Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli.
The nitrile hydratase (NHase) of Rhodococcus species N-774, which is composed of two subunits, alpha and beta, catalyzes the hydration of various nitrile compounds to the corresponding amides. The amino acid sequences of the NH2 termini and the fragments obtained by digesting each of the two subunits with lysyl endopeptidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kb SphI fragment which contained DNA sequences hybridizing to several of the probes was cloned in pBR322 in Escherichia coli. The nucleotide sequences together with the determined amino acid sequences indicated that the alpha and beta subunits of NHase consisted of 207 amino acids (Mr, 22918) and 212 amino acids (Mr, 23428), respectively. The open reading frame for the alpha subunit includes that for the beta subunit with a short interval of only 26 base pairs; the two genes are probably translated in a polycistronic manner. Although large amounts of the alpha- and beta-subunit proteins were produced as insoluble forms in E. coli when the cloned genes were placed under the control of the lac promoter, no enzymatic activity was detected. The activity of the enzyme was restored, to some extent, by solubilization of the proteins with 8 M urea and subsequent dialysis for refolding at pH 10 in the presence of Fe2+ and pyrroloquinoline quinone.
Ikehata
O
O
Department of Agricultural Chemistry, University of Tokyo, Japan.
Nishiyama
M
M
Horinouchi
S
S
Beppu
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000096:biosynthesis; D010449:Peptide Mapping; D010957:Plasmids; D012240:Rhodococcus / Q000201:enzymology*
D015342:DNA Probes; D009841:Oligonucleotides; D006836:Hydro-Lyases; C048831:nitrile hydratase