Probably cleaves the terminal D-Ala-D-Ala dipeptide of the peptidoglycan stem peptide (Probable). Shows significant D,D-carboxypeptidase activity in vitro (PubMed:22906310). Acts on the synthetic penta-peptide substrate Penta-DAP (L-Ala-gamma-D-Gln-DAP-D-Ala-D-Ala). Shows also weak activity on Penta-Lys (L-Ala-gamma-Glu-L-Lys-D-Ala-D-Ala) (PubMed:22906310). The catalytic domain binds weakly to peptidoglycan in vitro (PubMed:25551456). Plays an important role in the maintenance of colony morphology and cell wall permeability and integrity (PubMed:25467937). Inhibited by the beta-lactam antibiotic meropenem (PubMed:22906310). Inhibited by the non-specific inhibitor phenylmethylsulfonyl fluoride (PMSF) (Probable). Cell wall biogenesis; peptidoglycan biosynthesis. Deletion of the gene results in reduced growth in acidic medium under low oxygen conditions. The mutant shows better intracellular growth and survival inside THP-1 cells compared to wild-type and complemented strains. The colony morphology and antibiotic sensitivity of mutant and wild-type strains are similar (PubMed:22138550). The double knockout mutant pknI/dacB2 shows smoother colony morphology on solid agar and exhibits defective biofilm and cord formation. Double mutant is hypersensitive to cell wall damaging agents such as lysozyme, malachite green, ethidium bromide and to isoniazid, a first line anti-TB drug (PubMed:25467937). Overexpression in Mycobacterium smegmatis results in reduced growth, an altered colony morphology, and a defect in sliding motility and biofilm formation. Overexpression of the S69C mutant shows similar results, indicating that the effects produced are independent of protein's penicillin binding function. Belongs to the peptidase S11 family.