title: 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.
authors.affiliation: Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: George
authors.initials: G
authors.lastname: Nicola
authors.forename: Sridhar
authors.initials: S
authors.lastname: Peddi
authors.forename: Miglena
authors.initials: M
authors.lastname: Stefanova
authors.forename: Robert A
authors.initials: RA
authors.lastname: Nicholas
authors.forename: William G
authors.initials: WG
authors.lastname: Gutheil
authors.forename: Christopher
authors.initials: C
authors.lastname: Davies
mesh_terms: D000215:Acylation; D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry; D001665:Binding Sites; D001897:Boronic Acids / Q000737:chemistry*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D009842:Oligopeptides / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry; D046915:Penicillin-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D013296:Streptococcus pneumoniae / Q000201:enzymology; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D063065:Organophosphonates; D046915:Penicillin-Binding Proteins; D012694:Serine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases