Abstract
We have reported the localization on yeast microsomes for a modified P450c27 (mic-P450c27) that contains the microsomal targeting signal of bovine P450c17 in front of the mature form of rat mitochondrial P450c27 (Sakaki, T., Akiyoshi-Shibata, M., Yabusaki, Y., and Ohkawa, H. (1992) J. Biol. Chem. 267, 16497-16502). In this study, we found that mic-P450c27 could be reduced by NADPH in the yeast microsomes without supplement of its physiological redox partners, adrenodoxin and NADPH-adrenodoxin reductase. In order to elucidate the direct electron transfer from NADPH-P450 reductase to mic-P450c27, we carried out simultaneous expression of mic-P450c27 and yeast P450 reductase. The reduction rate of mic-P450c27 was increased by overproduction of yeast P450 reductase, roughly in proportion to the reductase content in the microsomes. In addition, we constructed a fused enzyme between mic-P450c27 and yeast P450 reductase. The reduction rate of heme iron in the fused enzyme was too rapid to be measured. These recombinant yeast microsomes showed a notable 27-hydroxylation activity toward 5beta-cholestane-3alpha,7alpha, 12alpha-triol in the absence of adrenodoxin and adrenodoxin reductase. Finally, we purified mic-P450c27 from the recombinant yeast microsomes and reconstituted the hydroxylation system in liposomal membranes using the purified mic-P450c27 and yeast NADPH-P450 reductase. Mic-P450c27 was reduced by NADPH and showed its monooxygenase activity on the reconstituted system. Therefore, yeast NADPH-P450 reductase alone was found to transfer two electrons from NADPH to mic-P450c27. These results clearly show that mic-P450c27 not only localizes on the microsomes but also functions as a microsomal cytochrome P450 that accepts electrons from NADPH-P450 reductase.
Highlights
Cytochrome P450 represents a large group of structurally related hemoproteins that catalyze the monooxygenase reactions of a wide variety of both endogenous and exogenous compounds
P450c27 was first purified from rat liver mitochondria by Okuda et al [24], who demonstrated the requirement of ADX and adrenodoxin reductase (ADR) for its activity in a reconstituted system
Jenkins and Waterman [5] reported the electron transfer from NADPH to heterologously expressed microsomal P450c17 through E. coli flavodoxin and flavodoxin reductase system, the 17␣hydroxylation activity supported by the E. coli redox system was 10-fold less efficient as compared with purified rat P450 reductase
Summary
Materials—DNA modifying enzymes, restriction enzymes, and DNA sequencing kit were purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). 5-[3H]Cholestane-3␣,7␣,12␣-triol (THC) and 125I-labeled Protein A were from Amersham Japan (Tokyo, Japan). Purification of Mic-P450c27 and Reconstitution of the Hydroxylation System in Liposomal Membranes—Microsomal fraction was prepared from the recombinant yeast strains as described previously [9]. The reaction mixture contained each of the microsomal fractions prepared from the recombinant yeast strains (0.025– 0.1 M P450), 100 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, and [3H]THC (20, 50, 100, or 200 M). The cellular THC 27-hydroxylation activity of the recombinant yeast was measured by incubation of the cell culture with 10 M [3H]THC at 30 °C as described previously [8]. Other Methods—Cytochrome c reductase activity derived from NADPH-P450 reductase or the fused enzyme in the microsomal fractions was measured as described previously [14, 18]. Protein concentration was determined by the method of Lowry et al [19], using bovine serum albumin as a standard
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