Differences in active-site microarchitecture explain the dissimilar behaviors of PBP5 and 6 in Escherichia coli.

Out of the four DD-carboxypeptidases (DD-CPases) in Escherichia coli, only penicillin-binding protein (PBP) 5 performs physiological functions such as maintaining cell shape; its nearest homolog, PBP6, cannot perform such functions. Moreover, unlike PBP6, PBP5 efficiently processes both beta-lactam, and peptide substrates. The crystal structure of PBP5 reveals strong inter-residue hydrogen-bonding interactions around the active site, which favor its catalytic activity. However, the recently solved crystal structure of PBP6 cannot explain the reason for the observed functional discrepancies between the two proteins. Enzymatic analyses indicate that moving the morphology maintenance domain from one protein to another can alter the affinities and activities of PBP5 and 6 toward their substrates. To determine why the activities of these enzymes differ, we used molecular modeling, and docking analyses with substrate-mimetic ligands to estimate how amino-acid alterations in the morphology maintenance domain would affect the structure of PBP and hence its substrate specificity. The results obtained from kinetic analyses were directly correlated to the three-dimensional structures of the PBPs determined through in silico analyses, indicating a change in the active-site microarchitectures of PBP5 and 6 as a plausible cause of the difference in their biochemical behaviors.