A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance.

Penicillin-binding protein 5 (PBP5), a dd-carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance in E. coli. A strain lacking PBP5 loses intrinsic beta-lactam resistance and simultaneous lack of two other PBPs results in aberrantly shaped cells. PBP5 expression in trans complements the shape and restores the lost beta-lactam resistance. PBP5 has an 'Ω-loop'-like region similar to that in class A beta-lactamases. It was previously predicted that Leu182 present in the 'Ω-like' loop of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and biochemical effects of the Leu182Glu mutation in PBP5. Upon overexpression in septuple PBP mutants, ~75 % of cells were abnormally shaped and intrinsic beta-lactam resistance maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5) retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and peptidoglycan mimetic substrates was ~10-fold less than that of the wild-type. Superimposition of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key residues may explain the low turnover numbers. Therefore, we establish the involvement of Leu182 in maintaining the physiological and biochemical behaviour of E. coli PBP5.