Purification and characterization of cytochrome P‐450sca from Streptomyces carbophilus

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Abstract
Prayastatin 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 μM and 229 μM, 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‐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.

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