Structural and biochemical insight into allosteric regulation of the human 2-aminoadipic semialdehyde synthase, a bifunctional enzyme involved in lysine catabolism

Structural and biochemical insight into allosteric regulation of the human 2-aminoadipic semialdehyde synthase, a bifunctional enzyme involved in lysine catabolism | 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 Structural and biochemical insight into allosteric regulation of the human 2-aminoadipic semialdehyde synthase, a bifunctional enzyme involved in lysine catabolism Michael Lazarus, Ruoxi Wu, Susmita Khamrui, Tetyana Dodatko, Yufei Xiang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9509715/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 Multiple clinically significant inborn errors of metabolism occur in the lysine degradation, even so the regulation of this biochemical pathway remains understudied. The initial rate-limiting step of lysine catabolism is catalyzed by the bifunctional enzyme 2-aminoadipic semialdehyde synthase (AASS). Therefore, understanding the regulation of AASS activity in normal and disease states will be critical for developing novel therapeutic approaches that modulate lysine degradation flux. Here, we report the cryo-EM structure of the full-length 400 kDa tetrameric AASS enzyme complex with substrates and products bound to both the lysine-2-oxoglutarate (LOR) and saccharopine dehydrogenase (SDH) catalytic domains. Our full-length structure shows that two SDH dimers are connected by a long alpha-helix group and flexible loops to a core LOR tetramer. This functional arrangement gives the SDH domain flexibility to move and readily access the products from the LOR domain. Through chemical screens, we also identified allosteric compounds that modulate activity of the AASS. Maleimides, a class of Michael-Addition chemistry substrates, activate the enzyme by reacting with cysteine 414 and shifting the pH optimum for biochemical activity. This observation can be explained mechanistically by pronounced changes in the affinity for NADPH as a function of pH. Remarkably, the mutagenesis of this allosteric Cys414 has dramatic effects on the enzyme’s conformation and activity despite being more than 12 Å from the active site. Simulations also show how changes at this distal site can affect the dynamics of the enzyme. Another compound, 1-methylnicotinamide, inhibits the enzyme by catalyzing the formation of a disulfide bond, which was observed through cryo-EM structural studies. Combined, our data indicate that AASS can be modulated at multiple allosteric sites through small molecules providing opportunities to regulate lysine catabolism by either inhibition or activation. Moreover, small changes near the hinge of an enzyme can have large effects on the catalytic properties of the enzyme. Lastly, targeting allosteric cysteines may be a general strategy for modulating enzyme activity by altering the dynamics of the enzyme. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biochemistry/Enzymes/Oxidoreductases Biological sciences/Chemical biology/Proteins/Mitochondrial proteins Full Text Additional Declarations There is NO Competing Interest. Supplementary Files LazarusAASSSupplementary2026.docx 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. 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-9509715","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":629026763,"identity":"b77daf32-1b24-45d8-87f8-28b5748e0aa4","order_by":0,"name":"Michael 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The initial rate-limiting step of lysine catabolism is catalyzed by the bifunctional enzyme 2-aminoadipic semialdehyde synthase (AASS). Therefore, understanding the regulation of AASS activity in normal and disease states will be critical for developing novel therapeutic approaches that modulate lysine degradation flux. Here, we report the cryo-EM structure of the full-length 400 kDa tetrameric AASS enzyme complex with substrates and products bound to both the lysine-2-oxoglutarate (LOR) and saccharopine dehydrogenase (SDH) catalytic domains. Our full-length structure shows that two SDH dimers are connected by a long alpha-helix group and flexible loops to a core LOR tetramer. This functional arrangement gives the SDH domain flexibility to move and readily access the products from the LOR domain. Through chemical screens, we also identified allosteric compounds that modulate activity of the AASS. Maleimides, a class of Michael-Addition chemistry substrates, activate the enzyme by reacting with cysteine 414 and shifting the pH optimum for biochemical activity. This observation can be explained mechanistically by pronounced changes in the affinity for NADPH as a function of pH. Remarkably, the mutagenesis of this allosteric Cys414 has dramatic effects on the enzyme’s conformation and activity despite being more than 12 Å from the active site. Simulations also show how changes at this distal site can affect the dynamics of the enzyme. Another compound, 1-methylnicotinamide, inhibits the enzyme by catalyzing the formation of a disulfide bond, which was observed through cryo-EM structural studies. Combined, our data indicate that AASS can be modulated at multiple allosteric sites through small molecules providing opportunities to regulate lysine catabolism by either inhibition or activation. 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