A molecular basis for stoichiometric enzyme encapsulation in the vitamin B2 biosynthesis compartment

A molecular basis for stoichiometric enzyme encapsulation in the vitamin B2 biosynthesis compartment | 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 Article A molecular basis for stoichiometric enzyme encapsulation in the vitamin B2 biosynthesis compartment Yusuke Azuma, Lukasz Koziej, Jedrzej Pankowski, Monika Stefanska, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8338872/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Encapsulating metabolic enzymes within protein cages enhances catalytic efficiency through substrate channeling. The vitamin B2 biosynthesis system, in which a dodecahedral lumazine synthase (LS) cage encapsulates a homotrimeric riboflavin synthase (RS), exemplifies this strategy, yet the molecular basis for this stoichiometric enzyme encapsulation have remained elusive. Here, cryogenic electron microscopy structures reveal a hierarchical assembly mechanism that ensures the defined host-guest ratio. RS C-terminal cage-localization signal peptides anchor at LS pentamer-pentamer interfaces early during assembly, stabilizing open intermediates that, together with delayed later-stage cage closure, extend the loading window until guest incorporation is complete. RS spatial occupancy prevents overloading, while a molecular lock upon final closure prevents disassembly. The elucidated anchoring mechanism enabled structure-based phylogenetic analysis across diverse organisms, suggesting multiple independent evolutionary origins of this modular encapsulation strategy. These structural insights provide design principles for engineering synthetic catalytic compartments with programmable stoichiometric control. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Evolution Biological sciences/Biochemistry/Enzymes/Multienzyme complexes Bacterial compartment Riboflavin biosynthesis Lumazine synthase Enzyme evolution Hierarchical assembly Full Text Additional Declarations Yes there is potential Competing Interest. Author J.T.H. is CEO of Nanometria, LLC, a cryo-EM data analysis service provider. Though the company has no direct financial interest or stake in these results, this affiliation may represent an indirect interest. We confirm no other conflicts of interest exist. Supplementary Files Koziejetal2025SupplementaryMaterialsYA20251206.pdf Extended Data Figs and Tables SupplementaryMovie1.mp4 Supplementary Movie 1 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version 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-8338872","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":562971269,"identity":"c6c9768e-6683-4ef3-a411-647571fe41f9","order_by":0,"name":"Yusuke Azuma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYJACxgYY6wMDG5BMAOICIrUwzoBrMSBSCzMPmCKgxeBG+jPJGRUM8vIzshM/27bxMfCz5xgwF+DVkmMmueEMg2HjjNzN0rltbAySPW8MmGfg18Im+bCNgbFZIncDWAtQxICZh5DDHv5jsG+TyN382xKoxZ6wlgQzyY0NDIk9ErnbpBlBtkgQ0CJ55o2x5YxjEskzeN5us+w5x8YjceZZwWF8fuE7nv7wZk+Nje389tzNN36UHZPjb0/e+LigArcWhQNgSgLGPwaOmsO4NTAwyDeg8mvAJDM+LaNgFIyCUTDiAABIV0vpeKysFgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3543-3159","institution":"Jagiellonian University","correspondingAuthor":true,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Azuma","suffix":""},{"id":562971270,"identity":"da995f25-e21b-4b6f-9446-4a4c5e1275cc","order_by":1,"name":"Lukasz Koziej","email":"","orcid":"https://orcid.org/0000-0003-3885-3845","institution":"Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Lukasz","middleName":"","lastName":"Koziej","suffix":""},{"id":562971271,"identity":"f408e976-b152-4907-9378-b703430fcec9","order_by":2,"name":"Jedrzej Pankowski","email":"","orcid":"https://orcid.org/0000-0003-3968-1790","institution":"Małopolska Centre of Biotechnology, Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Jedrzej","middleName":"","lastName":"Pankowski","suffix":""},{"id":562971272,"identity":"7221ac23-e0fb-443e-a71d-cb1fab42da7d","order_by":3,"name":"Monika Stefanska","email":"","orcid":"","institution":"Małopolska Centre of Biotechnology, Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Monika","middleName":"","lastName":"Stefanska","suffix":""},{"id":562971273,"identity":"b9ac8fe1-3bf5-4073-950d-3b5c2e463f7c","order_by":4,"name":"Daniel Jankowski","email":"","orcid":"","institution":"Małopolska Centre of Biotechnology, Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Jankowski","suffix":""},{"id":562971274,"identity":"0f14b914-e414-43cf-8003-0d71c5cab1e5","order_by":5,"name":"Agnieszka Gawin","email":"","orcid":"","institution":"Małopolska Centre of Biotechnology, Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Gawin","suffix":""},{"id":562971275,"identity":"dc5955de-6c49-4e93-bdef-cdfce817e1fa","order_by":6,"name":"V. 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We confirm no other conflicts of interest exist.","formattedTitle":"A molecular basis for stoichiometric enzyme encapsulation in the vitamin B2 biosynthesis compartment","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacterial compartment, Riboflavin biosynthesis, Lumazine synthase, Enzyme evolution, Hierarchical assembly","lastPublishedDoi":"10.21203/rs.3.rs-8338872/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8338872/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Encapsulating metabolic enzymes within protein cages enhances catalytic efficiency through substrate channeling. The vitamin B2 biosynthesis system, in which a dodecahedral lumazine synthase (LS) cage encapsulates a homotrimeric riboflavin synthase (RS), exemplifies this strategy, yet the molecular basis for this stoichiometric enzyme encapsulation have remained elusive. Here, cryogenic electron microscopy structures reveal a hierarchical assembly mechanism that ensures the defined host-guest ratio. RS C-terminal cage-localization signal peptides anchor at LS pentamer-pentamer interfaces early during assembly, stabilizing open intermediates that, together with delayed later-stage cage closure, extend the loading window until guest incorporation is complete. RS spatial occupancy prevents overloading, while a molecular lock upon final closure prevents disassembly. The elucidated anchoring mechanism enabled structure-based phylogenetic analysis across diverse organisms, suggesting multiple independent evolutionary origins of this modular encapsulation strategy. 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