Archives of biochemistry and biophysics最新文献

{"title":"Comparative molecular dynamics reveal the conformational dynamics of the IrtAB-cMBT complex in mycobacterial iron uptake","authors":"Gauri Shankar,&nbsp;Yusuf Akhter","doi":"10.1016/j.abb.2025.110477","DOIUrl":"10.1016/j.abb.2025.110477","url":null,"abstract":"<div><div>The IrtAB transporter in <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) is essential for iron acquisition through the import of iron-bound carboxymycobactin (cMBT), yet the molecular mechanisms governing substrate recognition and transport remain unresolved. Here, we employed 450-ns molecular dynamics simulations to elucidate the conformational dynamics of IrtAB in substrate-free (apo) and substrate-bound (holo) states, revealing key structural rearrangements and residue-specific interactions underpinning its transport cycle. Comparative analyses demonstrated enhanced conformational flexibility in the holo state, with asymmetric domain movements in IrtA and IrtB subunits facilitating cMBT translocation. Three histidine residues (His356, His402, His407) in IrtA undergone significant positional shifts (6–10 Å) upon substrate binding, forming a dynamic coordination network critical for cMBT recognition. The ligand exhibited complex behavior, including a 3.5–4.0 Å downward movement within the binding pocket and RMSD fluctuations (0.5–5.0 Å), indicative of multiple energetically favorable binding modes. Substrate-induced stabilization of the transmembrane domains correlates with progressive dehydration of the binding cavity, while RMSF profiles highlighted asymmetric flexibility in transmembrane helices during transport. These findings reveal how IrtAB's exporter-like architecture is repurposed for iron-siderophore import, balancing structural rigidity with conformational plasticity to enable efficient nutrient uptake. By delineating the mechanistic basis of IrtAB-mediated iron acquisition, this study provides a framework for targeting this pathway in <em>Mtb</em>, offering potential avenues for therapeutic intervention against tuberculosis.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110477"},"PeriodicalIF":3.8,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144168665","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":"Berberine triggers apoptosis through the PI3K/Akt pathways and Nrf2 by inducing ROS in papillary thyroid cancer.","authors":"Jing Li, Ziyi Zhu, Jing Ni, Lizi Ye, Xiaoyan Huang, Dong Jing, Yanhong Lu, Ling Yue","doi":"10.1016/j.abb.2025.110481","DOIUrl":"https://doi.org/10.1016/j.abb.2025.110481","url":null,"abstract":"<p><strong>Background: </strong>Nrf2 is highly expressed in papillary thyroid cancer (PTC) and is associated with negative outcomes. Research has indicated that Berberine (BBR) can lower Nrf2 levels and trigger apoptosis in cancer cells. However, the exact molecular mechanisms behind its anticancer effects in PTC are not fully understood.</p><p><strong>Methods: </strong>The effects of BBR on cell apoptosis were assessed using the MTT assay and flow cytometry. To evaluate BBR's in vivo antitumor efficacy, a xenograft model was used. Molecular and biochemical methods were applied to clarify the mechanisms through which BBR exerts its anticancer effects in PTC.</p><p><strong>Results: </strong>BBR has been shown to effectively inhibit the growth of PTC cells and promote programmed cell death. A higher dose of BBR administered via gavage significantly reduced the development of xenograft tumors. Mechanistically, BBR inhibits the Nrf2-dependent pathway of PI3K/Akt signaling pathway, resulting in the production of reactive oxygen species (ROS).</p><p><strong>Conclusions: </strong>Our results suggest indicate that BBR can target PTC by inhibiting the Nrf2 and PI3K/Akt pathways through ROS generation. This indicats that BBR may serve as a potential therapeutic agent for PTC treatment.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110481"},"PeriodicalIF":3.8,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144172495","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":"Triazole-quinoxaline attenuates epithelial-to-mesenchymal transition by suppressing the Wnt/β-catenin pathway in human colorectal cancer cells","authors":"Bada Yoon ,&nbsp;Rajaghatta N. Suresh ,&nbsp;Chakrabhavi Dhananjaya Mohan ,&nbsp;Kachigere B. Harsha ,&nbsp;C.S. Shivakumara ,&nbsp;Arunachalam Chinnathambi ,&nbsp;Sulaiman Ali Alharbi ,&nbsp;Gautam Sethi ,&nbsp;Kanchugarakoppal S. Rangappa ,&nbsp;Kwang Seok Ahn","doi":"10.1016/j.abb.2025.110476","DOIUrl":"10.1016/j.abb.2025.110476","url":null,"abstract":"<div><div>The Wnt/β-catenin signaling pathway regulates key cellular processes, including proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT). Dysregulation of this pathway has been implicated in various human cancers, including colorectal cancer (CRC), where it plays a critical role in promoting EMT and metastatic progression. In a recent study, triazole derivatives were shown to possess anti-EMT activity in cancer cells. Building on this finding, we synthesized a triazolyl-quinoxaline-based small molecule, SRN-18, and evaluated its impact on EMT in CRC cells. Specifically, we investigated the effect of SRN-18 on the mRNA and protein expression levels of CXCR4 and CXCR7, as well as its influence on the expression of MMP-2, MMP-9, and key EMT-associated proteins. As CXCL12 is a known ligand for both CXCR4 and CXCR7, we also examined the effects of CXCL12 stimulation on cell migration and invasion. Western blot analyses were conducted to determine whether SRN-18 modulates the expression of CXCR4/7, MMP-2/9, and EMT markers in the presence or absence of CXCL12 stimulation. Additionally, our findings revealed that the Wnt/β-catenin signaling pathway is involved in SRN-18-mediated EMT suppression. Since inhibition of the Wnt/β-catenin pathway has been shown to reduce the expression of CXCR4 and CXCR7, SRN-18-mediated suppression of this pathway led to the downregulation of CXCR4- and CXCR7-associated signaling proteins, including NF-κB and JNK. In summary, SRN-18 exerted its anti-EMT effects in colorectal cancer cells by targeting the Wnt/β-catenin signaling axis and its downstream signaling cascades.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110476"},"PeriodicalIF":3.8,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144138555","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":"High-intensity interval training elicits superior effects than continuous training to improve renal redox status via klotho and Nrf2 signaling in female rats with cisplatin nephrotoxicity","authors":"Érika Azenathe Barros Mercês ,&nbsp;Caroline Assunção Oliveira ,&nbsp;Fernanda Santos Portela ,&nbsp;Lara Fabiana Luz Malheiro ,&nbsp;Henrique Bruno Lopes Silva ,&nbsp;Júlia Spínola Ávila ,&nbsp;Bruna Santos Silva ,&nbsp;Ana Jullie Veiga Fernandes ,&nbsp;Laís Mafra De Benedictis ,&nbsp;Thiago Macêdo Lopes Correia ,&nbsp;Fabrício Freire de Melo ,&nbsp;Márcio Vasconcelos Oliveira ,&nbsp;Amélia Cristina Mendes de Magalhães ,&nbsp;Telma de Jesus Soares ,&nbsp;Liliany Souza de Brito Amaral","doi":"10.1016/j.abb.2025.110480","DOIUrl":"10.1016/j.abb.2025.110480","url":null,"abstract":"<div><div>Oxidative and nitrosative stress are the main mechanisms of nephrotoxicity induced by the chemotherapy cisplatin (CDDP). Exercise training can promote antioxidant effects by activating the Klotho and Nrf2 signaling pathways. We compared the effects of low (LIT), moderate (MIT), and high-intensity interval (HIIT) training protocols on the renal redox status via modulation of klotho and Nrf2 signaling in female rats with CDDP-induced AKI. Wistar rats were divided into five groups (n = 7): sedentary control (C + S); CDDP and sedentary (CDDP + S); CDDP and subjected to LIT (CDDP + LIT); CDDP and subjected to MIT (CDDP + MIT); and CDDP and subjected to HIIT (CDDP + HIIT). The training protocols were carried out on a motorized treadmill lasting 8 weeks. After this period, we administered CDDP (5 mg/kg) and, 7 days later, collected biological samples for analysis. Our data demonstrate that CDDP caused changes to the tubular structure and absorptive function, reduced klotho expression, and increased oxidative and nitrosative damage markers to cellular macromolecules, such as lipids (4-HNE and TBARS), proteins (carbonylation and 3-NT), and DNA (8-OHDG). However, training protocols reduced all of these changes in an intensity-dependent manner, with effects more prominent with HIIT. Our training protocols increased the renal antioxidant defenses (SOD-1, catalase, and GPx), probably through the activation of the Nrf2 pathway, also in an intensity-dependent manner. In conclusion, our results suggest that HIIT promoted more pronounced renoprotective effects than LIT and MIT, improving redox status via activating of the Klotho and Nrf2 signaling pathways in female rats with CDDP-induced nephrotoxicity.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110480"},"PeriodicalIF":3.8,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144138554","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":"Crystal structures of the phosphorylation mimics of human cytosolic branched chain aminotransferase","authors":"Elizabeth S. Dare ,&nbsp;Robert H. Newman ,&nbsp;Myra E. Conway ,&nbsp;Ming Dong","doi":"10.1016/j.abb.2025.110479","DOIUrl":"10.1016/j.abb.2025.110479","url":null,"abstract":"<div><div>The phosphorylation sites of the human cytosolic Branched Chain Aminotransferase (hBCATc) mediated by mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated-kinase 2 (ERK2, also known as MAPK1) were mapped. The crystal structures of the phosphorylation mimics at T33 and T36 were determined. The modified transaminase activity of these variants was analyzed. Although there were no major conformational changes in the phosphorylation mimics of hBCAT, a regional conformational change at the interdomain loop was observed mainly in mutant T33E. Consistently, when the catalytic turnovers of the T33E and T36E mutants were comparable to the wild type of hBCATc, the K_M dropped significantly compared to the wild type, indicating a shift of the substrate binding affinity in the mutants. Taken together, this indicated the phosphorylation of hBCATc by ERK2 is affecting the hBCATc's transaminase activity.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110479"},"PeriodicalIF":3.8,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144131212","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":"Activation fingerprints and allosteric modulation at the free fatty acid receptor 1 (FFAR1) revealed by molecular dynamics simulation","authors":"Rufaida Al Zoubi ,&nbsp;Samah Sokkar ,&nbsp;Israa H. Isawi ,&nbsp;Qanita Bani Baker","doi":"10.1016/j.abb.2025.110473","DOIUrl":"10.1016/j.abb.2025.110473","url":null,"abstract":"<div><div>The free fatty acid receptor 1 (FFAR1) is a transmembrane G-protein coupled receptor that mediates the metabolic and insulinotropic effects of endogenous free fatty acids in pancreatic cells while also exerting neuro-regulatory effects in the brain. The complexity of FFAR1 derives from its multiple binding sites and the absence of conventional activation motifs observed in class A GPCRs. This study uses molecular dynamics simulations to investigate the molecular mechanisms that underpin endogenous signaling and allosteric regulation in the FFAR1. We investigated and compared three ligand-bound states and the APO state. The ligand-bound simulations included FFAR1 in complex with γ-linolenic acid, FFAR1 in complex with γ-linolenic acid and TAK875, and a fully activated FFAR1 bundle complexed with docosahexaenoic acid and G-protein. The results highlight distinct protein contact fingerprints and dynamics in the ligand-bound states relative to the APO state. While ligand binding, in the absence of stabilizing G-protein, destabilizes the intracellular domain of the receptor, the second extracellular loop exhibits greater stability and salt bridge contact with the transmembrane domain. Notably, simulations of FFAR1 complexed with γ-linolenic acid, bound at the intracellular domain, revealed stable interactions between γ-linolenic acid and the receptor, as well as similar activation fingerprints when compared to FFAR1 in complex with docosahexaenoic acid and Gq. This suggests an effective allosteric regulation of the receptor following γ-linolenic acid binding to the intracellular domain. Finally, a set of hydrophobic amino acid residues at the intracellular and extracellular domains appears to function as potential rotameric switches, facilitating water-mediated receptor activation.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110473"},"PeriodicalIF":3.8,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144131803","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":"B(reath)e water my friend: hydration of pulmonary surfactant layers as analyzed by fourier transform-infrared spectroscopy","authors":"Ainhoa Collada ,&nbsp;Antonio Sebastián Rosa ,&nbsp;Jimena del Pilar Cejas ,&nbsp;Araceli Villalba ,&nbsp;Emma Batllori-Badia ,&nbsp;Alberto Galindo ,&nbsp;Antonio Cruz ,&nbsp;Edgardo Aníbal Disalvo ,&nbsp;Jesús Pérez-Gil ,&nbsp;María de los Ángeles Frías","doi":"10.1016/j.abb.2025.110474","DOIUrl":"10.1016/j.abb.2025.110474","url":null,"abstract":"<div><div>Water is essential for sustaining life, as well as the presence of a fully operational pulmonary surfactant (PS) system coating the respiratory surface. In this work, the interconnection between different hydration states and PS membrane structure is explored thanks to the use of ATR-FTIR spectroscopy. Thermotropic phase transitions and hydration level in complex membrane structures formed by two native surfactant systems, derived from animal and human sources, are compared with those found in synthetic mixtures used routinely to mimic PS composition and with re-assembled samples derived from porcine lavages. Native samples exhibited always a faster and higher hydration level than the synthetic ones under comparable conditions, notwithstanding their origin. This hydration degree was found to be crucial in modulating gel to fluid phase transitions. This new insight should be integrated to better comprehend structure-function relationships in PS natural and model membranes, and provides a novel framework to understand permeability and dynamics of alveolar surfactant coatings under physiological conditions.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110474"},"PeriodicalIF":3.8,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144116844","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":"Food-derived bioactive pigment phycocyanobilin binds to SARS-CoV-2 spike protein both covalently and noncovalently affecting its conformation and functionality","authors":"Ana Simovic ,&nbsp;Mirjana Radomirovic ,&nbsp;Nikola Gligorijevic ,&nbsp;Milos Milcic ,&nbsp;Masa Bicanin ,&nbsp;Simeon Minic ,&nbsp;Marijana Stojanovic ,&nbsp;Dragana Stanic-Vucinic ,&nbsp;Tanja Cirkovic Velickovic","doi":"10.1016/j.abb.2025.110475","DOIUrl":"10.1016/j.abb.2025.110475","url":null,"abstract":"<div><div>Phycocyanobilin (PCB), tetrapyrrole chromophore of Spirulina phycocyanin, is bilirubin analog and weak thiol-modifying agent. SARS-CoV-2 spike protein (SP) has bilirubin binding pocket, lacks free sulfhydryl, but it has two pairs of functionally important semi-stable disulfides reactive towards thiol-modifying agents. We investigated covalent and noncovalent binding of PCB to SP and its receptor-binding domain (RBD) and impact of covalent PCB conjugation to RBD on structure and binding to human angiotensin-converting enzyme 2 (ACE-2). PCB shows high-affinity for SP (Ka = 2.1 × 10⁷ M⁻¹), moderate-affinity for RBD (Ka = 8.4 × 10⁴ M⁻¹) and binds covalently to SP and RBD in reaction involving thiols. PCB binding alters RBD conformation. Molecular docking identified two binding sites of PCB to SP, bilirubin/biliverdin binding site and hydrophobic pocket of RBD in vicinity of Cys432, preferential target for covalent binding in <em>in silico</em> covalent docking of PCB to RBD. Redox proteomics mapped reactive Cys432, Cys391 and Cys525 in RBD. PCB-modified RBD exhibited reduced ability to bind to ACE-2. This is the first study demonstrating PCB reactivity towards semi-stable disulfides of proteins lacking free sulfhydryl groups. PCB may affect functionality and structure of SP and its RBD by noncovalent and covalent binding.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110475"},"PeriodicalIF":3.8,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144125394","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":"ZNF460 enhances HADHB level by activating its transcription to promote the progression and pulmonary invasion of cutaneous T cell lymphoma.","authors":"Qian He, Xingwen Tian, Qiong Mu","doi":"10.1016/j.abb.2025.110472","DOIUrl":"https://doi.org/10.1016/j.abb.2025.110472","url":null,"abstract":"<p><strong>Background: </strong>During the progression of primary cutaneous T-cell lymphoma (CTCL), malignant T cells acquire the capacity to invade extracutaneous sites.. However, the mechanisms driving CTCL progression and invasion remain poorly understood. This study aimed to investigate the role and specific mechanisms of ZNF460 and HADHB in the progression of CTCL.</p><p><strong>Methods: </strong>The viability, proliferation, apoptosis, migration, invasion, and fatty acid metabolism of malignant T lymphoma cells were assessed using CCK-8, EdU, TUNEL, Transwell, and ELISA assays. ChIP-qPCR, dual-luciferase reporter, qRT-PCR, and WB assays were utilized to elucidate the regulation of HADHB transcription by ZNF460. Tumor growth and pulmonary invasion in CTCL mouse models were evaluated through tumor growth curves and HE staining.</p><p><strong>Results: </strong>Knockdown of HADHB inhibited the viability, proliferation, migration, invasion, and fatty acid metabolism of malignant T lymphoma cells, whereas overexpression of HADHB exhibited the opposite effects. Furthermore, overexpression of HADHB accelerated the tumor growth rate and pulmonary invasion in CTCL mice. ZNF460 was found to upregulate HADHB levels in malignant T lymphoma cells by activating HADHB transcription. Additionally, knockdown of ZNF460 led to the reduction in viability, proliferation, and migration of malignant T lymphocytes and the inhibition of CTCL mice tumor growth and pulmonary invasion.</p><p><strong>Conclusion: </strong>Collectively, ZNF460 enhanced HADHB levels by activating its transcription, thereby promoting CTCL tumor growth and pulmonary invasion.</p>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110472"},"PeriodicalIF":3.8,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126172","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":"Structurally guided engineering of flavin-dependent nicotine dehydrogenase","authors":"Yuvarun Kapaothong,&nbsp;Panu Pimviriyakul","doi":"10.1016/j.abb.2025.110471","DOIUrl":"10.1016/j.abb.2025.110471","url":null,"abstract":"<div><div>Nicotine is a toxic alkaloid found in tobacco leaves that contaminates the environment when the leaves are smoked. In the present study, developed an enzyme technology for nicotine biodegradation and addressed biodetection applications. Recombinant wild-type nicotine oxidase/dehydrogenase from <em>Pseudomonas</em> sp. HZN6 (Nox-WT) was overexpressed and purified to ensure homogeneity. Nox-WT was clearly classified as a flavin adenine dinucleotide (FAD)-containing dehydrogenase, which catalyzes rapid nicotine oxidation in its reductive half-reaction; however, its oxidative half-reaction with O₂ was slow and was identified as the rate-limiting step. An imbalance in the rate between the two half-reactions limits the overall catalytic turnover of Nox-WT. According to kinetic behavior, incomplete flavin recovery and substrate inhibition were also identified as obstructed issues that limit the enzyme efficiency. Nox-WT engineering has been performed to address these problems. The modeled structure of Nox-WT was constructed using AlphaFold to design candidate residues for site-directed mutagenesis. Using systematic screening through rapid kinetic techniques, all the limitations were eliminated in the engineered triple mutated Nox-Y338F/H364V/W423H. The mutations at Y338F and H364V expanded the tunnel for O₂ accessibility, resulting in 21-fold faster FAD oxidation by O₂ in this mutant than in Nox-WT. Mutation at W423H disrupted the binding of nicotine; therefore, substrate inhibition was removed, and FAD was fully recovered. Nox-Y338F/H364V/W423H potentially transforms nicotine considerably faster than Nox-WT without loss of enzyme thermostability. Overall, using a rational design, we successfully engineered an effective mutant of Nox that would be useful for future applications.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"770 ","pages":"Article 110471"},"PeriodicalIF":3.8,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144107498","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}