Nucleotide sequence of the Pseudomonas putida cytochrome P-450(cam) gene and its expression in Escherichia coli

Cytochrome P-450(cam) catalyzes the sterospecific methylene hydroxylation of camphor to form 5-exo-hydroxycamphor and is encoded by the camC gene on the CAM plasmid of Pseudomonas putida, ATCC 17453. The cytochrome P-450(cam) structural gene has been cloned by mutant complementation in P. putida. We report the complete nucleotide sequence of the camC gene along with 155 base pairs of 5and 175 base pairs of 3' flanking sequence. Upon comparison of the amino acid sequence derived from the gene sequence to the one obtained from the purified protein, five differences were found. The most significant was the addition of a Trp and a Thr residue between Val-54 and Arg-55, thereby increasing the amino acid numbering scheme by 2 after Val-54, bringing the total number of amino acids to 414. Other differences were: Gln-274 → Glu-276, Ser-359 → His-361, and Asn-405 → Asp-407. N-terminal amino acid sequence analysis of the cloned cytochrome P-450(cam) enzyme expressed in Escherichia coli under the lac promoter showed a faithful translation to the hemoprotein, with the N-terminal Met removed by processing as found in P. putida. Purification to homogeneity of the cloned protein was accomplished by the method used for the CAM plasmid-encoded enzyme of P. putida. The G + C content of the camC gene was found to be 59.0%, caused by a preferred usage of G and C terminated codons. The gene encoding putidaredoxin reductase, camA, was located 22 nucleotides down-stream from the cytochrome P-450(cam) gene. The camA gene initiated with a novel GUG codon, the first such initiator documented in Pseudomonas.