Cloning of genes coding for the three subunits of thiocyanate hydrolase of Thiobacillus thioparus THI 115 and their evolutionary relationships to nitrile hydratase.
Thiocyanate hydrolase is a newly found enzyme from Thiobacillus thioparus THI 115 that converts thiocyanate to carbonyl sulfide and ammonia (Y. Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa, and H. Kuraishi, J. Biol. Chem. 267:9170-9175, 1992). We have cloned and sequenced the scn genes that encode the three subunits of the enzyme. The scnB, scnA, and scnC genes, arrayed in this order, contained open reading frames encoding sequences of 157, 126, and 243 amino acid residues, respectively, for the beta, alpha, and gamma subunits, respectively. Each open reading frame was preceded by a typical Shine-Dalgarno sequence. The deduced amino-terminal peptide sequences for the three subunits were in fair agreement with the chemically determined sequences. The protein molecular mass calculated for each subunit was compatible with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From a computer analysis, thiocyanate hydrolase showed significant homologies to bacterial nitrile hydratases known to convert nitrile to the corresponding amide, which is further hydrolyzed by amidase to form acid and ammonia. The two enzymes were homologous over regions corresponding to almost the entire coding regions of the genes: the beta and alpha subunits of thiocyanate hydrolase were homologous to the amino- and carboxyl-terminal halves of the beta subunit of nitrile hydratase, and the gamma subunit of thiocyanate hydrolase was homologous to the alpha subunit of nitrile hydratase. Comparisons of the catalytic properties of the two homologous enzymes support the model for the reaction steps of thiocyanate hydrolase that was previously presented on the basis of biochemical analyses.
Katayama
Y
Y
Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan. katayama@cc.tuat.ac.jp
Matsushita
Y
Y
Kaneko
M
M
Kondo
M
M
Mizuno
T
T
Nyunoya
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D005798:Genes, Bacterial / Q000235:genetics*; D015698:Genomic Library; D006836:Hydro-Lyases / Q000235:genetics; D006867:Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013855:Thiobacillus / Q000201:enzymology* / Q000235:genetics
D006867:Hydrolases; C074369:thiocyanate hydrolase; D006836:Hydro-Lyases; C048831:nitrile hydratase