New Cytochrome P-450 Family: Cytochrome P-4501in (CYP111) from Pseudomonas incognita

Cytochrome P-4501in catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source. By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-4501in gene (linC) was isolated. An open reading frame of 406 amino acids has been identified as that of P-4501in on the basis of amino acid sequence data from peptides of the native protein. Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter. The G+C content oflinC was found to be 55% overall and 58% in the third codon position. An optimized amino acid sequence alignment of P-4501in with cytochrome P-450cam shows that the two enzymes have only 25% identity. P-4501in was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an 02-binding pocket (T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195:687-700, 1987). The low amino acid sequence identity between P-4501in and all other P-450 sequences has shown that P45O1in is the first member of the CYPIll P-450 gene family.