Pravastatin (CS-514) is a tissue selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), a key enzyme in cholesterol biosynthesis. This compound is obtained by hydroxylation of ML-236B (mevastatin) in Streptomyces carbophilus catalyzed by a cytochrome P-450sca monooxygenase system. NADH-cytochrome P-450 reductase was purified to homogeneity from S. carbophilus as a single polypeptide chain with a molecular weight of 51 kDa, and reconstituted the hydroxylation in vitro with cytochrome P-450sca, NADH and O2. This protein contained FAD and FMN molecule. The FMN molecule was easily dissociated from the reductase, and had a Kd value of 5 x 10(-5) M. The cytochrome P-450sca monooxygenase system was present in the soluble fraction and consisted of only two components, cytochrome P-450sca and flavoprotein. Our results constitute the demonstration of a two component-type cytochrome P-450 system in a prokaryote.