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Mutational analysis of the zinc metalloprotease EmpA of Vibrio anguillarum

Yang, Hui, Chen, Jixiang, Yang, Guanpin, Zhang, Xiao-Hua, Li, Yun
FEMS microbiology letters 2007 v.267 no.1 pp. 56-63
Escherichia coli, Vibrio anguillarum, active sites, cytotoxicity, enzyme activity, flounder, genes, metalloproteinases, mutants, pathogenesis, pathogens, proteolysis, site-directed mutagenesis, virulence, zinc
The extracellular zinc metalloprotease, EmpA, is a putative virulence factor involved in pathogenicity of the fish pathogen Vibrio anguillarum. The 611-amino acid precursor of this enzyme is encoded by the empA gene. The residues His³⁴⁶, His³⁵⁰, Glu³⁷⁰, Glu³⁴⁷, His⁴²⁹, Tyr³⁶¹ and Asp⁴¹⁷ are highly conserved and putatively function together at the active site of the enzyme. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli strain BL21(DE3) as a 6 x His tagged protein (r-EmpA). All the conserved residues of EmpA mentioned above were individually mutated by site-directed mutagenesis and the mutants were also expressed (m-r-EmpAs). r-EmpA and m-r-EmpAs were purified, and assayed for their proteolytic activities with azocasein as the substrate and cytotoxicities on a flounder gill cell line. m-r-EmpAs that had been mutated at His³⁴⁶, His³⁵⁰, Glu³⁷⁰ and Glu³⁴⁷ almost completely lost their proteolytic activity and cytotoxicity, pointing towards the essential roles played by these residues. In contrast, those mutated at Tyr³⁶¹, His⁴²⁹ and Asp⁴¹⁷ still retained a partial proteolytic activity and cytotoxicity. Our results indicate that these conserved residues play important roles in enzymatic activity and that the proteolytic activity of the enzyme is involved in the pathogenesis of V. anguillarum.