MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and β-lactamase activities

Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. DD-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal D-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis, by showing that it exhibits both DD-CPase and beta-lactamase activities. Like conventional DD-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore similar to 75% of the cell population to their normal rod shape. Further, in vitro DD-CPase assay has confirmed its ability to release terminal D-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with D-Ala-D-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of beta-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitro DD-CPase and beta-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both DD-CPase and beta-lactamase activities.