Crystal structures of BapA complexes with β-lactam-derived inhibitors illustrate substrate specificity and enantioselectivity of β-aminopeptidases.

β-Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of β-amino acids, which serve as building blocks for the design of non-natural peptidomimetics. We have identified the β-lactam antibiotic ampicillin and the ampicillin-derived penicilloic acid as novel inhibitors of the β-aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K(i) values of 0.69 and 0.74 mM, respectively). We report high-resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA-catalyzed reactions with β-peptide substrates.