A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-elythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis
A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-elythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis
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DOI：
10.1073/pnas.95.17.9879
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发表时间：
1998-08-18
影响因子：
11.1
通讯作者：
Seto, H
中科院分区：
文献类型：
--
作者：
Takahashi, S;Kuzuyama, T;Seto, H
包括大肠杆菌在内的几种真细菌使用替代的非甲戊酸途径来生物合成二磷酸异戊酸，而不是普遍存在的甲戊酸途径。在替代途径中，2- c -甲基-d -赤藓糖醇或其4-磷酸是二磷酸异戊烯基的生物合成前体之一，由1-脱氧-d -木糖糖5磷酸经过分子内重排和还原过程形成。为了克隆负责合成2- c -甲基- d -赤藓糖醇4-磷酸的基因，我们制备并选择了生长和生存必需2- c -甲基赤藓糖醇的大肠杆菌突变体。这些突变体在合成2- c -甲基- d -赤藓糖醇4-磷酸过程中弥补缺陷的DNA片段均含有yaeM基因，该基因位于大肠杆菌染色体图谱上的4.2 min。该基因产物与流感嗜血杆菌、聚胞杆菌PCC6803、结核分枝杆菌、pyroobacter Helicobacter和枯草芽孢杆菌中存在的功能未知的假设蛋白具有显著的同源性。纯化的重组yaeM基因产物在大肠杆菌中过表达，并在NADPH存在下催化1-脱氧-d -木糖5-磷酸生成2- c -甲基-d -赤藓糖醇4-磷酸。用NADH替代NADPH使反应速率降低到原来的1%左右。这种酶也需要Mn2+、Co2+或Mg2+。这些数据清楚地表明，yaeM基因编码一种名为1-脱氧-d -木糖5-磷酸还原异构酶的酶，该酶通过分子内重排和还原一步合成2- c -甲基-d -赤藓糖醇4-磷酸，并且该基因负责大肠杆菌中萜类生物合成。
Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D -erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5 phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E, coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E, coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E, coli.