ENZYMOLOGY OF ISOPRENOID BIOSYNTHESIS

我计划研究形成异戊烯基二磷酸的酶学， 所有异戊二烯的通用前体，来自脱氧果糖磷酸 在链霉菌UC5319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 近年来丙酮酸/磷酸三糖合成异戊二烯的非经典途径 由路易斯·巴斯德大学的米歇尔·罗默教授于 法国斯特拉斯堡。基于我们自己长期以来对 萜类化合物的生物合成，以及生物化学和 特别是戊二烯生物合成的分子遗传学，我们计划 与罗默教授合作研究连接钥匙的途径 中间体1-脱氧果糖磷酸到异戊烯基二磷酸 链霉菌UC5319。拟议访问期间的具体目标 斯特拉斯堡将：1)从以下来源克隆1-脱氧果糖磷酸合酶 大肠杆菌将被用来制备同位素标记的1-脱氧果糖 从适当标记的丙酮酸和甘油醛-3-样品- 磷酸盐；2)标记的1-脱氧果糖磷酸将与 链霉菌UC5319的无细胞提取物及其产物分析 检测衍生代谢物，如2-C-甲基-D-红磷酸酯和 合成异戊烯基二磷酸的其他中间体；以及3) 大肠杆菌脱氧果糖磷酸合成酶基因的序列如下 用于设计用于扩增特定探针的聚合酶链式反应引物 从链霉菌基因组文库中分离相应基因 UC5319。访问时间：1999年2月1日-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