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

Abstract

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 terminald-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 ofMycobacterium smegmatis, by showing that it exhibits bothdd-CPase and β-lactamase activities. Like conventionaldd-CPase (PBP5 fromE. coli), upon ectopic complementation in a deformed seven PBP deletion mutant ofE. coli, MSMEG_2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further,in vitrodd-CPase assay has confirmed its ability to release terminald-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending withd-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 confirmedin vitrothrough nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of itsin vivoandin vitrodd-CPase and β-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses bothdd-CPase and β-lactamase activities.