Purification and characterization of a cytochrome P-450 from pravastatin-producing Streptomyces sp. Y-110

Abstract

Streptomyces sp. Y-110 cytochrome P-450, induced by the addition of compactin-Na into the culture medium, was purified from the cell extract to apparent homogeniety, mainly by DEAE-Sepharose, hydroxyapatite, and Mono Q column chromatography. The specific activity of purified enzyme on its substrate, compactin-Na, was determined to be 15 nmol of pravastatin per mg protein. The molecular mass of this enzyme on SDS-PAGE was 37±0.5 kDa, pI was 4.5, and its CO difference spectrum showed maximum absorption peaks at 452 and 550 nm, respectively. The N-terminal amino acid sequence was determined to be Met>Thr>Cys>Thr>Pro> 1.028 μM · min-1. The maximum substrate concentration (Ks) for reaction was 270 μM and thus 1/[Ks] was 3.7 μM. These physicochemical characteristics and kinetic behavior of this enzyme were compared and shown to be different from those of Streptomyces cytochrome P-450 enzymes reported, suggesting that this enzyme may be an additional member of the Streptomyces cytochrome P-450 family.