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

Several eubacteria including Esherichia coli use an alternative nonmev alonate pathway for the biosynthesis of isopentenyl diphosphate instea d of the ubiquitous mevalonate pathway. In the alternative pathway, 2- C-methyl-D -erythritol or its 4-phosphate, which is proposed to be for med from 1-deoxy-D-xylulose 5 phosphate via intramolecular rearrangeme nt followed by reduction process, is one of the biosynthetic precursor s of isopentenyl diphosphate. To clone the gene(s) responsible for syn thesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selecte d E. coli mutants with an obligatory requirement for 2-C-methylerythri tol for growth and survival. All the DNA fragments that complemented t he 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 c hromosomal map of E, coli. The gene product showed significant homolog ies to hypothetical proteins with unknown functions present in Haemoph ilus 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-xy lulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. Th e 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 i ntramolecular rearrangement and reduction and that this gene is respon sible for terpenoid biosynthesis in E, coli.