Biosynthesis of isoprenoids – studies on the mechanism of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase

2C‐Methyl‐d‐erythritol‐4‐phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalonate isoprenoid biosynthetic pathway. The reaction involves the isomerization of 1‐deoxy‐d‐xylulose 5‐phosphate, giving a branched‐chain aldose derivative that is subsequently reduced to 2C‐methyl‐d‐erythritol 4‐phosphate. The isomerization step has been proposed to proceed as an intramolecular rearrangement or a retroaldol–aldol sequence. We report the preparation of 13C‐labeled substrate isotopologs that were designed to optimize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol–aldol reaction sequence. In reaction mixtures containing large amounts of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidopsis thaliana, and a mixture of [1‐13C1]‐2C‐methyl‐d‐erythritol 4‐phosphate and [3‐13C1]2C‐methyl‐d‐erythritol 4‐phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorporated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1‐deoxy‐d‐xylulose 5‐phosphate to give 1‐deoxy‐l‐ribulose 5‐phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol–aldol mechanism cannot be ruled out conclusively, the data show that a retroldol–aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three genetically distant organisms.