Calcium Activation Mechanism of a Noncanonical Aromatic L-Amino Acid Decarboxylase from Psilocybin Mushroom

Calcium Activation Mechanism of a Noncanonical Aromatic L-Amino Acid Decarboxylase from Psilocybin Mushroom | 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 Calcium Activation Mechanism of a Noncanonical Aromatic L-Amino Acid Decarboxylase from Psilocybin Mushroom Yi Wang, Tianjie Li, Erin Reynolds, Ziqi Wang, Michael Torrens-Spence, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6329392/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Feb, 2026 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract PcncAAAD is a calcium-activatable noncanonical aromatic L-amino acid decarboxylase (AAAD) featuring a unique appendage C-terminal domain (CTD) and two metal-binding sites. In this study, we establish an in silico RMSD-based evaluation model through molecular dynamics simulations, validated by in vitro enzyme assays, to decipher the enzyme’s calcium activation mechanism. Between the two metal-binding sites, the site at the N-terminal domain/CTD interface (site A) is found to play a primary role in the calcium activation of PcncAAAD, whereas the secondary site within the unique CTD (site B) contributes to the calcium-mediated stabilization of enzyme structure. Binding of calcium, but not sodium, exerts a profound influence on PcncAAAD activity by stabilizing a "lid-rim" structure underlying site A, which in turn maintains the integrity of the substrate-binding environment. In silico mutations disrupting site A or the “lid-rim” structure show severe structural distortion of the active site, leading to reduced or even eliminated activity as demonstrated by in vitro assays. Collectively, our computational and experimental analyses pinpoint the molecular mechanism underlying the noncanonical activation of PcncAAAD by calcium. These findings deepen our understanding of metal-activatable enzymes and hold promise for the rational design of engineered enzymes for the synthesis of aromatic amino acid derivatives. Biological sciences/Biophysics/Computational biophysics Biological sciences/Chemical biology/Computational chemistry Full Text Additional Declarations Yes there is potential Competing Interest. J.K.W. is a member of the Scientific Advisory Board and a shareholder of DoubleRainbow Biosciences, Galixir, and Inari Agriculture, which develop biotechnologies related to natural products, drug discovery and agriculture. All other authors have no competing interests. Supplementary Files PcncAAADSI.pdf Cite Share Download PDF Status: Published Journal Publication published 26 Feb, 2026 Read the published version in Communications Biology → 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-6329392","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":447520587,"identity":"540f26fc-fa96-47bb-895c-2e2179b47401","order_by":0,"name":"Yi Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACZjBpw8AgwQakgdiAhzgtaaRogYDDJGgxOM5j+Lng13l5g9ttCQwfyg4zmPMcIKDlMI+x9My+24Yb7hw7wDjj3GEGy94GQlp4N0jz9txOMLiR3sDM23aYweA8IYcd5t38m7fnHETLXyK1bJPm+XEAqCXtADMjSMtZAg6TPMz/zZq3Idlw5o20hIM959J5DM4cwK+F7/yx5Ns8f+zk+W6kGT74UWYtZ3AmAb8WBZCRjG0QDohNOCLlwQ7/Q1DdKBgFo2AUjGQAADhWR/u+As7bAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4174-8790","institution":"Chinese University of Hong Kong","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wang","suffix":""},{"id":447520588,"identity":"f81ed29b-e99b-41ec-a4de-c1cc9a2bab88","order_by":1,"name":"Tianjie Li","email":"","orcid":"","institution":"Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Tianjie","middleName":"","lastName":"Li","suffix":""},{"id":447520589,"identity":"366c2ddf-2303-4461-a6e8-6c8f7bc71c6e","order_by":2,"name":"Erin Reynolds","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Erin","middleName":"","lastName":"Reynolds","suffix":""},{"id":447520590,"identity":"e8a2080e-210f-4c44-9994-8f363bac3614","order_by":3,"name":"Ziqi Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ziqi","middleName":"","lastName":"Wang","suffix":""},{"id":447520591,"identity":"eee31879-fb6e-4f99-987b-e499df9d83a4","order_by":4,"name":"Michael Torrens-Spence","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Torrens-Spence","suffix":""},{"id":447520592,"identity":"cb5735ca-470c-4a32-8c9e-9c44c810d765","order_by":5,"name":"Jing-Ke Weng","email":"","orcid":"https://orcid.org/0000-0003-3059-0075","institution":"Northeastern University","correspondingAuthor":false,"prefix":"","firstName":"Jing-Ke","middleName":"","lastName":"Weng","suffix":""}],"badges":[],"createdAt":"2025-03-28 15:27:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6329392/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6329392/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-026-09756-y","type":"published","date":"2026-02-26T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":106490030,"identity":"a8c7cb54-2c95-4530-a7ce-07262b7b58af","added_by":"auto","created_at":"2026-04-09 07:13:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1821537,"visible":true,"origin":"","legend":"","description":"","filename":"PcncAAADmaintext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6329392/v1_covered_f1d8e5b0-879b-4244-adc8-2c047896f5fa.pdf"},{"id":81497761,"identity":"70fe214d-3416-4d1e-8e57-4d5cf962849e","added_by":"auto","created_at":"2025-04-28 02:55:55","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4948345,"visible":true,"origin":"","legend":"","description":"","filename":"PcncAAADSI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6329392/v1/978aeac1da81098154090e60.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nJ.K.W. is a member of the Scientific Advisory Board and a shareholder of DoubleRainbow Biosciences, Galixir, and Inari Agriculture, which develop biotechnologies related to natural products, drug discovery and agriculture. All other authors have no competing interests.","formattedTitle":"Calcium Activation Mechanism of a Noncanonical Aromatic L-Amino Acid Decarboxylase from Psilocybin Mushroom","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6329392/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6329392/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"PcncAAAD is a calcium-activatable noncanonical aromatic L-amino acid decarboxylase (AAAD) featuring a unique appendage C-terminal domain (CTD) and two metal-binding sites. In this study, we establish an in silico RMSD-based evaluation model through molecular dynamics simulations, validated by in vitro enzyme assays, to decipher the enzyme’s calcium activation mechanism. Between the two metal-binding sites, the site at the N-terminal domain/CTD interface (site A) is found to play a primary role in the calcium activation of PcncAAAD, whereas the secondary site within the unique CTD (site B) contributes to the calcium-mediated stabilization of enzyme structure. Binding of calcium, but not sodium, exerts a profound influence on PcncAAAD activity by stabilizing a \"lid-rim\" structure underlying site A, which in turn maintains the integrity of the substrate-binding environment. In silico mutations disrupting site A or the “lid-rim” structure show severe structural distortion of the active site, leading to reduced or even eliminated activity as demonstrated by in vitro assays. Collectively, our computational and experimental analyses pinpoint the molecular mechanism underlying the noncanonical activation of PcncAAAD by calcium. 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