title: Characterization of tryptophanase from Vibrio cholerae.
abstract: Tryptophanase (Trpase) is a pyridoxal phosphate (PLP)-dependent enzyme responsible for the production of indole, an important intra- and interspecies signaling molecule in bacteria. In this study, the tnaA gene of Vibrio cholerae coding for VcTrpase was cloned into the pET-20b(+) vector and expressed in Escherichia coli BL21(DE3) tn5:tnaA. Using Ni(2+)-nitrilotriacetic acid (NTA) chromatography, VcTrpase was purified, and it possessed a molecular mass of ∼49 kDa with specific absorption peaks at 330 and 435 nm and a specific activity of 3 U/mg protein. The VcTrpase had an 80 % homology to the Trpase of Haemophilus influenzae and E. coli, but only around 50 % identity to the Trpase of Proteus vulgaris and Porphyromonas gingivalis. The optimum conditions for the enzyme were at pH 9.0 and 45 °C. Recombinant VcTrpase exhibited analogous kinetic reactivity to the EcTrpase with K m and k cat values of 0.612 × 10(-3) M and 5.252 s(-1), respectively. The enzyme catalyzed S-methyl-L-cysteine and S-benzyl-L-cysteine degradation, but not L-phenylalanine and L-serine. Using a site-directed mutagenesis technique, eight residues (Thr52, Tyr74, Arg103, Asp137, Arg230, Lys269, Lys270, and His463) were conserved for maintaining enzyme catalysis. All amino acid substitutions at these sites either eliminated or remarkably diminished Trpase activity. These sites are thus potential targets for the design of drugs to control the V. cholerae Trpase and to further investigate its functions.
authors.affiliation: Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla, Hat Yai, Thailand.
authors.forename: Taiyeebah
authors.initials: T
authors.lastname: Nuidate
authors.forename: Natta
authors.initials: N
authors.lastname: Tansila
authors.forename: Piraporn
authors.initials: P
authors.lastname: Chomchuen
authors.forename: Phattiphong
authors.initials: P
authors.lastname: Phattaranit
authors.forename: Supachok
authors.initials: S
authors.lastname: Eangchuan
authors.forename: Varaporn
authors.initials: V
authors.lastname: Vuddhakul
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D002384:Catalysis; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D014368:Tryptophanase / Q000235:genetics* / Q000378:metabolism; D014734:Vibrio cholerae / Q000201:enzymology* / Q000472:pathogenicity
chemical_list: D014368:Tryptophanase