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In this study, we characterize MSMEG_3978 (BlaE), a class C beta-lactamase from Mycobacterium smegmatis, providing the first biochemical and mechanistic evidence of its extended-spectrum activity. Heterologous expression of blaE in Escherichia coli \u0394ampC strain led to a two-to-four-fold increase in resistance to penicillins and a four-to-eight-fold increase in resistance against cephalosporins, including third-generation oxyimino-cephalosporins and imipenem. Purified BlaE enzyme efficiently hydrolysed a broad range of beta-lactam antibiotics and was resistant to clavulanic acid inhibition, consistent with group 1e class C beta-lactamases. Site-directed mutagenesis of the conserved Tyrosine 170 (Y170) to Phenylalanine (F) impaired deacylation but not acylation, implicating a specific catalytic role for the Y170 residue. Thermal shift assays and molecular dynamics simulations revealed reduced stability of the Y170F mutant protein relative to wild-type BlaE, although ligand binding remained largely unaffected. Collectively, our findings established MSMEG_3978 as an extended-spectrum class C beta-lactamase and identified the residue Y170 as a potential general base contributing to the deacylation process and strengthened our understanding of adaptive beta-lactam resistance in mycobacteria.<\/jats:p>","DOI":"10.1093\/femsle\/fnaf098","type":"journal-article","created":{"date-parts":[[2025,9,18]],"date-time":"2025-09-18T11:47:07Z","timestamp":1758196027000},"source":"Crossref","is-referenced-by-count":4,"title":["MSMEG_3978 (BlaE) from\n                    <i>Mycobacterium smegmatis<\/i>\n                    is an extended-spectrum beta-lactamase with a critical tyrosine residue governing its deacylation"],"prefix":"10.1093","volume":"372","author":[{"given":"Aditya Prasad","family":"Panda","sequence":"first","affiliation":[{"name":"Indian Institute of Technology Department of Bioscience and Biotechnology, \u00a0 Kharagpur, West Bengal 721302 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