| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
An industrial microorganism, anthelminthic antibiotic producer Streptomyces avermitilis, has many (33) kinds of genes encoded cytochrome P450 (CYP) by the genome analysis of this microorganism in 2003. In order to elucidate 33 kinds of these CYP, all genes were amplified by the PCR and their amplicons were introduced into T7-RNA promoted expression vector system in E. coli. Among of 26 of CYP genes could be expressed in E. coli and their gene products were obtained as soluble protein containing Fe-heme molecule. Out of 15 gene products expressed (SAV109, SAV412, SAV413, SAV 1308, SAV1987, SAV2061, SAV2894, SAV2999, SAV3704, SAV3882, SAV5841, SAV6249, SAV7130, SAV7186, SAV7469) have catalytic activities in the presence of ferredoxin, ferredoxin reductase, NADPH-generating system and substrate and structures of their reaction products were confirmed. From the structural elucidation of reaction products, eight CYPs catalyzed the hydroxylation at C4 position of carbazole, 4 CYPs hydroxylated the para-position of trans-stilbene and 5 CYPs hydroxylayed at omega position of fatty acids. Three CYPs, CYP154A2, CYP107L2 and CYP105D7, have the ability to catalyze the hydroxylation of many compounds. Especially, CYP105D7 could catalyze the hydroxylation of at least 8 different compounds (compactin, diclofenac, lauric acid, naringenin, tolubtamide, carbazole, milbemycin A3 and testosterone). The CYP105D6 was very similar to CYP105D7 by homology and alignment analyses. The difference between CYP105D6 and CYPIO5D7 is very interesting in the substrate specificity and structural importance in CYPs, because CYP105D6 and CYP105D7 were classified to the same family and CYP105D6 hydroxylated only the polyene-macrolide filipin.
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