Abstract
In spite of the enormous biological and clinical significance of penicillin-binding protein (PBP)/β-lactamase (βL), few of their many homologs (PBP)/βLs homologs) have been studied crystallographically, and have known functions. Herein, X-ray crystallographic study of a PBP/βL homolog (Rp46) from Ruegeria pomeroyi is described. Multiple sequence alignments indicate that Rp46 has a conserved serine residue within the S70-X-X-K73 motif (Motif I), acting as the catalytic nucleophile. Moreover, an invariant tyrosine residue (Tyr185) and a Trp365-X-Gly motif (Motif III) were also identified. The recombinant Rp46 protein was expressed in Escherichia coli and purified to homogeneity judging from the SDS-PAGE analysis. Rp46 was crystallized using a solution consisting of 20% (w/v) PEG 3000, 0.1 M Tris-HCl, pH 7.0, 0.2 M calcium acetate, and the X-ray diffraction data were collected to a resolution of 1.90 Å with an Rmerge of 7.4%. The crystals of Rp46 belong to the space group I422, with unit cell parameters a = b = 141.26 Å, and c = 119.75. The structure determination and biochemical characterization are in progress. (Synopsis: A penicillin-binding protein/β-lactamase homolog (Rp46) from Ruegeria pomeroyi was identified and crystallized in the space group I4, and the diffraction data were collected to a resolution of 1.90 Å.)
Highlights
Bacterial penicillin-binding proteins (PBPs) and β-lactamases form a large family of serine proteases [1,2]
The bacterial PBP/βL family is composed of three classes including low-molecular-weight penicillin-binding proteins, high-molecular-weight penicillin-binding proteins
We report the characterization, crystallization, and preliminary crystallographic analysis of a penicillin-binding protein/β-lactamase homolog (Rp46) from
Summary
Bacterial penicillin-binding proteins (PBPs) and β-lactamases (βLs) form a large family of serine proteases [1,2] These enzymes are characterized by phylogenetic analysis, primary sequences, functional properties, and structural similarities. Several bacterial enzymes with similar active sites to PBP/βL proteins, namely PBP-βL homologs, were identified from bacterial organisms including Klebsiella pneumoniae [8], Burkholderia gladioli [9], Arthrobacter nitroguajacolicus [10], Staphylococcus aureus [11], Marinobacter lipolyticus [12], Caulobacter crescentus [13], and Pseudomonas fluorescens [14]. The crystal structure of Rp46 will provide molecular understanding on the catalytic mechanism and structural features of PBP/βL homologs
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