Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) from Streptococcus pneumoniae

Abstract

Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single D-Ala,D-Ala (D,D)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 Å resolution. PBP3 folds into an NH 2 -terminal, D,Dcarboxypeptidase-like domain, and a COOH-terminal, elongated ␤-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel ␤-sheet surrounded by ␣-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (k cat /K m ‫؍‬ 50,500 M ؊1 ⅐s ؊1 ), an event that may be linked to its central role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to "skid" on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.Bacterial division is a complex phenomenon that requires the coordination of diverse processes including chromosomal segregation, FtsZ ring-dependent membrane constriction, and cell wall synthesis at the site of septation. The latter process involves the polymerization of glycan chains and transpeptidation of pentapeptidic moieties within the structure of the peptidoglycan, a highly cross-linked mesh that is crucial for maintaining bacterial shape and providing protection from osmotic shock and lysis (1). Both reactions are catalyzed by penicillinbinding proteins (PBPs), 1 membrane-associated molecules, which can be classified as high molecular mass (hmm; often bifunctional) and low molecular mass (lmm; monofunctional) and play key roles in the bacterial life cycle. The pathogenic bacterium Streptococcus pneumoniae offers a unique opportunity for the study of the relationship between cell division and cell wall synthesis, since it carries a relatively simple set of six PBPs, compared with other well studied organisms which present much higher complexity (2). In this organism, PBP1a, -1b, and -2a catalyze both glycosyltransfer and transpeptidation; PBP2b and -2x only catalyze the latter reaction, and PBP3, the single lmm PBP in S. pneumoniae, has been shown to act as a D-Ala,D-Ala (D,D) carboxypeptidase (3).The central role of hmm PBPs in the cell growth and division processes has been recently confirmed ...

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

“…pneumoniae PBP3 is another low-molecular-mass class A enzyme that has been studied to some degree both kinetically and structurally. 38 It appears to be more active in vitro than E. coli PBP5 (the k cat /K m for N,N′-diacetyl-L-lysyl-D-alanyl-D-alanine hydrolysis is 5700 s − 1 M − 1 ), 38 but still has weak affinity for peptide substrates (the K m for N,N′-diacetyl-L-lysyl-D-alanyl-D-alanine is 19 mM; the value of this parameter for E. coli PBP5 is at least this high). 25,39 This higher activity may reflect the multiple layers of peptidoglycan in Gram-positive bacteria.…”

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

“…33 The following peptides were purchased commercially and used as received: 10 (NeoMPS), 3 and 13 (Sigma-Aldrich), and 14−19 (New England Peptide). Peptides 7−9 were prepared by standard peptide methodology; the details are given in the Supporting Information.…”

“…33 In the DD-peptidase domain, these differences appear in loops surrounding the active site. In particular, the "Ω-like" loop in SpPBP3, comprising residues 156−181, is much larger than the corresponding loop, spanning residues 147−158, in EcPBP5.…”

“…Morlot et al 33 suggested that these differences may distinguish enzymes such as EcPBP5 that interact with peptidoglycan-bearing diaminopimelic acid as the third residue of the stem peptide 22 from those, such as SpPBP3, that bear lysine as this residue. 23 The extended structure in the latter case might indicate that the terminus of the stem peptide may directly interact with the protein, although no crystal structure showing this has been published.…”

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.…”