Characterization of Wild-type and a Novel Mutant of Arylamine N-acetyltransferase (NAT) from Mycobacterium Marinum as a Model for NAT from Mycobacterium Tuberculosis

Characterization of Wild-type and a Novel Mutant of Arylamine N-acetyltransferase (NAT) from Mycobacterium Marinum as a Model for NAT from Mycobacterium Tuberculosis | 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 Characterization of Wild-type and a Novel Mutant of Arylamine N-acetyltransferase (NAT) from Mycobacterium Marinum as a Model for NAT from Mycobacterium Tuberculosis Shreya Behl This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6605365/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 Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes linked to antibiotic resistance in Mycobacterium tuberculosis (MTB ), making them promising targets for therapeutic intervention. This research used Mycobacterium marinum’s NAT homolog as a model to evaluate the structural and functional effects of a site-directed missense mutation, T109Q, situated near the active site. The mutation substitutes threonine, a minor polar residue, with glutamine, a larger polar amino acid, which may influence substrate binding and catalytic performance. Both wild-type and T109Q mutant NAT proteins were produced in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) and ion exchange chromatography. SDS-PAGE and Western blotting were used to measure expression and solubility, with the wild-type protein outperforming the mutant in terms of both. Protein concentrations were measured using the Bradford and UV absorbance as-says. Enzymatic activity was assessed using acetyl-CoA and either isoniazid or hydralazine as substrates. The wild-type protein catalyzed isoniazid acetylation, but the mutant had no detectable activity for either drug. Thermal shift experiments revealed that the mutant was less thermally stable than the wild-type, indicating that folding or structural integrity had been disrupted. These results show that the T109Q mutation drastically reduced NAT expression, solubility, stability, and enzymatic performance, most likely due to disruption of active-site dynamics. The findings highlight NAT’s structural vulnerability to changes around its catalytic core and give mechanistic insights that might influence future inhibitor design targeting MTB NAT (Areej Abuhammad et al., 2010). Full Text Additional Declarations The authors declare no competing interests. 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. 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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-6605365","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452831028,"identity":"3a63a2f4-18d7-4306-9217-a311f738724a","order_by":0,"name":"Shreya Behl","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACCcYGEJXAIMHc+ADI4OEjQQtjswFICxthLRAKpKUNzCaoRX52c5vEjxqGPH7pxrbKrzl2MmwMzA8f3cCjxeDOwTbJnmMMxZJzDrbdlt2WDHQYm7FxDj4tEoltEkBliRtuJLbdltzGDGTzsEnj0yI/I7FN8s8/hsT9QC3FktvqCWthAKqU5m0D2gK0jvHjtsOEtRjcSGy2lu2TKJYAMqQZtx3nYWMm4Bf5GekPb775ZpPHPyP54Mef26rt+dmbHz7G6zAGBhYJWOww84BJ/MrBSj7AWIw/CKseBaNgFIyCEQgAGZJG1TzkpKoAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Shreya","middleName":"","lastName":"Behl","suffix":""}],"badges":[],"createdAt":"2025-05-06 17:16:49","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6605365/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6605365/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82226822,"identity":"4d4cc993-f381-4b2b-b330-acb543eb2081","added_by":"auto","created_at":"2025-05-08 04:41:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2457171,"visible":true,"origin":"","legend":"","description":"","filename":"SLRPFINALREPORT.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6605365/v1_covered_da43dddb-1ba1-415e-adf1-c6cad428939e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCharacterization of Wild-type and a Novel Mutant of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArylamine N-acetyltransferase (NAT) \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efrom \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMycobacterium Marinum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e as a Model for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNAT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMycobacterium Tuberculosis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6605365/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6605365/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eArylamine N-acetyltransferases (NATs)\u003c/em\u003e are xenobiotic-metabolizing enzymes linked to antibiotic resistance in \u003cem\u003eMycobacterium tuberculosis (MTB\u003c/em\u003e), making them promising targets for therapeutic intervention.\u003c/p\u003e\n\u003cp\u003eThis research used \u003cem\u003eMycobacterium marinum’s NAT \u003c/em\u003ehomolog as a model to evaluate the structural and functional effects of a site-directed missense mutation, \u003cem\u003eT109Q,\u003c/em\u003e situated near the active site. The mutation substitutes threonine, a minor polar residue, with glutamine, a larger polar amino acid, which may influence substrate binding and catalytic performance. Both wild-type and \u003cem\u003eT109Q\u003c/em\u003e mutant \u003cem\u003eNAT\u003c/em\u003e proteins were produced in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) and ion exchange chromatography. SDS-PAGE and Western blotting were used to measure expression and solubility, with the wild-type protein outperforming the mutant in terms of both. Protein concentrations were measured using the Bradford and UV absorbance as-says. Enzymatic activity was assessed using acetyl-CoA and either isoniazid or hydralazine as substrates. The wild-type protein catalyzed isoniazid acetylation, but the mutant had no detectable activity for either drug. Thermal shift experiments revealed that the mutant was less thermally stable than the wild-type, indicating that folding or structural integrity had been disrupted. These results show that the \u003cem\u003eT109Q\u003c/em\u003e mutation drastically reduced \u003cem\u003eNAT\u003c/em\u003e expression, solubility, stability, and enzymatic performance, most likely due to disruption of active-site dynamics. The findings highlight \u003cem\u003eNAT’s \u003c/em\u003estructural vulnerability to changes around its catalytic core and give mechanistic insights that might influence future inhibitor design targeting \u003cem\u003eMTB NAT (Areej Abuhammad et al., 2010).\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Characterization of Wild-type and a Novel Mutant of Arylamine N-acetyltransferase (NAT) from Mycobacterium Marinum as a Model for NAT from Mycobacterium Tuberculosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 04:32:57","doi":"10.21203/rs.3.rs-6605365/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e26f8d01-572a-4e0b-b5d8-65476bd71125","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-08T04:32:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-08 04:32:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6605365","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6605365","identity":"rs-6605365","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}