Purification and characterization of cytochrome <i>P</i>‐450_sca from <i>Streptomyces carbophilus</i>

Abstract

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.