14 非メバロン酸経路に関する研究 : 突然変異株の利用による反応機構の解明(口頭発表の部)

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Several eubacteria including Escherichia coli utilize an alternative mevalonate-independent pathway (nonmevalonate pathway) for the biosynthesis of isopentenyl diphosphate (IPP). The initial step of the nonmevalonate pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. In the second step, the intramolecular rearrangement and reduction of DXP occur simultaneously to yield 2-C-methyl-D-erythritol 4-phosphate (MEP). However, the following reactions leading to IPP from MEP remained unknown. To elucidate the remaining reactions, we employed a unique strategy for preparation of mutants of E. coli possessing a metabolic block(s) between MEP and IPP. Since such mutations would be lethal, we constructed an E. coli transformant expressing mevalonate kinase, phosphomevalonate kinase and diphosphomevalonate decarboxylase. Using this transformant as the parent strain, we prepared mutants with an obligatory requirement of mevalonate for growth. Thirty-three mutants from 60,000 colonies screened showed the expected phenotypes. With these mutants in hand, we were able to clone three genes, YACM, ychB and ygbB, that complemented the defect of each blocked mutant in synthesizing IPP from MEP. To identify the functions of these genes, plasmids for overexpression of these gene products were constructed. We found that the YACM gene product catalyzed a cytidylyl transfer to MEP to synthesize 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME). Next, the ychB gene product catalyzed phosphorylation of the tertiary hydroxy group in the CDP-ME molecule to produce 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME2P). Further, the ygbB gene product converted CDP-ME2P into 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP).
天然有機化合物討論会の論文
2000-10-01

14 非メバロン酸経路に関する研究 : 突然変異株の利用による反応機構の解明(口頭発表の部)

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