Cloning of genes coding for the three subunits of thiocyanate hydrolase of Thiobacillus thioparus THI 115 and their evolutionary relationships to nitrile hydratase

Abstract

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.

FIG. 1

Amino-terminal sequences of thiocyanate hydrolase subunits and degenerate primers for PCR. The peptide sequences were chemically determined from the purified enzyme of T. thioparus THI 115. For the γ subunit, the italicized sequence (Lys¹²-Pro¹³-Ala¹⁴) was not concordant with the His-Asp-His sequence that was deduced from the nucleotide sequence. The regions used for α, β, and γ primer sequences were chosen to minimize the degeneracy.

FIG. 2

Western blotting of the thiocyanate hydrolase subunits produced in E. coli clones carrying thiocyanate hydrolase genes. Positive E. coli clones SCN -1, -2, -3, and -5 in addition to a negative clone (N) that did not carry thiocyanate hydrolase genes were grown in LB medium with ampicillin. The bacterial cells were collected and denatured in Laemmli’s buffer. A partially purified enzyme sample prepared from T. thioparus THI 115 grown in TC10 medium was used as a positive control (T). Reported sizes for the three subunits (11) were 19 (α), 23 (β), and 32 kDa (γ).

FIG. 3

Schematic representation of PCR products and gene organization of scnA, scnB, and scnC. pUC118/T3 is a plasmid with a 5-kb EcoRI fragment derived from cosmid clone SCN-2. The annealing sites for universal (P1) and reverse (P3) primers of the pUC118 vector were located 16 bp downstream and 10 bp upstream, respectively, of the multicloning site. The α, β, and γ primers are shown in Fig. 1. Thick bars represent the sizes of the PCR products.

FIG. 4

Nucleotide sequence of scnB-scnA-scnC series of genes. The deduced amino acid sequences for β (scnB), α (scnA), and γ (scnC) subunits are shown under each coding sequence. Asterisks indicate termination codons. The consensus Shine-Dalgarno sequences are underlined. Possible −35 and −10 regions are indicated by broken lines above the sequences. An EcoRI site was present at nucleotide 1606.

FIG. 5

Alignment of amino acid sequences of each subunit of thiocyanate hydrolase and nitrile hydratases of different bacteria. Conserved amino acids among three (β- and α-subunits) or six (γ-subunit) sequences were boxed. Gaps (hyphens) were introduced to maximize the homology. The residue numbers of the carboxyl termini are indicated by -COOH. The accession numbers for each nitrile hydratase sequence are as follows: P. chlororaphis α and β subunits, D90216 (18); R. erythropolis α and β subunits, D14454 (7): Brevibacterium sp. α subunit, M60264 (17); R. rhodochrous α subunits of high- and low-molecular-mass enzymes (nhhA and nhlA), X64359 and X64360, respectively (14); P. putida α subunit, U89363 (20); Klebsiella sp. α subunit, E08305 (3).

FIG. 6

Evolutionary relationships among the γ subunit of thiocyanate hydrolase and the α subunits of nitrile hydratases of different bacteria. The phylogenetic tree was made by the unweighted pair group method by arithmetic averaging with the computer program GENETYX. The bar indicates the genetic distance for 0.1 amino acid substitution/site.

FIG. 7

Structural homology and functional similarity between thiocyanate hydrolase and nitrile hydratase. Homologous subunits (boxes) are aligned with bars indicating positions of identical amino acids. The P. chlororaphis enzyme was chosen as the representative for nitrile hydratase. The lengths of the boxes and the spaces between bars are proportional to the actual numbers of amino acid residues. The array of the thiocyanate hydrolase subunits was drawn to fit the order of scn genes, but the gene organization of nitrile hydratase subunits varies among different bacteria. In the case of P. chlororaphis, the gene coding for the β subunit is downstream of that coding for the α subunit. Thiocyanate hydrolase converts thiocyanate (SCN⁻) to ammonia (NH₃) and COS (SCO) through several intermediates; two intermediates assumed by Katayama et al. (11) are omitted in the figure for simpler comparison. Nitrile hydratase converts nitrile (RCN) to amide (RCONH₂), which is hydrolyzed by a different enzyme, amidase, to produce carboxylic acid (RCOOH) and ammonia (2).