Cytochrome P460 genes from the methanotroph Methylococcus capsulatus bath

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They h ave been isolated from the ammonia-oxidizing bacterium Nitrosomonas europae a (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231-244, 197 2) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879-5887, 1994). A degenerate oligonucleot ide probe was synthesized based on the N-terminal amino acid sequence of cy tochrome P460 and used to identify a DNA fragment from M. capsulatus Bath t hat contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (cyp) encoding cytoch rome P460. The products of the first two ORFs have no apparent similarity t o any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are p resent in the two proteins. Both cytochromes have a C-terminal, c-heme bind ing motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457-460, 1997). Gene probing using cyp indicated that a cyt ochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis par vus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Met hylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immnnoblo t analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected eit her by expression of the two different methane monooxygenases or by additio n of ammonia to the culture medium.