Open AccessMutational analysis of the zinc metalloprotease EmpA of Vibrio anguillarum
Author(s) -
Yang Hui,
Chen Jixiang,
Yang Guanpin,
Zhang XiaoHua,
Li Yun
Publication year - 2007
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2006.00533.x
Subject(s) - empa , metalloproteinase , vibrio anguillarum , zinc , microbiology and biotechnology , vibrio , chemistry , biology , matrix metalloproteinase , bacteria , biochemistry , genetics , mineralogy , electron microprobe , organic chemistry
Abstract 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 His 346 , His 350 , Glu 370 , Glu 347 , His 429 , Tyr 361 and Asp 417 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 × 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 His 346 , His 350 , Glu 370 and Glu 347 almost completely lost their proteolytic activity and cytotoxicity, pointing towards the essential roles played by these residues. In contrast, those mutated at Tyr 361 , His 429 and Asp 417 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 .