Crystal Structure of a Deacylation-defective Mutant of Penicillin-binding Protein 5 at 2.3-Å Resolution

Abstract

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a D-alanine carboxypeptidase, cleaving the C-terminal D-alanine residue from cell wall peptides. Like all PBPs, PBP 5 forms a covalent acyl-enzyme complex with ␤-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acyl-enzyme complex (t1 ⁄2 ϳ 9 min). A Gly-105 3 Asp mutation in PBP 5 markedly impairs this ␤-lactamase activity (deacylation), with only minor effects on acylation, and promotes accumulation of a covalent complex with peptide substrates. To gain further insight into the catalytic mechanism of PBP 5, we determined the three-dimensional structure of the G105D mutant form of soluble PBP 5 (termed sPBP 5) at 2.3 Å resolution. The structure is composed of two domains, a penicillin binding domain with a striking similarity to Class A ␤-lactamases (TEM-1-like) and a domain of unknown function. In addition, the penicillin-binding domain contains an active site loop spatially equivalent to the ⍀ loop of ␤-lactamases. In ␤-lactamases, the ⍀ loop contains two amino acids involved in catalyzing deacylation. This similarity may explain the high ␤-lactamase activity of wild-type PBP 5. Because of the low rate of deacylation of the G105D mutant, visualization of peptide substrates bound to the active site may be possible.Penicillin and other ␤-lactam antibiotics exert their lethal effect by inhibiting the proteins that synthesize bacterial cell wall peptidoglycan (1). These proteins, known as penicillinbinding proteins or PBPs, 1 utilize lipid-linked disaccharide peptide substrates to catalyze both the polymerization of glycan chains (transglycosylation) and cross-linking of peptide chains (transpeptidation) during cell wall synthesis. In the latter reaction, a serine residue on the PBP reacts with the acyl-D-Ala-D-Ala C terminus of the peptide chain to form a transient acyl-enzyme complex, releasing the C-terminal Dalanine residue. This complex reacts with an amino group from another peptide chain to form a cross-link, which is crucial to the integrity and rigidity of the cell wall. An additional activity catalyzed by some PBPs, carboxypeptidation, occurs when the acyl-enzyme complex reacts with water. Penicillin and other ␤-lactam antibiotics mimic the structure of the acyl-D-Ala-D-Ala C terminus of the peptide chain (2) and react with PBPs to form an acyl-enzyme complex. Unlike the transient nature of the PBP-peptide complex, the acyl-enzyme complex formed between PBPs and ␤-lactam antibiotics is much more stable and results in prolonged inhibition of the enzyme.In Escherichia coli, at least 10 PBPs have been identified. These PBPs can be split into two classes: the high molecular mass PBPs (PBPs 1A, 1B, 1C, 2, and 3) and the low molecular mass PBPs (PBPs 4, 5, 6, 6b, and 7) (3). High molecular mass PBPs are essential for cell viability and are involved in the physiological processes of cell elongation, cell division, and the maintenance of cell shape (4). The role of low molecular mass PBPs in bacterial ...

The 2.4-Å crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin

“…The C-terminal domain shows structural similarities with the transpeptidase domain of the penicillin-sensitive PBP2x and more generally with the penicillin-recognizing enzymes (b-lactamases of classes A, C or D [35], E. coli PBP5 [36], and the Streptomyces R61 [37] and K15 [38] DD-peptidases). The active-site serine 422 is located between two subdomains, one a five-stranded b sheet covered by helices, the other being all a helices.…”

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

“…Examination of the superimposed backbones showed them to be virtually indistinguishable, except for one region comprising residues 242-248, inclusive. In the wildtype enzyme, this region forms a distorted helical region immediately preceding α10, which starts at residue 249 (see Davies et al 31 for secondary structure assignments). In the cephalosporin-6-bound structure, however, these residues adopt a more canonical helical conformation such that α10 now starts earlier at residue 245 (Fig.…”

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

“…Other Ser/Lys peptidases include the penicillin binding proteins 5 D-Ala-D-Ala carboxypeptidase B (Davies et al 2001), the viral VP4 protease of the Birnaviridae family (Feldman et al 2006;Lee et al 2007), signal peptide peptidase A (Kim et al 2007), and Lactoferrin (Table 1; Anderson et al 1989). The Ser/Lys dyad active site arrangement has arisen many times during evolution.…”