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 β-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 ~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 β-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 β-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd -CPase and β-lactamase activities.