Cloning and expression of IspDF from Mesorhizobium loti. Characterization of a bifunctional protein that catalyzes non-consecutive steps in the methylerythritol phosphate pathway.
Cloning and expression of IspDF from Mesorhizobium loti. Characterization of a bifunctional protein that catalyzes non-consecutive steps in the methylerythritol phosphate pathway.
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中索根瘤菌 IspDF 的克隆和表达。
DOI：
10.1016/j.bbapap.2005.08.006
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Poulter,CDale
中科院分区：
文献类型：
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作者：
Testa,CharlesA;Lherbet,Christian;Pojer,Florence;Noel,JosephP;Poulter,CDale
革兰氏阴性细菌、植物叶绿体、绿色藻类和一些革兰氏阳性细菌利用2-C-甲基-d-甲基-磷酸（MEP）途径来生物合成类异戊二烯。ispD、ispE和ispF编码在MEP途径中的异戊烯基二磷酸和二甲基烯丙基二磷酸的生物合成期间将MEP转化为2-C-甲基-d-d2，4-环二磷酸（cMEDP）所需的酶。在分析中慢生根瘤菌基因组时，ORF mll 0395显示与ispD和ispF两者的同源性，并且似乎编码融合蛋白。M. loti ispE位于染色体的其他位置。纯化的重组IspDF蛋白主要是同源二聚体，分子量约为46 kDa/亚基。将IspDF与MEP、CTP和ATP一起孵育，得到4-二磷酸胞苷酰-2-C-甲基-d-cytosidyl（CDP-ME）作为唯一产物。当将大肠杆菌IspE蛋白加入孵育混合物中时，形成cMEDP。此外，M. loti ORF mll 0395补充鼠伤寒沙门氏菌中ispD和ispF的致死性破坏。这些结果表明，IspDF是一种双功能蛋白，其催化MEP转化为cMEDP的第一步和第三步。
Gram-negative bacteria, plant chloroplasts, green algae and some Gram-positive bacteria utilize the 2-C-methyl-d-erythritol phosphate (MEP) pathway for the biosynthesis of isoprenoids. IspD, ispE, and ispF encode the enzymes required to convert MEP to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) during the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate in the MEP pathway. Upon analysis of the Mesorhizobium loti genome, ORF mll0395 showed homology to both ispD and ispF and appeared to encode a fusion protein. M. loti ispE was located elsewhere on the chromosome. Purified recombinant IspDF protein was mostly a homodimer, MW ∼46 kDa/subunit. Incubation of IspDF with MEP, CTP, and ATP gave 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) as the only product. When Escherichia coli IspE protein was added to the incubation mixture, cMEDP was formed. In addition, M. loti ORF mll0395 complements lethal disruptions in both ispD and ispF in Salmonella typhimurium. These results indicate that IspDF is a bifunctional protein, which catalyzes the first and third steps in the conversion of MEP to cMEDP.