Biochimica et biophysica acta. Proteins and proteomics最新文献

{"title":"Biochemical characterization of a glycoside hydrolase family 10 β-galactosidase from a haloalkaliphilic archaeon","authors":"Nawee Jantarit ,&nbsp;Chamaipon Beagbandee ,&nbsp;Yothin Teethaisong ,&nbsp;Pornpat Sam-Ang ,&nbsp;Kanjana Wongkrajang ,&nbsp;James R. Ketudat-Cairns","doi":"10.1016/j.bbapap.2026.141124","DOIUrl":"10.1016/j.bbapap.2026.141124","url":null,"abstract":"<div><div>Extremophilic archaea have garnered attention as valuable sources of robust enzymes appropriate for industrial applications. This study involved the cloning and heterologous expression of a gene initially designated as encoding a putative glycoside hydrolase family 10 (GH10) endo-1,4-β-xylanase from the haloalkaliphilic archaeon <em>Natronococcus occultus</em> and biochemical characterization of the protein. Surprisingly, substrate specificity screening revealed exclusive β-galactosidase activity, indicating a possible misannotation. The enzyme, designated NoBGAL, had the highest activity toward <em>p</em>-nitrophenyl-β-<span>d</span>-galactopyranoside (pNPβGal) and β-1,3-galactobiose. NoBGAL exhibited the optimum catalytic efficacy at pH 7.5 and 50 °C and retained high activity and stability under elevated temperatures and at NaCl concentrations up to 1 M, reflecting its extremophilic origin. It had higher catalytic specificity for pNPβGal than for lactose. Notably, NoBGAL possessed a relatively high inhibition constant (<em>K</em>_i = 64 mM) for galactose, suggesting low product inhibition. Enzyme activity was markedly enhanced by Na⁺, K⁺, Mg²⁺, and Mn²⁺ ions, whereas it was strongly suppressed by Cu²⁺, Zn²⁺, and Hg²⁺. Sequence and structural analyses indicated that NoBGAL retains the conserved key residues found in GH10 xylanases; however, biochemical assays revealed β-galactosidase activity rather than xylanase activity, highlighting functional divergence despite a conserved active-site architecture. To our knowledge, this is the first biochemical characterization of a β-galactosidase from GH10 enzymes. Its halotolerant and alkaliphilic properties make it a promising candidate for industrial applications, particularly for lactose hydrolysis processes under high-salt and alkaline conditions.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 3","pages":"Article 141124"},"PeriodicalIF":2.3,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145905559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ornithine racemase uses a catalytic cysteine","authors":"Robert S. Phillips ,&nbsp;Xuan Tran ,&nbsp;Brooke Mangano","doi":"10.1016/j.bbapap.2025.141120","DOIUrl":"10.1016/j.bbapap.2025.141120","url":null,"abstract":"<div><div><em>Salmonella enterica</em> serover typhimurium expresses an ornithine racemase (OrnR), located in a small operon containing amino acid transporter homologues and D-ornithine/<span>d</span>-lysine decarboxylase. This OrnR is part of a clade that has high identity (&gt;75 %) with other sequences in enterobacteria, but is distant (∼40 % identity) from another clade of OrnR found in anaerobic Firmicutes and actinobacteria. OrnR is a member of the pyridoxal-5′-phosphate (PLP)-dependent alanine racemase (AlaR) superfamily (Fold III), but sequence alignments show that it lacks the catalytic Tyr acid/base of the other racemases. However, the sequences show a conserved cysteine. OrnR shows hyperbolic kinetics in the L → D direction, but exhibits sigmoid kinetics in the D → L direction, with <em>n</em> = 1.8. The structure of OrnR predicted by AlphaFold3 was complexed with PLP-DL-ornithine in silico, minimized, and subjected to molecular dynamics simulation. The resulting structure shows that Cys-164 is in the active site, about 4 Å from the Cα of the external aldimine of L-ornithine, while the NZ of Lys-36 is located about 4 Å below Cα on the D-face. The C164A mutant enzyme has no measurable racemization activity, and does not exchange the α-H of L- or D-ornithine in D₂O, consistent with the function of Cys-164 as an acid/base catalyst. These data are consistent with a concerted mechanism for the racemization. Gel filtration of OrnR shows a mixture of dimeric and tetrameric assemblies.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 2","pages":"Article 141120"},"PeriodicalIF":2.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145793133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calcium dependence and biochemical characterization of metacaspase-3 from Trypanosoma cruzi","authors":"Ane Caroline Moreira Duarte,&nbsp;Vinicius Henrique de Oliveira,&nbsp;João Pedro Martins Silva Costa,&nbsp;Mariana Nascimento Romero Trujilho,&nbsp;Wagner Alves de Souza Judice,&nbsp;Maurício Ferreira Marcondes Machado","doi":"10.1016/j.bbapap.2025.141122","DOIUrl":"10.1016/j.bbapap.2025.141122","url":null,"abstract":"<div><div>Metacaspases are cysteine proteases structurally related to caspases, widely distributed in plants, fungi, and protozoa, but absent in metazoans. In <em>Trypanosoma cruzi</em>, the etiological agent of Chagas disease, the functional and biochemical properties of metacaspases remain poorly understood. In this study, we performed a detailed characterization of the metacaspase TcMCA3, focusing on its expression, purification, calcium-dependent processing, and enzymatic activity. SDS-PAGE and western blotting revealed that TcMCA3 is primarily expressed in a processed form and undergoes further autoproteolytic cleavage in the presence of calcium. Kinetic analyses showed that calcium activates TcMCA3, enhancing catalytic efficiency (k_cat/K_M) and turnover rate (k_cat) up to 1 mM CaCl₂, beyond which activity decreases, likely due to autodegradation. Optimal activity was observed at pH 8.5 and 25 mM NaCl, suggesting a requirement for mildly alkaline and moderate ionic strength environments. Fluorescence spectroscopy confirmed that TcMCA3 undergoes conformational changes upon calcium binding, with a high-affinity site responsible for structural activation. We observed that calcium-dependent processing correlates with changes in catalytic activity, suggesting structural and functional differences between TcMCA3 isoforms. Overall, our findings reveal that TcMCA3 activation is tightly regulated by calcium, pH, and ionic strength, and that structural rearrangements induced by calcium binding are essential for its enzymatic function. These findings advance our understanding of the regulatory mechanisms of metacaspases in protozoan parasites and may help inform future evaluation of TcMCA3 as a potential target for therapeutic strategies against <em>T. cruzi</em>.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 2","pages":"Article 141122"},"PeriodicalIF":2.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145833100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nitrated products formed on α-synuclein are preferentially incorporated into oligomers but excluded from fibrils: A mechanism for accumulation of neurotoxic species","authors":"Daniel E. Otzen ,&nbsp;Luke F. Gamon ,&nbsp;Per Hägglund ,&nbsp;Janni Nielsen ,&nbsp;Jannik N. Pedersen ,&nbsp;Tina Nybo ,&nbsp;Jan S. Nowak ,&nbsp;Søren K. Amstrup ,&nbsp;Mitra Pirhaghi ,&nbsp;Michael J. Davies","doi":"10.1016/j.bbapap.2025.141118","DOIUrl":"10.1016/j.bbapap.2025.141118","url":null,"abstract":"<div><div>Post-translational modifications (PTMs) such as nitration of Tyr (Y) residues and di-tyrosine (DT) formation are known to impact the aggregation behavior of α-synuclein (α-syn), a protein closely linked to Parkinson's disease. Using tetranitromethane (TNM) as a model nitrating agent, we systematically investigated the chemical modifications of α-syn and their consequences for aggregation. Mass spectrometry analysis revealed site-selective nitration of all four Tyr residues, with Y39 and Y125 being most susceptible. DT crosslinks were also observed, primarily involving Y39, but were disfavored at higher TNM concentrations, indicating competition between nitration and crosslinking pathways. Higher TNM concentrations favored nitration over crosslinking, consistent with common radical intermediates. Even sub-stoichiometric amounts of TNM-modified α-syn significantly inhibited fibril elongation, suggesting that nitration disrupts the templated addition of monomers into the ordered fibrillar structure. Consistent with this, TNM-modified α-syn was strongly excluded from fibrillar assemblies. In contrast, they were preferentially incorporated into soluble oligomers, underlining the promiscuous ability of oligomers to act as a sink for chemically modified α-syn monomers, with potential implications for neurotoxicity.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 2","pages":"Article 141118"},"PeriodicalIF":2.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145736613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MetIoR: A meta predictor to predict intrinsic disorder in RNA binding proteins","authors":"Swarnadip Mitra ,&nbsp;Ranjit Prasad Bahadur","doi":"10.1016/j.bbapap.2025.141119","DOIUrl":"10.1016/j.bbapap.2025.141119","url":null,"abstract":"<div><div>Intrinsic disorder in proteins is ubiquitous in nature across various proteomes. The association of intrinsically disordered proteins (IDP) with a large variety of biomolecules is essential in many cellular functions. Moreover, IDPs are involved in many macromolecular assemblies including ribosome and spliceosome. Intrinsic disorder has been reported in several RNA binding proteins (RBPs), which makes the study of disordered regions in RBPs crucial. Experimental difficulties in accurately determining the structure of proteins with disordered regions using biophysical techniques necessitates their computational prediction. A large number of available computational tools have been developed to predict disordered residues in proteins, but the majority of them are generic in nature. In this study, we have addressed the lack of specific intrinsic disorder predictors in RBPs. We have developed MetIoR, a prediction tool specifically trained on RBPs, to predict intrinsically disordered regions in RBP using a meta-approach. We have developed our meta predictor, which requires a protein sequence, using five individual disorder predictors. We have employed a number of machine learning classifiers with stratified ten-fold cross validation and selected the best method in terms of prediction metrics. We have also examined the most optimal combination of individual predictors. MetIoR developed using Random Forest is fast, accurate and outperforms all its individual component predictors achieving an accuracy of 93.87 %, and a high AUC value of 0.91. The developed meta predictor is deployed as the MetIoR webserver, which can be freely accessed at <span><span>http://www.csb.iitkgp.ac.in/applications/MetIoR/index.php</span><svg><path></path></svg></span></div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 2","pages":"Article 141119"},"PeriodicalIF":2.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145793177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Drug–biomolecular interaction: Spectroscopic and computational insights into esomeprazole binding with human serum albumin","authors":"Anna Tanuja Safala Bodapati ,&nbsp;Ragaiahgari Srinivas Reddy ,&nbsp;Kandikonda Lavanya ,&nbsp;Shravya Rao Madku ,&nbsp;Rajdeep Chowdhury ,&nbsp;Bijaya Ketan Sahoo","doi":"10.1016/j.bbapap.2025.141109","DOIUrl":"10.1016/j.bbapap.2025.141109","url":null,"abstract":"<div><div>Cellular process relies on specific binding of drug molecules with desired biological receptors. Biomolecular receptors and drugs exhibit their biological functions upon their interactions. Understanding the binding parameters and energetics of the binding provides a plethora of information that may be helpful in drug design and discovery. Further, drug interaction with carrier proteins affects the pharmacokinetics and dynamics of other exogenous and endogenous drugs. In this work, we report the binding behavior of esomeprazole with human serum albumin using several biophysical techniques. Qualitative and quantitative aspects of the binding along with the binding energetics has been focused in extracting the thermodynamics parameters and key interaction force. The binding constants (<em>K</em>_<em>b</em>)obtained from Scatchard analysis are-1.17 × 10⁵, 7.49 × 10⁴, and 3.23 × 10⁴ M⁻¹ at 298, 303, 308 K respectively. Esomeprazole binds preferentially at site 1 in subdomain IIA of human serum albumin and was confirmed, supported by site-specific marker displacement studies and docking simulations. Negative value of change in free energy (∆G⁰) -32 <em>kJ</em>/<em>mol</em> at 298 K showed thermodynamically favourable interaction and outweigh of entropic factor (T∆S⁰ = 230.76 ± 3 <em>kJ</em> for <em>T</em> = 298 K) over the enthalpic contribution (∆H⁰ = −261.99 <em>kJ</em>/<em>mol</em>) revealed an entropy-driven process. Binding of esomeprazole affected the helical structure of human serum albumin. Molecular docking studies (theoretical) shows the binding pocket of ESM at site1(IIA), which is in accordance with the experimental result. Further, the interface residues involved in the binding were analysed from the 2D diagram and ligplot of the docked complex.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 1","pages":"Article 141109"},"PeriodicalIF":2.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145430373","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure and analysis of a virulent chitinase from Listeria monocytogenes","authors":"Saima Rehman ,&nbsp;Maria Baczynska ,&nbsp;Beichang Zhang ,&nbsp;Christian.D. Lorenz ,&nbsp;James A. Garnett","doi":"10.1016/j.bbapap.2025.141106","DOIUrl":"10.1016/j.bbapap.2025.141106","url":null,"abstract":"<div><div><em>Listeria monocytogenes</em> is the causative agent of <em>Listeriosis</em>, a serious foodborne illness that primarily affects pregnant women, new-borns, the elderly, and immunocompromised individuals. <em>L. monocytogenes</em> secretes proteins that bind and degrade chitin, a linear polysaccharide formed of β1,4-linked <em>N</em>-acetylglucosamine residues, and although humans do not produce chitin, these enzymes act as virulence factors that promote bacterial growth during host infection. The chitinase ChiA is a major contributor to virulence, and it can modulate host immunity through downregulating the expression of host inducible nitric oxide synthase (iNOS), although this precise mechanism has yet to be determined. Here we present the X-ray crystal structure of <em>L. monocytogenes</em> ChiA at 1.95 Å resolution, complemented by solution small angle X-ray scattering analysis and molecular dynamics simulations. Our comparative analyses reveal structural conservation with homologous bacterial chitinases and highlight potential alternative ligand-binding sites beyond the canonical chitin-binding channel. Molecular dynamics simulations of an N-glycopeptide model demonstrate stable interactions between LmChiA and the mannose-rich N-glycan core near these putative sites. These findings suggest that LmChiA may interact with branched host glycans rather than exclusively processing chitin-derived substrates, and this provides a potential explanation for its role in modulating host immune responses.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 1","pages":"Article 141106"},"PeriodicalIF":2.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct cryo-EM visualization of the β-sheet structure in curved amyloid protofibril","authors":"Naoki Yamamoto ,&nbsp;Jin Inoue ,&nbsp;Ritsumi Saito ,&nbsp;Kei Nanatani ,&nbsp;Ai Takahashi ,&nbsp;Seizo Koshiba ,&nbsp;Ryo Kanno","doi":"10.1016/j.bbapap.2025.141105","DOIUrl":"10.1016/j.bbapap.2025.141105","url":null,"abstract":"<div><div>Protofibrils are key intermediates to explore effective therapeutic strategies for amyloid-related diseases; however, their structural features remain largely ambiguous. Here, we report a direct visualization of the intermolecular β-sheet structure of amyloid protofibrils using cryo-electron microscopy. We analyzed the protofibrils formed by an insulin-derived peptide and observed 4.7 Å regularly spaced lines at their centers surrounded by fuzzy regions, consistent with the hydrogen-bonded β-strand structure. We propose a model in which short β-strands form a β-sheet core in the center, whereas the outer fuzzy regions are composed of random coiled structures. This structure is different from that of mature amyloid fibrils, where β-sheets span the entire structure, suggesting that the partial β-sheet formation in protofibrils is responsible for their curvy noodle-like appearance. Overall, this study highlights cryo-electron microscopy as a powerful tool for visualizing seemingly flexible structures, such as protofibrils, and establishes a conceptual framework for the rational design of diagnostic and therapeutic agents targeting the protofibril β-sheet structure.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 1","pages":"Article 141105"},"PeriodicalIF":2.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of fatty acid biosynthesis in microalga Scenedesmus - from the perspective of biofuel production","authors":"Harshit Kumar Sharma ,&nbsp;Ma. Belén Velázquez ,&nbsp;Noelia Marchetti ,&nbsp;Ma. Victoria Busi ,&nbsp;Ajay Kumar ,&nbsp;Julieta Barchiesi ,&nbsp;Chitralekha Nag Dasgupta","doi":"10.1016/j.bbapap.2025.141097","DOIUrl":"10.1016/j.bbapap.2025.141097","url":null,"abstract":"<div><div><em>Scenedesmus quadricauda</em>, a freshwater microalga, has gained attention for its high lipid accumulation potential. However, information on fatty acid (FA) biosynthesis pathways in <em>Scenedesmus</em> species remains limited. Biomass (1.010 gL⁻¹) and lipid content (404 mgL⁻¹) in <em>S. quadricauda</em> were found to be higher compared to other microalgal isolates under autotrophic nutrition. All biodiesel indices were found within the defined range of biodiesel (EN14214) and petro-diesel (EN590:2013) standards. The predominant fatty acid was palmitic acid (16:0), accounting for 33.201 % of the total. Further, homology study and 3D models of all subunits of <em>S. quadricauda</em> enzymes acetyl-CoA carboxylase (ACC) – the key enzyme of the FA biosynthetic pathway - identified distinct structural features. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), β-carboxyltransferases (β-CT) subunits of the heteromeric ACC of <em>S. quadricauda</em> showed homology with its eukaryotic counterparts, whereas the α -carboxyltransferases (α-CT) subunit showed homology with prokaryotic carboxyltransferase. In contrast, homologous enzymes in other <em>Scenedesmus</em> species were found to resemble prokaryotic forms exclusively. Conserved motifs such as the glycine-rich loop, ERYV motif, and AAAP motif were identified in the BC and BCCP enzymes. Malonyl-CoA:ACP transacylase (MAT) enzyme of <em>S. quadricauda</em> was of prokaryotic origin but showed structural divergence from its homologs in other <em>Scenedesmus</em> species. Fatty-acyl thioesterases (FAT) enzyme contained a duplication of two 4-hydroxybenzoyl-CoA thioesterase-like domains (4HBT). These unique sequences and binding sites in the fatty acid biosynthesis enzymes of <em>S. quadricauda</em> may contribute to the distinct regulation of carbon flux and lipid assembly compared to other species.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 1","pages":"Article 141097"},"PeriodicalIF":2.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145013758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Flexibility in acidophilic thioredoxins: Insights from Asp43 substitutions in E. coli thioredoxin","authors":"Oanh Mai Ho,&nbsp;Mohammed Shazaly A. Elhassan,&nbsp;Khang Nguyen,&nbsp;ChangWoo Lee","doi":"10.1016/j.bbapap.2025.141115","DOIUrl":"10.1016/j.bbapap.2025.141115","url":null,"abstract":"<div><div>Thioredoxins (Trxs) are small, highly conserved oxidoreductases characterized by a redox-active CXXC motif. While the structural adaptation of modern Trxs such as <em>Escherichia coli</em> Trx (EcTrx) has been described, the mechanisms underlying Trx adaptation to acidic environments remain unclear. In EcTrx, Asp43 stabilizes the active-site region through a water-mediated hydrogen bond with Lys57, which modulates the protonation state of Cys32 in cooperation with Asp26. Comparative sequence analysis shows that small, nonpolar amino acids—such as Gly and Ala—are frequently found at position 43 in acidophilic Trxs, indicating that structural flexibility at this position may contribute to acidic adaptation. To test how substitutions at position 43 affect Trx stability and function, we generated EcTrx mutants (D43G, D43A, D43S, D43N, D43L, and D43E). Thermal shift and guanidinium chloride-induced unfolding assays revealed that D43A and D43S markedly reduced stability, while D43G retained moderate stability, likely due to tighter N-terminal packing as observed in the acidophilic <em>Acetobacter aceti</em> Trx. These three mutants also displayed increased conformational flexibility, whereas D43N and D43L conferred partial stabilization through polar or hydrophobic side chains. In contrast, D43E—mimicking ancestral Glu—restored hydrogen bonding and enhanced thermal stability, resulting in the most rigid structure. Most mutants retained catalytic activity in DTNB assays, except D43S, which showed 50% of wild-type activity. Overall, our results demonstrate that Asp43 is critical for maintaining EcTrx structural stability and suggest that enhanced, but not excessive, flexibility at this position facilitates Trx adaptation to acidic environments.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1874 1","pages":"Article 141115"},"PeriodicalIF":2.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145572846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}