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

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-450,,, after its origin. Cytochrome P-450,,, was purified by successive chromatography on anion-exchange, gel filtration and hydroxyapatite columns. On hydroxyapatite cytochrome P-450,,, was further separated into minor and major peaks, designated cytochrome P-450,,,_1 and cytochrome P-450,,,-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-450,,,-1 and P-450,,,.2 for sodium ML-236B carboxylate were 179 pM and 229 pM, 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-450,,,-1 and P-450,,,-2 contained 46% and 47% hydrophobic residues, respectively. On Western blotting, cytochromes P-45OS,,-and P-450,,,-2 were immunologically identical.Pravastatin sodium (the generic name for CS-514, hereafter referred to as pravastatin) [l -41 is obtained by hydroxylation of sodium ML-236B carboxylate [ 5 , 6 ] at the 68 position (Fig.

Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase

“…Rhodococcus cells resuspended in 50 mM phosphate buffer (pH 7) containing 0.3 mM phenylmethylsulfonyl fluoride were broken by three passages through a French pressure cell (SLM-Aminco) at 25,000 lb/in 2 . The supernatants obtained after centrifugation at 12,000 ϫ g at 4ЊC (15 min) were used for recording difference spectra in the presence of CO or EPTC (48). For the A 452 peak, an extinction coefficient of 91 mM Ϫ1 cm Ϫ1 was used to quantify cytochrome P-450 in crude extracts (49).…”

Aerobic Double Dehydrogenative Cross Coupling between Cyclic Saturated Ketones and Simple Arenes

“…A number of MCMOs play important roles as biocatalysts in commercially important biotechnological applications. This includes the hydroxylase from Streptomyces carbophilus SANK 62585 employed for the bioconversion of mevastatin into the widely used hypocholestemic drug pravastatin (Matsuoka et al 1989;Serizawa and Matsuoka 1991) and a number of luciferases exploited both directly and indirectly in various applications due to their characteristic bioluminescent properties (Hastings et al 1985;Meighen 1991).…”

Directed Evolution of the Actinomycete Cytochrome P450 MoxA (CYP105) for Enhanced Activity

“…For example, P450sca from Streptomyces carbophilus has successfully been applied to the bioconversion of ML236-B (compactin) to produce pravastatin. 9,10) Recently, we identified P450 MoxA (P450moxA), a class I P450, from the actinomycete Nonomuraea recticatena NBRC 14525, which catalyzes the hydroxylation of fatty acids, steroids, and aromatic compounds. [11][12][13] Many of these reactions were human P450-like, indicating that the P450moxA-catalyzed transformation system would be applicable in the preparation of human or actinomycete-specific P450 metabolites from a diverse set of pharmaceuticals in the early stages of drug development.…”