Abstract
The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified. This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue.
Highlights
The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly
The bacterial cell wall peptidoglycan assembly machinery comprises, among other components, multimodular membrane enzymes that catalyze the polymerization of lipid-transported disaccharide-pentapeptide
The second step involves the breakdown of the peptidyl enzyme and the formation of a new peptide bond between the carbonyl of the D-alanyl moiety and the -amino group of another peptide unit acting as amino acceptor
Summary
Crystallization and Data Collection—The K15 enzyme is synthesized in the form of a 291-amino acid precursor possessing a cleavable 29amino acid signal peptide. The crystals belong to the orthorhombic space group P212121, with cell dimensions of a ϭ 46.59 Å, b ϭ 54.53 Å, and c ϭ 108.70 Å They contain 51% solvent and one molecule/asymmetric unit (21). The calculation of the MHP along the peptide sequence was based upon the concept introduced by Furet et al (30) and Fauchere et al (31) for a small molecule. This approach was extended to a three-dimensional representation of the molecular envelope with isopotential contour lines around an 18-residue helical peptide (32). A plane was moved every 3 Å, and the MHP was computed for all points in the plane defined by a 3-Å side grid
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