Crystal Structure of Escherichia coli Penicillin-Binding Protein 5 Bound to a Tripeptide Boronic Acid Inhibitor:  A Role for Ser-110 in Deacylation

Abstract

Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.

Crystal Structures of Complexes of Bacterial dd-Peptidases with Peptidoglycan-Mimetic Ligands: The Substrate Specificity Puzzle

“…Of further interest, a conformational change that occurred in a complex of PBP5 with a boronic acid peptide mimetic-a shift in residues 152-154 near the active site 32 -was not observed in either of the ligand-bound structures presented here. Thus, it appears that different compounds reacting with PBP5 induce different and specific conformational changes in the protein (see below).…”

“…Striking also are the smaller distances in the PBP5-boronate inhibitor complex where the boronate moiety is hydrogen-bonded to the protein in the manner of a transition-state analogue. 32 The complexes of PBP5 with β-lactams 5 and 6 show no such tightening of the structure, which might have been expected to accompany specific recognition. It may be that PBP5, in crystalline form, in solution, and perhaps when resting in vivo, relaxes to an open inactive conformation, unless stimulated by a specific-and perhaps extended-substrate.…”

Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

“…It ends with Ser87, the equivalent of Ser78 in Pa sPBP5. The O-␥ atom of Ser87 is only 3.6 Å away from the boronate oxygen of a transitionstate analog for the acylation event, which is suggested to mimic the leaving-group nitrogen in carboxypeptidase catalysis (26). The equivalent serine residue in E. coli PBP6 places its O-␥ atom 3.8 Å away from this leaving group (the backbone nitrogen of the terminal D-Ala) in the Michaelis complex structure with a substrate analog (28).…”

“…Additional conserved active-site motifs for both classes of enzymes include the Ser-X-Asn and Lys-Thr-Gly triads. Mechanistic understanding has benefited from microbiological (20), kinetic (21)(22)(23), structural (including complexes with various ligands along the reaction coordinate) (24)(25)(26)(27)(28), and computational (29,30) studies of E. coli PBP5 and PBP6. In addition to the catalytic serine, the lysine in the Ser-X-X-Lys motif has an essential function as a general base in both the acylation and deacylation steps (23).…”

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

“…As previously noted, solubilized E. coli PBP5 rests in an active site-distorted conformation in the crystalline solid state, and, most likely, also in membranes 52 and in solution. 35 It is pertinent to note at this point that a nonspecific boronate has been shown to induce organization of the reaction center, 53 but this boronate did not have a peptidoglycan-mimetic side chain; thus, no direct evidence about how a specific side chain might bind was obtained. These results indicate that none of the structural motifs in the substrates tested was able to induce a catalytically effective conformation.…”