Biochemistry Biochemistry最新文献

{"title":"Discovery of Bacterial Unspecific Peroxygenases","authors":"Esteban Lopez-Tavera,&nbsp;Anton A. Stepnov,&nbsp;Nikolai S. Ersdal,&nbsp;Marta Barros-Reguera,&nbsp;Ronja Marlonsdotter Sandholm,&nbsp;Sabina Leanti La Rosa,&nbsp;Morten Sørlie,&nbsp;Vincent G. H. Eijsink and Gustav Vaaje-Kolstad*,&nbsp;","doi":"10.1021/acs.biochem.6c00230","DOIUrl":"10.1021/acs.biochem.6c00230","url":null,"abstract":"<p >Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds because of their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have been reported in prokaryotic organisms. Here, we describe the discovery of a diverse family of bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial proteins, termed bacterial UPOs (BUPOs), display clear structural homology to family I (short) fungal UPOs despite low sequence identity (&lt;20%). Expression of one of these proteins (<i>Hyd</i>BUPO) in its native host (<i>Hydrogenophaga</i> <i>sp</i>. A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in <i>Escherichia coli</i>. In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, as well as two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol, and 16-hydroxypalmitic acid, using hydrogen peroxide as a cosubstrate. These enzymes thus represent a previously unknown group of bacterial heme-thiolate peroxygenases that share key structural and functional features with eukaryotic UPOs, offering potential candidates for the development of industrially relevant biocatalysts.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2283–2299"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.6c00230","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148349877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"How Medically Important Antimicrobials Bind to the 30S Ribosomal Subunit in a Bacterial Pathogen","authors":"Swati R. Manjari,&nbsp;Caleb Mallery and Nilesh K. Banavali*,&nbsp;","doi":"10.1021/acs.biochem.6c00008","DOIUrl":"10.1021/acs.biochem.6c00008","url":null,"abstract":"<p >Ribosomes translate the genetic code in mRNA to synthesize proteins in all living organisms. Decoding of mRNA occurs in the small subunit of the ribosome and is mediated by tRNA anticodons. Regions near the decoding center are a target for antibiotics, such as aminoglycosides and tetracyclines, where their presence results in errors in protein synthesis. More than two decades of high-resolution structural studies have shown how such medically important antimicrobials (MIAs) bind to the small subunit of the bacterial ribosome. Here, we comprehensively analyze these previously reported structures to help understand the variability with which MIAs bind to small subunits of bacterial ribosomes. We previously solved the hibernating 70S ribosome structure of the bacterial pathogen <i>Borrelia burgdorferi</i> (<i>Bbu</i>), the causative agent of Lyme disease, but there is no structure of any MIA bound to this ribosome reported. Our structural analysis makes it possible to use inexpensive computational methods to predict the binding of these MIAs to the <i>Bbu</i> ribosomal 30S small subunit. For this, we used structural analogy, restrained energy minimization, and single-point binding free energy computations. We find the single-point binding free energy of the MIAs to be very sensitive to small conformational changes in the MIA and its environment. Incorporating this knowledge in structure-guided design could aid in the development of narrow-spectrum MIAs targeting specific bacterial pathogens.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2250–2258"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147757391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Differential Hydroxypyruvate Processing by E. coli and P. aeruginosa DXP Synthases Reveals Preferential Xylulose 5-Phosphate Formation by the P. aeruginosa Enzyme","authors":"Stephanie Henriquez,&nbsp;Charles R. Nosal,&nbsp;Lydia J. Kramer,&nbsp;Eucolona M. Toci,&nbsp;Sara A. Trauth,&nbsp;Analisa M. Lott and Caren L. Freel Meyers*,&nbsp;","doi":"10.1021/acs.biochem.6c00231","DOIUrl":"10.1021/acs.biochem.6c00231","url":null,"abstract":"<p >The thiamine diphosphate (ThDP)-dependent enzyme 1-deoxy-<span>d</span>-xylulose 5-phosphate synthase (DXPS) converts pyruvate and <span>d</span>-glyceraldehyde 3-phosphate (<span>d</span>-GAP) to the essential branchpoint metabolite, DXP. DXPS exhibits a ligand-gated mechanism where the predecarboxylation intermediate derived from pyruvate persists until <span>d</span>-GAP triggers decarboxylation. Together with its substrate/catalytic promiscuity and other features of multifunctionality, ligand gating on DXPS may poise this enzyme to catalyze alternate activities that aid in bacterial adaptation. We previously discovered alternative donors for <i>Escherichia coli</i> DXPS, with donor processing and acceptor specificity influenced by the donor. Here, we demonstrate species differences in DXPS substrate usage and processing which could signify that DXPS homologues support bacterial adaptation in distinct ways. We showed that hydroxypyruvate (HPA) is a donor for <i>Pseudomonas aeruginosa</i> DXPS (<i>Pa</i>DXPS), similar to <i>Ec</i>DXPS, yet HPA processing and donor–acceptor specificity differs on these homologues; HPA reacts with ThDP on <i>Pa</i>DXPS to form a longer-lived intermediate in the absence of added trigger, relative to <i>Ec</i>DXPS. Notably, <i>Pa</i>DXPS preferentially catalyzes xylulose 5-phosphate formation from HPA and <span>d</span>-GAP, in contrast to <i>Ec</i>DXPS, suggesting a potential connection between DXPS activity and pentose phosphate pathway metabolism in <i>P. aeruginosa</i>. Finally, both <i>Ec</i>DXPS and <i>Pa</i>DXPS were found to generate a 5-carbon β-ketoacid via a one-substrate reaction of HPA, distinguishing DXPS from transketolase (TK) which produces erythrulose under similar conditions. This study illustrates species differences in DXPS donor usage and processing, and donor–acceptor specificity that supports distinct multifunctionality across bacterial pathogens and the plausibility of species-specific targeting.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2300–2317"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148373790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"N-Terminal Amine Chemistry Influences α-Synuclein Interactions with Lipid Bilayers","authors":"Emily Brackhahn,&nbsp;Ming-Hao Li,&nbsp;Paola Miranda-Castrodad,&nbsp;Hudson Lee,&nbsp;Jonathan Musila,&nbsp;David Eliezer and Elizabeth Rhoades*,&nbsp;","doi":"10.1021/acs.biochem.6c00382","DOIUrl":"10.1021/acs.biochem.6c00382","url":null,"abstract":"<p >α-Synuclein (αSyn) is an intrinsically disordered protein whose reversible membrane binding via amphipathic α-helix formation is central to its function and may also contribute to the pathology of Parkinson’s disease. In mammals, αSyn is constitutively N-terminally acetylated, a modification that alters its structural propensity and interactions with lipid bilayers. Here, we decouple the structural and chemical contributions of N-terminal acetylation by engineering several αSyn variants that systematically vary N-terminal helix propensity and physicochemical properties: unmodified αSyn (αSyn_Unmod), αSyn with an N-terminal Asn (αSyn_Asn), N-terminally acetylated αSyn (αSyn_Acetyl), and N-terminally succinylated αSyn (αSyn_Succinyl). Circular dichroism and NMR measurements show that all three modifications increase α-helical sampling within the first ∼10–12 residues, establishing a hierarchy of N-terminal helicity (αSyn_Succinyl &gt; αSyn_Acetyl &gt; αSyn_Asn &gt; αSyn_Unmod). However, fluorescence correlation spectroscopy reveals that enhanced helicity in solution does not strictly correlate with lipid bilayer affinity across several different lipid compositions. Instead, membrane binding is more strongly influenced by N-terminal chemistry, lipid composition, and bilayer curvature. Collectively, these results underscore how modifications tune αSyn’s interactions with binding partners, providing mechanistic insight into how this modification modulates αSyn function. For lipid bilayers, N-terminal acetylation tunes both the structural and physicochemical properties of αSyn to enhance binding.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2272–2282"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.6c00382","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148416989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bilirubin Reductase from Mediterraneibacter gnavus: Positional Reduction Preferences and Transient-State Analysis","authors":"Corine O. Smith,&nbsp; and ,&nbsp;Graham R. Moran*,&nbsp;","doi":"10.1021/acs.biochem.6c00365","DOIUrl":"10.1021/acs.biochem.6c00365","url":null,"abstract":"<p >The first committed step for the excretion of bilirubin is reduction of four vinylic bonds to form urobilinogen. This reaction is catalyzed by bilirubin reductase (BilR) that uses four NADHs to accomplish the transformation. Due to the asymmetry of bilirubin, BilR must either recognize 15 different oxidant substrates or have a preferred reduction order. AlphaFold structural prediction indicates structural homology with bacterial 2,4-dienoyl-CoA reductase and 2-napthoyl-CoA reductase, both of which are single-subunit proteins with a two-domain structure that houses three cofactors: an FAD, an Fe₄S₄ cluster, and an FMN. In this study, anaerobic kinetic and mechanistic analysis of bilirubin reductase from <i>Mediterraneibacter gnavus</i> (MgBilR) is presented. Anaerobic solvent exchange studies define the stereochemistry of hydride transfer from NADH as ProS. Transient-state kinetics of the reductive half-reaction demonstrate that MgBilR can become reduced by four electrons with either NADH or NADPH, but that NADH is the preferred reductant by four orders of magnitude (based on <i>k</i>_red/<i>K</i>_d values). Only the first hydride transfer from NADH occurs at a rate that is catalytically relevant. Single-turnover experiments show two dominant phases corresponding to two-electron flavin reduction and subsequent rate-limiting bilirubin reduction that occurs with flavin reoxidation. Single-turnover data correlated with steady-state assays and NMR tracking of the steady-state reaction confirm that BilR can reduce both types of vinylic bonds of bilirubin but appears to have a 4-fold preference for reduction of α-vinylic over enamine bonds that is defined almost entirely by the rate of hydride transfers to the oxidant.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2330–2341"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148402961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural and Functional Characterization of Heterologous Nitrogenase Complexes","authors":"Yizhou Li,&nbsp;Sarah M. Narehood,&nbsp;Brian D. Cook,&nbsp;Kelly L. McGuire,&nbsp;Mark A. Herzik Jr.* and F. Akif Tezcan*,&nbsp;","doi":"10.1021/acs.biochem.6c00360","DOIUrl":"10.1021/acs.biochem.6c00360","url":null,"abstract":"<p >Nitrogenase is the only known enzyme that catalyzes the reduction of dinitrogen to ammonia. The most prevalent isozyme, molybdenum nitrogenase, comprises the catalytic molybdenum–iron protein (MoFeP) and the ATP-dependent reductase iron protein (FeP). Although Mo-nitrogenases are widespread across bacteria and archaea and appear to share conserved mechanistic and structural features, FeP and MoFeP show considerable sequence variability across diazotrophs. This raises questions about the conservation of chemomechanical mechanisms coupling FeP-dependent ATP hydrolysis and electron transfer to MoFeP, and about the functional compatibility of nitrogenase components from divergent species. Previous studies showed that some heterologous FeP−MoFeP pairs can functionally complement each other, whereas other pairs lack catalytic activity, but the absence of structural information on such heterologous pairs has limited mechanistic understanding. To this end, we investigated the functional and structural compatibility of FeP and MoFeP from <i>Azotobacter vinelandii</i> (<i>Av</i>) and <i>Gluconacetobacter diazotrophicus</i> (<i>Gd</i>), two phylogenetically and ecologically distinct species. Building on our prior work with <i>Gd</i>-nitrogenase and recently developed cryogenic electron microscopy (cryoEM) protocols, we determined the ADP·BeF_<i>x</i>-trapped structure of the homologous <i>Gd</i>FeP–<i>Gd</i>MoFeP complex and showed that it adopted the same geometry as its <i>Av</i> counterpart. Activity measurements showed that heterologous <i>Gd/Av</i> combinations retained 60–80% of homologous catalytic activities despite 30–50% sequence divergence in FeP and MoFeP. High-resolution cryoEM structures of <i>Gd</i>FeP–<i>Av</i>MoFeP and <i>Av</i>FeP–<i>Gd</i>MoFeP corroborated these activities and revealed that functional complementation tolerates substantial sequence variation when the core structural elements supporting ATP binding/hydrolysis, protein–protein interaction, electron transfer, and substrate reduction are conserved.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2318–2329"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148366166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Aromatic Cage-Directed Azide-Methyllysine Photochemistry for Profiling Nonhistone Interacting Partners of the MeCP2 Methyl-CpG-Binding Domain","authors":"Jyotirmayee Padhan,&nbsp;Sayar Ghosh,&nbsp;Soumen Barman and Babu Sudhamalla*,&nbsp;","doi":"10.1021/acs.biochem.6c00309","DOIUrl":"10.1021/acs.biochem.6c00309","url":null,"abstract":"<p >Methyl-CpG-binding protein 2 (MeCP2) is a canonical reader of DNA methylation and a key chromatin regulator implicated in neurodevelopment and cancer. Beyond DNA binding, MeCP2 recognizes trimethylated histone H3 lysine 27 (H3K27me3) through an aromatic cage within its methyl-CpG-binding domain (MBD). Whether this methyllysine-binding interface also mediates interactions with nonhistone methyllysine-containing proteins remains unknown. Here, we developed an aromatic cage-directed chemoproteomic strategy to capture methyllysine-dependent MeCP2 interactions in cells. By site-specifically incorporating the photocrosslinkable unnatural amino acid 4-azido-<span>l</span>-phenylalanine (AzF) into the MeCP2-MBD, we enabled covalent capture of transient MeCP2-binding partners. Engineered MeCP2 variants efficiently crosslinked to methylated histone ligands, validating preserved methyllysine recognition. Proteomic analysis of crosslinked complexes from human cell lysates uncovered previously unrecognized MeCP2-associated proteins involved in chromatin regulation, RNA processing, translation, and metabolism. Integration with single-cell transcriptomic data revealed coordinated expression of <i>MECP2</i> and its interacting partners in defined cellular populations. Imaging and chromatin-based analyses further demonstrated nuclear colocalization and shared genomic occupancy with selected interactors at transcriptionally relevant loci. Together, these findings establish the MeCP2 aromatic cage as a versatile interaction interface and expand the functional landscape of MeCP2 beyond DNA methylation reading to encompass chromatin-associated regulatory networks.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2259–2271"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148373793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"From First Principles to Function: How AI Is Reshaping Enzyme Design","authors":"Sebastian Lindner,&nbsp;Florence J. Hardy and Donald Hilvert*,&nbsp;","doi":"10.1021/acs.biochem.6c00356","DOIUrl":"10.1021/acs.biochem.6c00356","url":null,"abstract":"<p >Artificial intelligence is rapidly transforming everyday life and driving major advances across science. De novo enzyme design is an area of particular promise, with implications for medicine, biotechnology, and industry. Recent applications of AI-enhanced methodologies have yielded a range of enzymes with catalytic activities that were previously unattainable through conventional approaches. This perspective surveys emerging strategies and computational models that are redefining enzyme engineering and examines the opportunities and challenges on the path toward truly on-demand enzyme design.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2202–2212"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148409660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metal Binding to Tau Protein: Physiological and Pathological Relevance","authors":"Liliana Quintanar*,&nbsp;Gerardo U. Juárez-Romero and Gala R. López-Herrera,&nbsp;","doi":"10.1021/acs.biochem.6c00323","DOIUrl":"10.1021/acs.biochem.6c00323","url":null,"abstract":"<p >The microtubule-associated protein tau is an intrinsically disordered protein that exhibits a remarkable diversity of functions, while its amyloid aggregation is the hallmark of tauopathies, including Alzheimer′s disease. This perspective article discusses recent advances in understanding copper and zinc binding to tau, the nature of the metal binding sites, and its physiological and pathological relevance. First, an argument for a potential role of metal binding in the interactome and diverse functionality of tau is provided, since metal coordination occurs in regions of tau engaged in its interaction with key binding partners. Then, the role of metal binding to tau in modulating its structural dynamics and amyloid aggregation behavior is discussed, with an emphasis on a potential role of metal ions in the morphological diversity of tau fibrils. Finally, the interplay between metal binding and post-translational modifications (PTMs) of tau is examined in physiological and pathological contexts, discussing how PTMs may influence metal coordination and how metals may modulate PTM processing of tau. Overall, this perspective delineates the exciting future of bioinorganic research related to tau protein, underscoring the importance of investigating the role of metal ions in tau biology as functional modulators and/or drivers of pathological transitions, linking metal homeostasis to tau physiology and disease.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 14","pages":"2213–2226"},"PeriodicalIF":2.7,"publicationDate":"2026-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.6c00323","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148416983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gut Bacterial 20-Hydroxysteroid Dehydrogenases Modify Endogenous Glucocorticoids and Corticosteroid Drugs","authors":"Sean Coyne,&nbsp;Robert Ghergurovich,&nbsp;Francis Sacco,&nbsp;Francesca Lombardi,&nbsp;Kailey Paar,&nbsp;Jackson DeMartino,&nbsp;Annick A. Kenfack and Tyler M. M. Stack*,&nbsp;","doi":"10.1021/acs.biochem.5c00661","DOIUrl":"10.1021/acs.biochem.5c00661","url":null,"abstract":"<p >Bacterial 20-hydroxysteroid dehydrogenases (20-HSDHs) from the human gut are known to reduce the C-20 carbonyl group of cortisol, producing either 20α- or 20β-dihydrocortisol. Two 20-HSDHs from <i>Bifidobacterium adolescentis</i> L2–32 and <i>Agathobaculum desmolans</i> ATCC 43058, both members of the short-chain dehydrogenases/reductases superfamily, are known to produce 20β-dihydrocortisol, while the 20-HSDH from <i>Clostridium scindens</i> ATCC 35704, belonging to the zinc-containing alcohol dehydrogenase family, produces 20α-dihydrocortisol. These three enzymes were characterized for their activity toward cortisol and structurally related therapeutic corticosteroids. Kinetic analyses revealed narrow substrate specificity, with all enzymes preferring cortisol but maintaining significant activity for prednisone and prednisolone (<i>k</i>_cat/<i>K</i>_M values between 10³–10⁶ M^–1 s^–1 for the 20β-HSDH enzymes), and some detectable activity of the 20β-HSDHs to reduce triamcinolone. The 20β-HSDHs exhibited pH-dependent substrate inhibition, influencing their activity profile. Structural docking studies indicated that suitable substrates occupy a single productive binding mode within the 20β-HSDH enzyme active site. Our findings show that enzymes in the gut microbiome can metabolize corticosteroid drugs by reducing the 20-keto group, which could have implications for drug efficacy and side effects. This work highlights the importance of gut microbial enzymes in the biotransformation of both endogenous and therapeutic steroids, informing future research into drug-microbiome interactions and personalized medicine.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"65 13","pages":"2180–2189"},"PeriodicalIF":2.7,"publicationDate":"2026-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.5c00661","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148306320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}