-
Journal of Basic Microbiology Jun 2024NAD-dependent (2 R,3 R)‑2,3‑butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium-chain...
The critical role of residues Phe120 and Val161 of (2 R,3 R)‑2,3‑butanediol dehydrogenase from Neisseria gonorrhoeae as probed by molecular docking and site-directed mutagenesis.
NAD-dependent (2 R,3 R)‑2,3‑butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium-chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 R,3 R)‑2,3‑butanediol (RR-BD) and meso-2,3‑butanediol (meso-BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent K values of the Phe120Ala mutant for RR-BD and meso-BD increased 36- and 369-fold, respectively; the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 586- and 3528-fold, respectively; and the apparent K values of the Val161Ala mutant for RR-BD and meso-BD increased 4- and 37-fold, respectively, the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 3- and 28-fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR-BD; 7.3-fold with meso-BD) due to an increase in K (sixfold for RR-BD; 24-fold for meso-BD) and a slight increase (2.8-fold with RR-BD; 3.3-fold with meso-BD) in k. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.
Topics: Molecular Docking Simulation; Neisseria gonorrhoeae; Mutagenesis, Site-Directed; Alcohol Oxidoreductases; Kinetics; Butylene Glycols; Phenylalanine; Binding Sites; Substrate Specificity; Bacterial Proteins; Valine; Catalytic Domain; Hydrophobic and Hydrophilic Interactions
PubMed: 38644586
DOI: 10.1002/jobm.202300751 -
Molecular Biology Reports Apr 2024Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the...
BACKGROUND
Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice.
METHODS AND RESULTS
We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains.
CONCLUSIONS
The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.
Topics: Saccharomyces cerevisiae; Vitis; DNA, Complementary; Transaminases; Fermentation; RNA; Metschnikowia
PubMed: 38642187
DOI: 10.1007/s11033-024-09444-2 -
Angewandte Chemie (International Ed. in... Jul 2024Here we demonstrate the preparation of enzyme-metal biohybrids of NAD reductase with biocatalytically-synthesised small gold nanoparticles (NPs, <10 nm) and core-shell...
Here we demonstrate the preparation of enzyme-metal biohybrids of NAD reductase with biocatalytically-synthesised small gold nanoparticles (NPs, <10 nm) and core-shell gold-platinum NPs for tandem catalysis. Despite the variety of methods available for NP synthesis, there remains a need for more sustainable strategies which also give precise control over the shape and size of the metal NPs for applications in catalysis, biomedical devices, and electronics. We demonstrate facile biosynthesis of spherical, highly uniform, gold NPs under mild conditions using an isolated enzyme moiety, an NAD reductase, to reduce metal salts while oxidising a nicotinamide-containing cofactor. By subsequently introducing platinum salts, we show that core-shell Au@Pt NPs can then be formed. Catalytic function of these enzyme-Au@Pt NP hybrids was demonstrated for H-driven NADH recycling to support enantioselective ketone reduction by an NADH-dependent alcohol dehydrogenase.
Topics: Metal Nanoparticles; NAD; Biocatalysis; Gold; Platinum; Hydrogen; Alcohol Dehydrogenase; Oxidation-Reduction
PubMed: 38641561
DOI: 10.1002/anie.202404024 -
Frontiers in Microbiology 2024The versatility of plastic has resulted in huge amounts being consumed annually. Mismanagement of post-consumption plastic material has led to plastic waste pollution....
The versatility of plastic has resulted in huge amounts being consumed annually. Mismanagement of post-consumption plastic material has led to plastic waste pollution. Biodegradation of plastic by microorganisms has emerged as a potential solution to this problem. Therefore, this study aimed to investigate the microbial communities involved in the biodegradation of polypropylene (PP). Mangrove soil was enriched with virgin PP sheets or chemically pretreated PP comparing between 2 and 4 months enrichment to promote the growth of bacteria involved in PP biodegradation. The diversity of the resulting microbial communities was accessed through 16S metagenomic sequencing. The results indicated that Xanthomonadaceae, unclassified Gaiellales, and Nocardioidaceae were promoted during the enrichment. Additionally, shotgun metagenomics was used to investigate enzymes involved in plastic biodegradation. The results revealed the presence of various putative plastic-degrading enzymes in the mangrove soil, including alcohol dehydrogenase, aldehyde dehydrogenase, and alkane hydroxylase. The degradation of PP plastic was determined using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), and Water Contact Angle measurements. The FTIR spectra showed a reduced peak intensity of enriched and pretreated PP compared to the control. SEM images revealed the presence of bacterial biofilms as well as cracks on the PP surface. Corresponding to the FTIR and SEM analysis, the water contact angle measurement indicated a decrease in the hydrophobicity of PP and pretreated PP surface during the enrichment.
PubMed: 38638899
DOI: 10.3389/fmicb.2024.1347119 -
Food Chemistry Aug 2024Radix puerariae thomsonii (RPT) contains many phenolics and exhibits various health benefits. Although the free phenolics in RPT have been identified, the composition...
Radix puerariae thomsonii (RPT) contains many phenolics and exhibits various health benefits. Although the free phenolics in RPT have been identified, the composition and content of bound phenolics, which account for approximately 20% of the total phenolic content, remain unknown. In this study, 12 compounds were isolated and identified from RPT-bound phenolic extracts, of which 2 were novel and 6 were reported first in RPT. ORAC and PSC antioxidant activities of 12 compounds, as well as their effects on alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), α-glucosidase, and α-amylase were evaluated. Genistein exhibited the highest ORAC activity, while daidzin demonstrated superior PSC activity. Five compounds, including two new compounds, exhibited the ability to activate both ADH and ALDH. All the compounds except 4-hydroxyphenylacetic acid methyl ester and 2,4,4'-trihydroxydeoxybenzoin demonstrated inhibitory effects on α-glucosidase and α-amylase. Alkaline hydrolysis and stepwise enzymatic hydrolysis revealed that bound phenolics in RPT mainly exist within starch.
Topics: Pueraria; Phenols; alpha-Amylases; alpha-Glucosidases; Plant Extracts; Binding Sites; Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Antioxidants; Plant Roots; Enzyme Inhibitors; Molecular Structure; Drugs, Chinese Herbal
PubMed: 38636386
DOI: 10.1016/j.foodchem.2024.139323 -
Plant Physiology and Biochemistry : PPB May 2024Climate change is increasing flooding in provinces of the south of the Yangtze River, posing challenges for promoting Styrax tonkinensis seedlings in these areas. To...
Climate change is increasing flooding in provinces of the south of the Yangtze River, posing challenges for promoting Styrax tonkinensis seedlings in these areas. To understand the physiological reasons for this species' intolerance to waterlogging, we observed biochemical parameters in one-year-old S. tonkinensis seedlings during two seasons. For 4 and 12 days in summer and winter experiments, respectively, we subjected seedlings to a pot-in-pot waterlogging treatment. Control groups were established at 0 h and 0 days. We examined indicators related to root vigor, reactive oxygen species (ROS), antioxidant enzymes, fermentative pathways, and more. The results displayed that decreased abscisic acid accumulation in roots inhibited water transport. Increased dehydrogenase and lactate dehydrogenase activity in roots promoted alcohol and lactate fermentation, causing toxic damage and reduced root vigor, impeding water absorption. In leaves, high ROS levels led to lipid peroxidation, exacerbating water loss from continuous transpiration. The high relative electric conductivity and low leaf relative water content indicated water loss, causing leaf wilting and shriveling. Conversely, winter seedlings, devoid of leaves, significantly reduced transpiration, and dormancy delayed root fermentation. With less ROS damage in roots, winter seedlings exhibited greater waterlogging tolerance. In summary, excessive water loss from leaves and inhibited vertical water transport contributed to low summer survival rates, while winter leafless dormancy and reduced ROS damage enhanced tolerance. Our findings provide insights for enhancing waterlogging resistance in S. tonkinensis amidst climate change challenges.
Topics: Seedlings; Reactive Oxygen Species; Plant Roots; Water; Stress, Physiological; Floods; Plant Leaves; Abscisic Acid
PubMed: 38636255
DOI: 10.1016/j.plaphy.2024.108587 -
ACS Catalysis Apr 2024Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this...
Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (Δ) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of Δ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.
PubMed: 38633402
DOI: 10.1021/acscatal.3c05584 -
Chembiochem : a European Journal of... Jun 2024Chiral alcohols are not only important building blocks of various bioactive natural compounds and pharmaceuticals, but can serve as synthetic precursors for other...
Chiral alcohols are not only important building blocks of various bioactive natural compounds and pharmaceuticals, but can serve as synthetic precursors for other valuable organic chemicals, thus the synthesis of these products is of great importance. Bio-catalysis represents one effective way to obtain these molecules, however, the weak stability and high cost of enzymes often hinder its broad application. In this work, we designed a biological nanoreactor by embedding alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) in metal-organic-framework ZIF-8. The biocatalyst ADH&GDH@ZIF-8 could be applied to the asymmetric reduction of a series of ketones to give chiral alcohols in high yields (up to 99 %) and with excellent enantioselectivities (>99 %). In addition, the heterogeneous biocatalyst could be recycled and reused at least four times with slight activity decline. Moreover, E. coli containing ADH and GDH was immobilized by ZIF-8 to form biocatalyst E. coli@ZIF-8, which also exhibits good catalytic behaviours. Finally, the chiral alcohols are further converted to marketed drugs (R)-Fendiline, (S)-Rivastigmine and NPS R-568 respectively.
Topics: Alcohol Dehydrogenase; Metal-Organic Frameworks; Ketones; Enzymes, Immobilized; Biocatalysis; Escherichia coli; Glucose 1-Dehydrogenase; Oxidation-Reduction; Stereoisomerism
PubMed: 38629211
DOI: 10.1002/cbic.202400147 -
Journal of Gastroenterology and... Apr 2024We previously identified that ever-smoking and severe gastric atrophy in pepsinogen are risk factors for synchronous gastric cancers (SGCs). This study aimed to...
BACKGROUND AND AIM
We previously identified that ever-smoking and severe gastric atrophy in pepsinogen are risk factors for synchronous gastric cancers (SGCs). This study aimed to determine the association of alcohol drinking status or alcohol-related genetic polymorphism with SGCs and also stratify their risk.
METHODS
This multi-center prospective cohort study included patients who underwent endoscopic submucosal dissection for the initial early gastric cancers at 22 institutions in Japan. We evaluated the association of alcohol drinking status or alcohol dehydrogenase 1B (ADH1B) and acetaldehyde dehydrogenase 2 (ALDH2) genotypes with SGCs. We then stratified the risk of SGCs by combining prespecified two factors and risk factors identified in this study.
RESULTS
Among 802 patients, 130 had SGCs. Both the ADH1B Arg and ALDH2 Lys alleles demonstrated a significant association with SGCs on multivariate analysis (odds ratio, 1.77), although alcohol drinking status showed no association. The rates of SGCs in 0-3 risk factors in the combined evaluation of three risk factors (ever-smoking, severe gastric atrophy in pepsinogen, and both the ADH1B Arg and ALDH2 Lys alleles) were 7.6%, 15.0%, 22.0%, and 32.1%, respectively. The risk significantly increased from 0 to 3 risk factors on multivariate analysis (P for trend <0.001).
CONCLUSIONS
Both the ADH1B Arg and ALDH2 Lys alleles were at high risk for SGCs. The risk stratification by these three factors may be a less invasive and promising tool for predicting their risk.
PubMed: 38628101
DOI: 10.1111/jgh.16570 -
Colloids and Surfaces. B, Biointerfaces Jun 2024In this study, the encapsulation and structural characteristics of the self-assembled liposome formed by epigallocatechin gallate (EGCG) and alcohol dehydrogenase (ADH)...
In this study, the encapsulation and structural characteristics of the self-assembled liposome formed by epigallocatechin gallate (EGCG) and alcohol dehydrogenase (ADH) were studied. According to the results, EGCG significantly increased the catalytic activity of ADH with a 33.33 % activation rate and the liposomes were able to entrap EGCG-ADH with an effectiveness of 88.94 %. The self-assembled monolayers had nanometer-sized particles, and the excellent self-assembled system was demonstrated by the low PDI value and high surface absolute potential. The scanning electron microscope showed that the self-assembled liposome was honeycomb, groove-shaped, and rough. The spectroscopic results showed that EGCG-ADH complex was formed through hydrogen bond, which changed the secondary structure of the liposome, and verified EGCG-ADH liposome system was successfully prepared. In vitro digestion experiments showed that the gastrointestinal tolerance and antioxidant activity of EGCG-ADH liposomes were significantly higher than those of free EGCG-ADH.
Topics: Liposomes; Catechin; Alcohol Dehydrogenase; Antioxidants; Particle Size; Hydrogen Bonding
PubMed: 38615391
DOI: 10.1016/j.colsurfb.2024.113917