Crystal Structure of Wild-type Penicillin-binding Protein 5 from Escherichia coli

Abstract

1 Cross-linking of the peptide chains confers rigidity to the peptidoglycan and viability to the bacterial cell. The related carboxypeptidases, which hydrolyze the C-terminal D-Ala moiety from the peptide chain, may modulate the degree of cross-linking.In E. coli at least 10 PBPs have been identified. These enzymes fall into two categories: the high molecular mass PBPs, which are essential for cell viability and catalyze transpeptidase and sometimes transglycosylase activity, and the low molecular mass PBPs, which are non-essential and catalyze D,D-carboxypeptidase (CPase) and sometimes D,D-endopeptidase activity (1). Regardless of the type of PBP, all of these enzymes react with peptide substrates and ␤-lactam antibiotics by a similar mechanism. The initial step in the reaction of PBPs with their peptide substrates is a nucleophilic attack of the D-Ala-D-Ala peptide bond by a conserved serine residue, leading to acylation of the serine hydroxyl side chain and the concomitant release of the C-terminal D-Ala. In the subsequent deacylation step, the acyl-enzyme complex can react with either an amino group (from m-DAP) of another peptide to form a cross-link (transpeptidation) or it can react with water to release the peptide (carboxypeptidation). Penicillin and other ␤-lactam antibiotics inactivate these enzymes by mimicking the structure of the D-Ala-D-Ala C terminus of the peptide chain (2, 3) and reacting with the same serine nucleophile to form an analogous acyl-enzyme complex (4). Unlike the complex formed with peptide substrates, however, the ␤-lactam-PBP complex is long-lived and renders the enzyme inactive.PBPs and other penicillin-interacting enzymes (e.g. class A ␤-lactamases) are characterized by a set of conserved motifs that are clustered in their respective active sites (5). These motifs include the Ser-X-X-Lys (SXXK) tetrad that contains the serine nucleophile, the Ser-X-Asn (SXN) triad, and the LysThr(Ser)-Gly (KTG) triad. In all serine-based PBPs and ␤-lactamases of known structure, these motifs adopt a strikingly similar conformation to the extent that the active site of one PBP or ␤-lactamase can look very much like another. In addition to these three motifs, class A ␤-lactamases have a fourth motif, Glu-X-X-X-Asn, present on the so-called ⍀ loop, that is responsible for the extremely high rates of deacylation of the * This work was supported by National Institutes of Health Grants AI36901 (to R. A. N.) and GM66861 (to C. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence may be addressed: Dept.

Peptidoglycan Hydrolases of Escherichia coli

“…3f) revealed two distinct, roughly perpendicular domains (46,189). The active site located in domain 1 showed high structural similarity with those of other PBPs and ␤-lactamases.…”

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

“…22 In vivo, PBP5 is attached to the E. coli inner membrane by a C-terminal α-helix, 23 removal of which affords the solubilized protein used for kinetics studies and crystal structure determination. 24 The solubilized construct is inhibited by β-lactams and also catalyzes the hydrolysis and aminolysis of small D-alanyl peptides and esters, 25 although much less efficiently than the R39 enzyme. The general organization of catalytic functional groups in PBP5 is very similar to that in the R39 DD-peptidase, and both resemble that of a class A β-lactamase; 16 neither, however, contains an analogue of Glu166 of the β-lactamase-the β-lactam deacylation catalyst.…”

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

“…The crystal structures of several high-and low-molecularweight PBPs from various organisms have been determined (33,89,110,112,121,133,137,167,168). Acyl-PBP complexes formed with antibiotics have generated structural insight into PBP-substrate binding and provided understanding of the development of antibiotic resistance as well as a possible starting point for the development of novel antibiotics.…”