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In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.<\/jats:p>","DOI":"10.3390\/biom16091231","type":"journal-article","created":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T08:00:48Z","timestamp":1787644848000},"page":"1231","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structural and Functional Interrogation of Active Streptococcus pneumoniae Sortase A"],"prefix":"10.3390","volume":"16","author":[{"given":"Eunjeong","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"},{"name":"Biopharmaceutical Research Center, Korea Basic Science Institute, 162 Yeongudanji-Ro, OchangEup, Cheongju-Si 28119, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Blaine Hunter","family":"Gordon","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jasmina S.","family":"Redzic","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6890-512X","authenticated-orcid":false,"given":"Anthony J.","family":"Saviola","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean P.","family":"Maroney","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven","family":"Shaw","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2014Head & Neck Surgery, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mila","family":"Cordero","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2000-6106","authenticated-orcid":false,"given":"Shaun","family":"Bevers","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2258-6490","authenticated-orcid":false,"given":"Angelo","family":"D\u2019Alessandro","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kirk C.","family":"Hansen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5975-5032","authenticated-orcid":false,"given":"Sarah E.","family":"Clark","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2014Head & Neck Surgery, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6967-0892","authenticated-orcid":false,"given":"Elan","family":"Eisenmesser","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Breijyeh, Z., Jubeh, B., and Karaman, R. 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