A stereoselective cobalt-containing nitrile hydratase.
Nitrile hydratase from Pseudomonas putida NRRL-18668 has been purified and characterized. The purified enzyme catalyzes the hydration of 2(S)-(4'-chlorophenyl)-3-methylbutyronitrile at least fifty times faster than that of 2(R)-(4'-chlorophenyl)-3-methylbutyronitrile. This enzyme is a member of the class of nitrile hydratase that contains cobalt. Visible absorption and CD spectra suggest the cobalt exists as a non-corrin low-spin Co3+ ion in a tetragonally-distorted octahedral ligand field. Chemical reduction of the native enzyme results in a species with the EPR signature of a low-spin Co2+ complex. Like the other cobalt-containing nitrile hydratases, this enzyme is relatively stable, maintaining its activity below 35 degrees C, and it shows a broad activity optimum between pH 7.2 and 7.8. The structural genes for this enzyme have been cloned and sequenced. The deduced amino acid sequences for the alpha and beta subunits show 48-63% and 35-41% homology, respectively, to other sequenced nitrile hydratases. In particular, the cysteine residues in the alpha subunit that have been suggested to coordinate the metal ion in the iron-containing nitrile hydratases [Brennan, B. A., Cummings, J. G., Chase, D. B., Turner, I. M., Jr., & Nelson, M. J. (1996) Biochemistry 35, 10068-10077] are conserved in this enzyme, suggesting that this nitrile hydratase, like the enzyme from Rhodococcus rhodochrous J1, is a member of a newly described class of metalloenzymes with Co3+-thiolate ligation [Brennan, B. A., Alms, G., Nelson, M. J., Durney, L. T., & Scarrow, R. C. (1996) J. Am. Chem. Soc. 118, 9194-9195].
Payne
M S
MS
Central Research and Development and Agricultural Products Department, DuPont, Wilmington, Delaware 19880-0328, USA.
Wu
S
S
Fallon
R D
RD
Tudor
G
G
Stieglitz
B
B
Turner
I M
IM
Nelson
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009570:Nitriles / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism; D013696:Temperature
C106216:2-(4'-chlorophenyl)-3-methylbutyronitrile; D009570:Nitriles; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase