Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus. ML-236B (compactin) induces a cytochrome P-450sca in Streptomyces carbophilus that hydroxylates ML-236B to pravastatin sodium (CS-514), a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase.
Pravastatin sodium (CS-514) is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis. This compound is obtained by microbial hydroxylation of sodium ML-236B (compactin) carboxylate. The soluble cytochrome P-450 was induced by sodium ML-236B carboxylate in Streptomyces carbophilus of Actinomycetes as detected in its cell-free extract. This cytochrome P-450 was designated as cytochrome P-450sca after its origin. Cytochrome P-450sca was purified by successive chromatography on anion-exchange, gel filtration and hydroxyapatite columns. On hydroxyapatite cytochrome P-450sca was further separated into minor and major peaks, designated cytochrome P-450sca-1 and cytochrome P-450sca-2, respectively. Each peak yielded a single band on sodium dodecyl sulfate/polyacrylamide gels with molecular masses of 46 +/- 1 kDa. The activity hydroxylating sodium ML-236B carboxylate to pravastatin sodium was reconstituted in the presence of an electron transport system, an NADPH-generating system and oxygen. The Ks values of the cytochromes P-450sca-1 and P-450sca-2 for sodium ML-236B carboxylate were 179 microM and 229 microM, respectively. The CO versus reduced difference spectra of both cytochromes P-450 showed an absorption maximum at 448.5 nm. Their substrate difference spectra with sodium ML-236B carboxylate showed an absorption maximum at 386 nm. Amino acid analysis indicated that cytochrome P-450sca-1 and P-450sca-2 contained 46% and 47% hydrophobic residues, respectively. On Western blotting, cytochromes P-450sca-1 and P-450sca-2 were immunologically identical.
Matsuoka
T
T
Fermentation Research Laboratories, Sankyo Co. Ltd, Tokyo, Japan.
Miyakoshi
S
S
Tanzawa
K
K
Nakahara
K
K
Hosobuchi
M
M
Serizawa
N
N
D000596:Amino Acids / Q000032:analysis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006538:Heptanoic Acids / Q000378:metabolism*; D006900:Hydroxylation; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008148:Lovastatin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D009281:Naphthalenes / Q000378:metabolism*; D017035:Pravastatin; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*
D000596:Amino Acids; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D009281:Naphthalenes; C012258:mevastatin; D003577:Cytochrome P-450 Enzyme System; D008148:Lovastatin; D017035:Pravastatin