{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T21:02:19Z","timestamp":1770757339877,"version":"3.50.0"},"posted":{"date-parts":[[2026,2,6]]},"group-title":"Biochemistry","reference-count":97,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,6]],"date-time":"2026-02-06T00:00:00Z","timestamp":1770336000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000057","name":"NIGMS","doi-asserted-by":"crossref","award":["R35GM124893"],"award-info":[{"award-number":["R35GM124893"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by\n                  <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                  (\n                  <jats:italic>Mtb<\/jats:italic>\n                  ) and non-tuberculous mycobacteria (NTM). L,D-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of \u03b2-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.\n                <\/jats:p>","DOI":"10.64898\/2026.02.05.698683","type":"posted-content","created":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T02:40:13Z","timestamp":1770432013000},"source":"Crossref","is-referenced-by-count":0,"title":["Activity Profiling of Mycobacterial L,D-Transpeptidases"],"prefix":"10.64898","author":[{"given":"Karl L.","family":"Ocius","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel E.","family":"Sanborn","sequence":"additional","affiliation":[{"name":"Chemistry Department, United States Naval Academy, Annapolis, MD 21402, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ananya","family":"Naick","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leighanne A. 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