Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor.
Pravastatin, a drug for treating hypercholesterolemia, is produced by hydroxylation of ML-236B-Na in Streptomyces carbophilus (Sc) catalyzed by the cytochrome P-450sca (CytP-450sca) monooxygenase system. The gene (cytP-450sca-2) encoding CytP-450sca-2 was cloned from Sc. The gene had an open reading frame of 1233 bp, encoding a 410-amino-acid protein. The partial sequencing of the purified CytP-450sca-2 revealed that the N-terminal Met had been removed. CytP-450sca-2 contained the heme-binding HR2 region characteristic of all CytP-450, as well as the putative oxygen-binding site proposed in CytP-450cam from Pseudomonas putida. ML-236B.Na enhanced transcription of cytP-450sca-2, suggesting that substrate induction in Sc is transcriptionally regulated. S. lividans (Sl) transfected with cytP-450sca-2 converted ML-236B.Na to pravastatin, indicating the cloned gene to be functional in Sl.
Watanabe
I
I
Biomedical Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan.
Nara
F
F
Serizawa
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017035:Pravastatin / Q000378:metabolism*; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D004791:Enzyme Inhibitors; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D009838:Oligodeoxyribonucleotides; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C069505:mevastatin hydroxylase; D017035:Pravastatin