2006 Fiscal Year Final Research Report Summary
Studies on isoprenoid biosynthetic genes isolated from prokaryotes
| Project/Area Number |
16310153
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | Toyama Prefectural University |
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Principal Investigator |
DAIRI Tohru Toyama Prefectural University, Biotechnology Research Center, Associate professor, 工学部, 助教授 (70264679)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUURA Nobuyasu Okayama University of Science, Department of Life Science, Associate professor, 理学部, 講師 (60281250)
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| Project Period (FY) |
2004 – 2006
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| Keywords | isoprenoid / cyclase / Actinomycetes / ent-copaly1 diphosphate / pimara-9 (11),15-diene / furaquinocin A / furaquinocin A |
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| Research Abstract |
We have cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp.strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-copalyl diphosphate(CDP) synthase and a geranylgeranyl diphosphate(GGDP) synthase, respectively, by experiments using recombinant enzymes. ORF3,that did not show similarities with any other known proteins was also expressed in E.coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into pimara-9(11),15-diene. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg^<2+> and Zn^<2+> for its activity. The optimum pH and temperature were 5.5 and 35℃. The Km value was calculated to be 13.7±1.0μM for GGDP and the kcat value was 3.3 x 10^<-2>/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30℃. The Km value for ent-CDP was estimated to be 2.6±0.2μM and the kcat value was 1.4 x 10^<-3>/sec.
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