CYP51 has been recognized as a unique CYP family that consists of one isolated molecular species, a sterol 14-demethylase essential for sterol biosynthesis. However, another CYP51 gene classified as the CYP51H subfamily has been identified in higher plants, in addition to a sterol 14-demethylase gene, CYP51G1. To shed light on the function of this "second CYP51", oat CYP51H10 was introduced into the β-amyrin-producing yeast cells, and the effect of the expressed CYP51H10 on β-amyrin metabolism in the host cells was examined. In the CYP51H10-introduced cells, β-amyrin was converted to a metabolite with 12,13-epoxy and one additional hydroxyl group. Since the 12,13-epoxy group introduced into β-amyrin ring is an essential structure of avenacin A-1, a triterpene glycoside produced in oat from β-amyrin, the present findings indicate the contribution of CYP51H10 to avenacin A-1 biosynthesis from β-amyrin. This is the first study showing a second function of the CYP51 family.Key words CYP51; P450; sterol 14-demethylation; β-amyrin metabolism; new function P450 has diversified into huge numbers of different monooxygenases through evolution. These variants have been adapted to diversified and specific metabolisms occurring in individual biological species.1-7) P450 classified as the CYP51 family is considered to be unique. It is distributed widely in eukaryotes with conserved function as a sterol 14-demethylase, [8][9][10][11][12][13] and is considered to exist as an isolated molecular species, CYP51A1 in animals, CYP51B1 in bacteria, CYP51E1 in protozoa, CYP51D1 in slime molds, and one of the CYP51F subfamily members in fungi (Nelson DR: http:// drnelson.uthsc.edu/cytochromeP450.html). In higher plants, however, another CYP51 genes classified as the CYP51H subfamily have been identified in addition to the conserved sterol 14-demethylase gene, CYP51G1 (Nelson DR: http://drnelson. uthsc.edu/biblioD.html). This is an interesting finding that suggests that diversification occurred in the extremely wellconserved CYP51.Recently, it was reported that Sad2, one of the essential genes for avenacin A-1 synthesis in oat, Avena strigosa, is synonymous with CYP51H10.14) Avenacin A-1 is a triterpene glycoside synthesized from β-amyrin, and the Sad2 deletion mutant of A. strigosa was found to accumulate β-amyrin in roots.14) This finding suggests that CYP51H10 is an essential enzyme for the bioconversion of β-amyrin to avenacin A-1. To obtain more direct evidence for this genetically assumed role of CYP51H10, the effect of the heterologous expression of oat CYP51H10 on the metabolism of β-amyrin in β-amyrinproducing yeast cells was examined. The results indicated that CYP51H10 converted β-amyrin to a metabolite having 12,13-epoxy and one hydroxyl group, which might be an intermediate of avenacin A-1 biosynthesis. MATERIALS AND METHODS Cloning of the DNA Fragment Encoding CYP51H10and Its Expression in Yeast Cells DNA fragments corresponding to exons 1 and 2 of CYP51H10 were prepared by polymerase chain reaction (PCR) using the DNA obt...

Cytochrome P450 (P450) is a group of heme-protein monooxygenases that catalyze the oxidation of a wide variety of organic compounds including steroids, fatty acids, xenobiotics, and so forth. P450 requires two electrons to activate one oxygen molecule in its catalytic cycle, and the electrons are provided from reduced nicotinamide-adenine dinucleotide phospahte (NADPH) or reduced nicotinamide-adenine dinucleotide (NADH) by means of a specific electrontransferring system, such as NADPH-P450 reductase (P450 reductase), cytochrome b 5 plus NADH-cytochrome b 5 reductase, or iron-sulfur protein plus its reductase. 1) Although most P450 and electron-transferring proteins are independent proteins encoded by different genes, there are a few self-sufficient monooxygenases that contain both P450 and P450 reductase in a single polypeptide chain. A typical example of such a self-sufficient P450 monooxygenase is the long-chain fatty acid hydroxylase P450 BM-3 of Bacillus megaterium. 2)This monooxygenase consists of P450 (CYP102A1) and P450 reductase domains and shows extremely high molecular turnover (Ͼ1500/min).3) This high activity is considered to be due to efficient intramolecular electron transfer from the reductase domain to the P450 domain. This has prompted many P450 investigators to construct recombinant self-sufficient drug and steroid metabolizing P450s using various mammalian P450 species and P450 reductase, and many recombinant self-sufficient P450 monooxygenases have been reported. [4][5][6][7][8][9][10][11][12] These studies indicate that a self-sufficient artificial P450 monooxygenase can be constructed by joining P450 and P450 reductase by protein engineering.Some specific P450 species such as CYP19 and CYP51 catalyze oxidative cleavage of C-C bond through three successive monooxygenation reactions, 13) and it would be interesting to construct a self-sufficient C-C bond-cleaving P450. Sterol 14-demethylase P450 (CYP51) is a unique P450 that is widely distributed among five biological kingdoms as a conserved P450 species, 14) and a fusion protein consisting of CYP51 and an iron-sulfur protein that displays sterol 14-demethylase activity in the presence of ferredoxin reductase has been found in Methylococcus capsulatus.15) However, the electron transfer system to eukaryotic CYP51 is different. Eukaryotic CYP51 receives electrons from P450 reductase, although the bacterial enzyme receives electrons via an electron-transfer system consisting of an iron-sulfur protein and a flavoprotein. The authors will attempt to construct a selfsufficient lanosterol 14-demethylase fusion protein by using yeast CYP51 and P450 reductase. This paper reports the construction of two kinds of recombinant self-sufficient lanosterol 14-demethylase and their molecular and catalytic properties. Two forms of a self-sufficient lanosterol 14-demethylase fused enzyme consisting of Saccharomyces cerevisiae CYP51 and S. cerevisiae reduced nicotinamide-adenine dinucleotide phospahte (NADPH)-P450 reductase were constructed and characterized. Th...