A Single Substitution of Valine to Glutamic Acid in the Omega-Like Loop Introduces a Penicillinase-Like Activity in MSMEG_6194 by Compromising its Role as DD-Carboxypeptidase.

The genome of Mycobacterium smegmatis encodes numerous penicillin-interacting enzymes, and analysing their functions provides insights into the evolutionary mechanisms behind beta-lactam resistance in mycobacteria. In this study, we characterised one such enzyme, MSMEG_6194, annotated as a putative beta-lactamase. Although MSMEG_6194 shares structural similarity with class A beta-lactamases, it showed no detectable beta-lactamase activity under the tested conditions. Heterologous expression of MSMEG_6194 in Escherichia coli and Δmsmeg_6194 deleted strains of M. smegmatis did not confer significant resistance to beta-lactams, and the purified protein failed to hydrolyse nitrocefin either. However, ectopic expression of MSMEG_6194 partially restores morphological defects in seven PBP-deleted E. coli strains, and the purified enzyme cleaves the terminal D-alanine from a pentapeptide substrate, supporting its DD-carboxypeptidase activity. Structural analysis revealed the absence of a conserved glutamic acid residue in the omega-loop, which is critical for beta-lactamase catalysis in class A beta-lactamase. Substituting this residue (V139E mutant) imparts beta-lactamase activity, though it significantly reduces DD-carboxypeptidase function. Overall, these findings suggest that MSMEG_6194 act as a DD-carboxypeptidase and illustrate how a single amino acid change can alter catalytic preference, shedding light on the evolutionary transition from DD-carboxypeptidases to beta-lactamases in mycobacteria.