ENZYMOLOGY OF ISOPRENOID BIOSYNTHESIS

我计划研究异戊烯基二磷酸形成的酶学， 所有类异戊二烯的通用前体，来自磷酸脱氧木酮糖 在链霉菌UC 5319中，倍半萜抗生素的生产者 戊烯内酯。 这种原核生物已被证明使用 非经典丙酮酸/磷酸丙糖途径合成类异戊二烯 由大学的米歇尔·罗默教授发现 路易·巴斯德在 法国，斯特拉斯堡。 基于我们长期以来对 萜类化合物的生物合成，以及生物化学和 特别是并环戊二烯生物合成的分子遗传学，我们计划 与Rohmer教授合作研究连接关键的途径 中间体1-脱氧木酮糖磷酸转化为异戊烯基二磷酸， 链霉菌UC 5319. 拟议访问期间的具体目标是 斯特拉斯堡将是：1）克隆的1-脱氧木酮糖磷酸合酶来自 e.大肠杆菌将用于制备同位素标记的1-脱氧木酮糖 从适当标记的丙酮酸和甘油醛-3- 2）将标记的1-脱氧木酮糖磷酸与以下物质一起孵育： 链霉菌UC 5319的无细胞提取物和分析的产物， 检测衍生的代谢物，如2-C-甲基-D-赤藓糖磷酸， 其他中间体导致异戊烯基二磷酸;和 E.大肠杆菌脱氧木酮糖磷酸合酶基因将被 用于设计PCR引物，用于扩增预期的特异性探针 从链霉菌的基因组文库中分离相应的基因 UC5319访问期间：1999年2月1日至1999年6月30日

I plan to study the enzymology of formation of isopentenyl diphosphate, the universal precursor of all isoprenoids, from deoxyxylulose phosphate in Streptomyces UC5319, the producer of the sesquiterpene antibiotic pentalenolactone. This prokaryotic organism has been shown to use the non-classical pyruvate/triose phosphate pathway to isoprenoids recently discovered by Prof. Michel Rohmer of the Universit Louis Pasteur in Strasbourg, France. Based on our own long-standing interest in the biosynthesis of terpenoids in general, and the biochemistry and molecular genetics of pentalenene biosynthesis in particular, we plan to collaborate with Prof. Rohmer to study the pathway linking the key intermediate, 1-deoxyxylulose phosphate, to isopentenyl diphosphate in Streptomyces UC5319. The specific goals during the proposed visit to Strasbourg will be: 1) Cloned 1-deoxyxylulose phosphate synthase from E. coli will be used to prepare isotopically labeled 1-deoxyxylulose from appropriately labeled samples of pyruvate and glyceraldehyde-3- phosphate; 2) Labeled 1-deoxyxylulose phosphate will be incubated with cell-free extracts of Streptomyces UC5319 and the products analyzed to detect derived metabolites such as 2-C-methyl-D-erythrose phosphate and other intermediates leading to isopentenyl diphosphate; and 3) The sequence of the E. coli deoxyxylulose phosphate synthase gene will be used to design PCR primers for amplification of specific probes intended to isolate the corresponding gene from a genomic library of Streptomyces UC5319. Period of visit: February 1, 1999 - June 30, 1999