Testa Charles A, Lherbet Christian, Pojer Florence, Noel Joseph P, Poulter C Dale
Department of Chemistry, University of Utah, Salt Lake City, 84112, USA.
Biochim Biophys Acta. 2006 Jan;1764(1):85-96. doi: 10.1016/j.bbapap.2005.08.006. Epub 2005 Aug 31.
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 approximately 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.
革兰氏阴性菌、植物叶绿体、绿藻以及一些革兰氏阳性菌利用2-C-甲基-D-赤藓糖醇磷酸（MEP）途径进行类异戊二烯的生物合成。在MEP途径中异戊烯基二磷酸和二甲基烯丙基二磷酸的生物合成过程中，IspD、IspE和IspF编码将MEP转化为2-C-甲基-D-赤藓糖醇2,4-环二磷酸（cMEDP）所需的酶。在分析百脉根中生根瘤菌基因组时，开放阅读框mll0395与ispD和ispF均显示出同源性，并且似乎编码一种融合蛋白。百脉根ispE位于染色体的其他位置。纯化的重组IspDF蛋白主要是同型二聚体，分子量约为46 kDa/亚基。将IspDF与MEP、CTP和ATP一起孵育，得到的唯一产物是4-二磷酸胞苷-2-C-甲基-D-赤藓糖醇（CDP-ME）。当将大肠杆菌IspE蛋白添加到孵育混合物中时，会形成cMEDP。此外，百脉根开放阅读框mll0395可弥补鼠伤寒沙门氏菌中ispD和ispF的致死性缺失。这些结果表明，IspDF是一种双功能蛋白，它催化MEP转化为cMEDP过程中的第一步和第三步。
