MSMEG_3978 (BlaE) from Mycobacterium smegmatis is an extended-spectrum beta-lactamase with a critical tyrosine residue governing its deacylation.

Abstract

The growing success of beta-lactam and beta-lactamase inhibitor combinations against mycobacterial infections emphasizes the need to deepen our understanding of beta-lactamase-mediated resistance in mycobacteria. 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 ΔampC 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.