Purification and biochemical characterization of a novel glutamyl endopeptidase secreted by a clinical isolate of Staphylococcus aureus

Abstract

Staphylococcus aureus (S. aureus) is a ubiquitous Gram-positive pathogenic bacterium responsible for a majority of skin infections and toxic shock syndromes. In this study, a 34-kDa glutamate-specific serine protease (named VSPase) secreted by a clinical isolate of S. aureus sp. strain C-66 was purified and characterized, and VSPase-encoding gene was also cloned by PCR. VSPase enzyme purified from culture supernatant and its recombinant enzyme expressed in E. coli exhibited a proteolytic activity over a broad range of pH (6.0-8.5) and showed an optimal activity at 45°C. The enzyme activity was completely inhibited by DFP. The N-terminal sequence of native VSPase showed that the enzyme was produced as a form of zymogen and activated to a functional enzyme by losing its N-terminal 68 amino acid residues. VSPase specifically cleaved peptide bonds at the carboxyl sides of glutamate residues in a protein substrate such as prothrombin and exhibited its amidolytic activity towards a chromogenic substrate, Z-Phe-Leu-Glu-pNA (L-2135). The Km, kcat and kcat/Km values for VSPase were estimated to be 1.48±0.156 mM, 44.4±2.66/sec and 30/mM/sec, respectively, when L-2135 was used as a substrate. It was revealed by site-directed mutagenesis that one of substitution mutations resulted in His119, Asp 161 and Ser237 residues of VSPase abolished the enzyme activity dramatically, suggesting that the three amino acid residues may compose a catalytic triad in VSPase as in typical serine proteases. Taken together, the results obtained by the present study demonstrate that VSPase is a typical glutamate-specific serine endopeptidase.