Inter-domain flexibility and AI-guided sequence optimization enhance de novo enzyme function

Inter-domain flexibility and AI-guided sequence optimization enhance de novo enzyme function | 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 Inter-domain flexibility and AI-guided sequence optimization enhance de novo enzyme function Paula Wagner Egea*, Florent Delhommel*, Ghulam Mustafa, Florian Leiss-Maier, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6744712/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Incorporating metal cofactors into computationally designed protein scaffolds is a powerful strategy to catalyze new-to-nature reactions. However, a major challenge in de novo enzyme design is the optimization of conformational equilibria and protein dynamics crucial for catalysis. Here we show that a modular scaffold architecture with flexible inter-domain linkers enables opening/closing motions in otherwise rigid de novo proteins. Furthermore, we modified the scaffold’s metal-binding specificity and conformational behavior by rational point mutations. Structural and biophysical analyses revealed that a lanthanide-specific variant was initially trapped in an inactive conformational state, which impaired efficient metal coordination and cerium-dependent photocatalytic activity. Stabilization of the active conformation by AI-guided sequence optimization led to accelerated lanthanide binding and a 10-fold increase in k cat / K m for a photoenzymatic model reaction. Our results underscore the importance of inter-domain flexibility and AI-guided sequence redesign in de novo enzyme engineering. *Paula Wagner Egea & Florent Delhommel contributed equally. Biological sciences/Biochemistry/Biocatalysis Biological sciences/Structural biology/X-ray crystallography Biological sciences/Structural biology/NMR spectroscopy Biological sciences/Structural biology/Molecular modelling Physical sciences/Chemistry/Biochemistry/Enzymes Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformationVideoS1.mp4 Supplementary video 1 SupplementaryInformationVideoS2.mp4 Supplementary video 2 SupplementaryInformationVideoS3.mp4 Supplementary video 3 SupplementaryInformationVideoS4.mp4 Supplementary video 4 SupplementaryInformationVideoS5.mp4 Supplementary video 5 SupplementaryInformation250520final.pdf Supplementary Information Cite Share Download PDF Status: Posted 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. 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