Evolvable Carbohydrate-Binding Modules Shape Function and Engineering of a Bacterial Galactose Oxidase

Domain Architecture Shapes Function and Engineering in a Modular Bacterial Galactose Oxidase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Domain Architecture Shapes Function and Engineering in a Modular Bacterial Galactose Oxidase Ligia Martins, André Taborda, Tomás Frazão, Tiago Lopes, Carolina Dias, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8880588/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Galactose oxidases (GalOxs) are copper radical oxidases that catalyze the selective oxidation of primary alcohols and constitute attractive platforms for carbohydrate biocatalysis. Although fungal AA5_2 enzymes have been extensively characterized, bacterial representatives remain unexplored, and the contribution of appended carbohydrate-binding modules (CBMs) to redox enzyme function is poorly understood. Here, we report the first biochemical and structural characterization of a bacterial galactose oxidase, Ps GalOx from Pseudarthrobacter siccitolerans , revealing a previously unexplored bacterial architectural solution within the AA5_2 family. While the catalytic Cu(II)–tyrosyl radical center is conserved, Ps GalOx displays a divergent modular architecture comprising two tandem N-terminal CBM32 domains. Functional analyses reveal pronounced asymmetry between these duplicated modules: one enhances galactan binding but compromises solubility, whereas the second primarily contributes to structural stabilization. Removal of the N-terminal CBM markedly increases soluble expression without affecting intrinsic catalytic parameters toward D-galactose, demonstrating that enzyme performance can be tuned independently of the conserved catalytic core. Consistently, adaptive engineering trajectories preferentially target domain-scale rearrangements within the CBM region, indicating that selection initially acts on modular architecture to improve physicochemical properties such as solubility, rather than directly enhancing catalytic turnover. These findings expand the structural landscape of AA5_2 oxidases and establish domain architecture as a primary determinant of solubility, substrate recognition, and catalytic output in modular copper radical oxidases. Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Biochemistry/Biocatalysis Copper radical oxidases AA5_2enzymes Modular enzyme architecture Directed evolution Redox Enzymology Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SIGalox3Mar.docx Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8880588","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596722482,"identity":"716f2238-9442-4728-855f-fa11e9c9ef13","order_by":0,"name":"Ligia Martins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIie3SIQvCQBTA8TcezDK3Jsp9icmBZWFfZVdcERlYjIJgk9X7GIJgPnnB4jeYQcuyYDW4m0Est60J3j+88ODH4+AAbLYfzK9nCBAgAH42hlw9VEVGa3yTauOsmok+pFqTAZWPe3aJ+Sk4FhnQ3IX+6WokbMqlCktxIMRIAi1c8IX5Ckt49RZKJoQu84DEBrxxA0kfmsR83Z7M6ivODjuQBZxDEpKQRzJMxQZ9IU0kYOkelk+Kg/x4K7JlJPLeVt1N5Kuh/gaAXmugSV0XYrPZbP/QC/i1Pn+izcryAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0082-9591","institution":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa","correspondingAuthor":true,"prefix":"","firstName":"Ligia","middleName":"","lastName":"Martins","suffix":""},{"id":596722483,"identity":"b9bdf3da-f3de-4d11-8dc1-fd1c31825a43","order_by":1,"name":"André Taborda","email":"","orcid":"https://orcid.org/0000-0002-3003-0580","institution":"nstituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"","lastName":"Taborda","suffix":""},{"id":596722484,"identity":"f3746874-8822-4fb5-a9b5-75876277c98a","order_by":2,"name":"Tomás Frazão","email":"","orcid":"https://orcid.org/0000-0001-5522-4085","institution":"nstituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Tomás","middleName":"","lastName":"Frazão","suffix":""},{"id":596722485,"identity":"d5a8da94-6f7a-4096-94cb-a56a64a201a5","order_by":3,"name":"Tiago Lopes","email":"","orcid":"","institution":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"","lastName":"Lopes","suffix":""},{"id":596722486,"identity":"0a4b7aaf-425e-4a7b-b8a1-d8c9abd100df","order_by":4,"name":"Carolina Dias","email":"","orcid":"","institution":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Dias","suffix":""},{"id":596722487,"identity":"e1c48cdc-a5e3-4f5b-bf12-ebf49ef50e29","order_by":5,"name":"Felipe Folgosa","email":"","orcid":"","institution":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"","lastName":"Folgosa","suffix":""},{"id":596722488,"identity":"bc3b93b7-5e65-4596-8100-950fec5c1165","order_by":6,"name":"Marcia Renio","email":"","orcid":"","institution":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Marcia","middleName":"","lastName":"Renio","suffix":""},{"id":596722489,"identity":"69b3308f-98c5-4972-a777-1759c7c43832","order_by":7,"name":"M. 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Although fungal AA5_2 enzymes have been extensively characterized, bacterial representatives remain unexplored, and the contribution of appended carbohydrate-binding modules (CBMs) to redox enzyme function is poorly understood. Here, we report the first biochemical and structural characterization of a bacterial galactose oxidase, \u003cem\u003ePs\u003c/em\u003eGalOx from \u003cem\u003ePseudarthrobacter siccitolerans\u003c/em\u003e, revealing a previously unexplored bacterial architectural solution within the AA5_2 family. While the catalytic Cu(II)–tyrosyl radical center is conserved, \u003cem\u003ePs\u003c/em\u003eGalOx displays a divergent modular architecture comprising two tandem N-terminal CBM32 domains. Functional analyses reveal pronounced asymmetry between these duplicated modules: one enhances galactan binding but compromises solubility, whereas the second primarily contributes to structural stabilization. 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