R2 and VOSviewer 1.6.18 were used to visually analyze the authors, institutions, journals, and keywords of the published articles. Secondly, by thoroughly reading the top 100 most cited articles and focusing on research hotspots such as cytochrome P450 2E1 (CYP2E1), gut microbiota, acetaldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH), the pathogenesis of ALD was preliminarily explored. Finally, the pathogenesis of ALD was further analyzed based on disease databases. A total of 1521 articles were retrieved from the WOS database, and 384 of these were selected for in-depth reading. From GeneCards, 9084 genes related to ALD were identified. KEGG enrichment analysis was performed using DAVID, and the hsa04936: Alcoholic liver disease pathway was selected for visualization. This study preliminarily elucidates the pathogenesis of ALD, which may be associated with the release of acetaldehyde, reactive oxygen species (ROS), and various pro-inflammatory factors during alcohol metabolism. It is also closely related to gut microbiota dysbiosis and increased intestinal permeability induced by multiple factors.

Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli.

Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts. In this study, we expand on the existing MoClo toolkits by constructing and characterizing compatible low- (p15A) and medium-copy (pBR322) destination vectors. Together with existing high-copy vectors, these backbones enable a protein expression range covering a 500-fold difference in normalized fluorescence output. We further characterize the expression- and burden profiles of each vector and demonstrate their use for the optimization of growth-coupled enzyme expression. The optimal expression of adhE (encoding alcohol dehydrogenase) for ethanol-dependent growth of Escherichia coli is determined using randomized Golden Gate Assembly, creating a diverse library of constructs with varying expression strengths and plasmid copy numbers. Through selective growth experiments, we show that relatively low expression levels of adhE facilitated optimal growth using ethanol as the sole carbon source, demonstrating the importance of adding low-copy vectors to the MoClo vector repertoire. This study emphasizes the importance of varying vector copy numbers in selection experiments to balance expression levels and burden, ensuring accurate identification of optimal conditions for growth. The vectors developed in this work are publicly available via Addgene (catalog #217582-217609).

Protein thermal stability in the undergraduate biochemistry laboratory: Exploring protein thermal stability with yeast alcohol dehydrogenase.

We created a novel laboratory experience where undergraduate students explore the techniques used to study protein misfolding, unfolding, and aggregation. Despite the importance of protein misfolding and aggregation diseases, protein unfolding is not typically explored in undergraduate biochemistry laboratory classes. Yeast alcohol dehydrogenase (YADH) is used in the undergraduate biochemistry laboratory course at Miami University as the model system to explore protein overexpression and purification, bioinformatics, and enzyme characterization. Using one model protein across the entire semester allows the students to independently link topics introduced in the individual experiments; for example, students might draw connections between the thermal denaturation experiment and the requirement to keep the enzyme cold during a kinetics experiment. Students quantitated changes in secondary structure resulting from thermal denaturation by analyzing circular dichroism data. Monitoring the turbidity of a YADH solution with a temperature-controlled UV-Vis spectrometer was a reliable and easy method for undergraduate students to observe the thermally-induced aggregation of YADH. Together these experiments provide undergraduate students with first-hand experience in techniques to study protein unfolding and aggregation.

Short-Time High-Oxygen Pre-Treatment Delays Lignification of Loquat (Eriobotrya japonica Lindl.) During Low-Temperature Storage.

Lignification often occurs during low-temperature storage in loquat fruit, leading to increased firmness and lignin content, water loss, and changes in flavor. As induced stress factors, short-time high-oxygen pre-treatment (SHOP) can initiate resistant metabolism and regulate the physicochemical qualities during fresh fruit storage. However, the effect of SHOP on the lignification and quality of loquat has been reported less. In the present study, loquat fruit was immersed in oxygen concentrations of 70%, 80%, and 90% for 30 min before being stored at 4 ± 1 °C. The results showed that the 80% SHOP samples had lower lignin accumulation and firmness, showing reductions of 23.1% and 21.1% compared to the control at 50 days. These effects were associated with the inhibition of the activities of lignin synthesis-related enzymes, including phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD). Meanwhile, 80% SHOP improved the antioxidant enzyme system and maintained the structural integrity of the cells. Furthermore, SHOP retained the color and suppressed decay and weight loss and the decline in the soluble solids content (SSC) and titratable acidity (TA). As a convenient and cheap physical approach, SHOP is a promising technology for delaying lignification by regulating lignin synthesis in loquat storage.

Alcohol dehydrogenase 1 acts as a scaffold protein in mitophagy essential for fungal pathogen adaptation to hypoxic niches within hosts.

Fungi have evolved diverse physiological adaptations to hypoxic environments. However, the mechanisms mediating such adaptations remain obscure for many filamentous pathogenic fungi. Here, we show that autophagy mediated mitophagy occurs in the insect pathogenic fungus Beauveria bassiana under hypoxic conditions induced by host cellular immune responses. Mitophagy was essential for fungal evasion from insect hemocyte encapsulation, allowing for fungal proliferation and colonization in the host hemocoel. Our data showed that B. bassiana autophagy-related protein 11 (Atg11) interacts with Atg8 as a scaffold mediating mitophagy. The mitochondrial protein Atg43 was demonstrated to act as a receptor for the selective mitophagy, directly interacting with Atg8 for the autophagosomal targeting. Alcohol dehydrogenase BbAdh1, as a novel scaffold protein, participates in mitophagy through interacting with Atg8 and Atg11 under hypoxic stress. BbAdh1 was critical for fungal intracellular redox homeostasis and energy metabolism under hypoxic conditions. These data provide a pathway for mitochondrial degradation via metabolism linked autophagosome- to-vacuole targeting during hypoxic stress. This mitophagy results in depletion of oxidative mitochondrial dependent functions as a cellular adaptation to the low oxygen levels.

The Use of Dansyl Chloride to Probe Protein Structure and Dynamics.

Dansyl labeling is a widely used approach for enhancing the detection of small molecules by UV spectroscopy and mass spectrometry. It has been successfully applied to identify and quantify a variety of biological and environmental specimens. Despite clear advantages, the dansylation reaction has found very few applications in the study of proteins. We reasoned that the mild labeling conditions, small size, and rapid reaction could be beneficial for studying protein structure and dynamics. To test this, we investigated the impact of dansylation on protein fold, stability, protein-protein, and protein-cofactor interactions. We selected two model proteins, myoglobin and alcohol dehydrogenase, for analysis using native mass spectrometry and ion mobility mass spectrometry. Our work establishes the utility of dansyl chloride as a covalent probe to study protein structure and dynamics under native conditions.

A handheld biofluorometric system for acetone detection in exhaled breath condensates.

As a marker of human metabolism, acetone is important for lipid metabolism monitoring and early detection of diabetes. In this study, we developed a handheld biosensor for acetone based on fluorescence detection by utilizing the enzymatic reaction of secondary alcohol dehydrogenase (S-ADH) with β-nicotinamide adenine dinucleotide (NADH, λex = 340 nm, λem = 490 nm). In the reaction, NADH is oxidized when acetone is reduced to 2-propanol by S-ADH, and the acetone concentration can be measured by detecting the amount of NADH consumed in this reaction. First, we constructed a compact and light-weight fluorometric NADH detection system (209 g for the sensing system and 342 g for the PC), which worked using battery power. Then, sensor characteristics were evaluated after optimization of the working conditions. The developed system was able to quantify acetone in a range of 510 nM-1 mM within 1 minute. The developed battery-operated acetone biosensor demonstrated its ability to measure the acetone concentration in the exhaled breath condensate of 10 healthy subjects at rest (23.4 ± 15.1 μM) and after 16 h of fasting (37.7 ± 14.7 μM) and it distinguished the results with significant differences (p = 0.011). With the advantages of handheld portability, and high levels of sensitivity and selectivity, this sensor is expected to be widely used in clinical diagnosis and wearable biochemical sensors in the future.

Artificial cell-free system for the sustainable production of acetoin from bioethanol.

The present work introduces and validates an artificial cell free system for the synthesis of acetoin from ethanol, representing a greener alternative to conventional chemical synthesis. The one pot multi-enzymatic system, which employs pyruvate decarboxylase from Zymobacter palmae (ZpPDC), alcohol dehydrogenase from Saccharomyces cerevisiae (ScADH), and NADH oxidase from Streptococcus pyogenes (SpNOX), achieves nearly 100 % substrate conversion and reaction yield within 6 h under optimal conditions (pH 7.5, enzyme activities: ZpPDC 100 U·mL-1, ScADH 50 U·mL-1, SpNOX 127 U·mL-1, and 1 mM NAD+). Using air for oxygen supply mitigates enzyme inactivation while effectively accelerating the regeneration of NAD+. The use of bioethanol as a substrate demonstrates the robustness and sustainability of the bioprocess, enabling the production of natural acetoin from renewable resources. This environmentally friendly approach offers significant advantages for industrial applications, aligning with green chemistry principles.

Association of haloacid dehydrogenase and alcohol dehydrogenase with vegetative growth, virulence and stress tolerance during tea plant infection by Didymella segeticola

Tea leaf spot, caused by the fungus Didymella segeticola, occurs in the high-mountain tea plantations of Southwest China. Due to a limited understanding of the disease's epidemiology and the lack of comprehensive control measures, it has a significant negative impact on tea yield and quality. In this study, we revealed that D. segeticola infection begins when conidia germinate to form a germ tube on the leaf surface. The fungus then grows in the intercellular spaces of the leaf epidermal cells, invading tea tissue and causing necrotic lesions. This infection leads to significant alterations in the cell walls of spongy and palisade mesophyll cells, severely damaging chloroplasts. We employed transcriptomic and metabolomic analyses based on an in vitro infection model using matcha powder to uncover two key genes of D. segeticola: DsHAD (encoding holoacid dehydrogenase) and DsADH (encoding alcohol dehydrogenase). These genes are the first to be associated with conidiation, virulence, and sensitivity to oxidative stress. DsHAD regulates the virulence of D. segeticola by modulating glutamate homeostasis. Our results elucidate the infection strategy of D. segeticola on tea leaves and provide valuable data for future research on control measures for tea leaf spot.

Effects of Shade Stress on the Synthesis of Cellulose and Lignin in Maize Nodal Roots

ABSTRACTLow solar radiation is an important factor affecting maize root growth and development. Roots have an anchoring function, and their important components are cellulose and lignin. Here, shade experiments were conducted using shade nets with 50% light transmittance (L50). The experiment was conducted in 2021 and 2022 using the ‘Xianyu 335’ maize variety under two nitrogen conditions (N1 = 180 kg ha−1 and N2 = 240 kg ha−1) to investigate the effect of shading on the structural carbohydrate content of maize nodal roots. The results showed that light had a highly significant effect on cellulose and lignin contents. Compared with normal light (L100), the cellulose content significantly decreased by 10.36%–13.87% and the lignin content significantly decreased by 12.96%–18.68% under shading (L50). Shading decreased the sucrose and soluble sugar contents and the cellulose and lignin‐related enzyme activities. The cellulose and lignin contents were significantly positively correlated with the sucrose content. The cellulose content at the silking (R1) stage was significantly positively correlated with the soluble sugar content and sucrose synthase (SS) and sucrose phosphate synthase activities at the 15th leaf (V15) stage; the lignin content at R1 was significantly positively correlated with the soluble sugar content and SS, acid invertase, tyrosine ammonia‐lyase, cinnamyl alcohol dehydrogenase, polyphenol oxidase and peroxidase activities at V15. Genes related to cellulose synthesis, including sucrose synthase (SS), cellulose synthase (CESA), cellulose synthase‐interactive protein 1 (CSI1), Chitinase‐Like1 (CTL1) and STELLO2 (STL2), were downregulated under shading, as were the lignin synthesis‐related phenylalanine/tyrosine ammonia‐lyase (PTAL), 4‐coumarate‐CoA ligase (4CL) and peroxidase (POD) genes. Auxin and jasmonic acid were significantly affected by light and decreased under shading, thereby reducing cellulose and lignin synthesis. These findings provide theoretical support for the development of appropriate maize cultivation practices under reduced solar radiation.

Structure prediction of alcohol dehydrogenase from Gluconobacter frateurii and its application in efficient biotransformation of D-allulose from allitol

Structure prediction of alcohol dehydrogenase from Gluconobacter frateurii and its application in efficient biotransformation of D-allulose from allitol

Novel alcohol dehydrogenase activation by the choline component of Deep eutectic solvents

Novel alcohol dehydrogenase activation by the choline component of Deep eutectic solvents

Bioanalytical method for NAD+ detection in blood plasma utilizing solution-phase Candida boidinii formate dehydrogenase and electrochemical detection.

Nicotinamide adenine dinucleotide is a crucial coenzyme in cellular metabolism and is implicated in various diseases. This work introduces an electrochemical bioanalytical method utilizing solution-phase Candida boidinii formate dehydrogenase (CbFDH) for detecting its oxidized form (NAD+) in human blood plasma samples. The detection mechanism involves the catalytic conversion of NAD+ to NADH, facilitated by CbFDH in the presence of formate. This NADH is then quantified by electrochemical measurements at disposable carbon screen-printed electrodes. The reaction is completed within one minute. The assay exhibits a linear response range from 3.74 μM to 2.00 mM, a sensitivity of 8.98 ± 0.18 μA mM-1, and a limit of detection (3sb/m) of 1.12 μM. It demonstrates selectivity against common interferences found in plasma samples, including glucose, urea, creatinine, guanosine 5'-monophosphate, cytidine 5'-monophosphate, flavin adenine dinucleotide, adenosine 5'-triphosphate, and lactate, with interference levels below 5% relative to the unperturbed NAD+ signal. Recovery studies showed 98.0-104.4% recoveries, with further validation against a colorimetric alcohol dehydrogenase assay confirming accuracy in plasma samples.