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

Abstract

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.

Mechanistic insights into 1‐deoxy‐d‐xylulose 5‐phosphate reductoisomerase, a key enzyme of the MEP terpenoid biosynthetic pathway

“…The subsequent release of MEP and further discharge of NADP + and M 2+ terminate the catalytic cycle. The reverse reaction sequence would take place in the opposite direction, although the equilibrium largely favors the formation of MEP [15,16].…”

“…Given the C3-C4 DXP binding mode and that (a) DXR requires M 2+ for catalytic activity [1,16,32], (b) DXR follows an ordered mechanism in which NADPH and M 2+ bind first preceding the binding of the substrate DXP [16,33], (c) all the reaction segments remain tightly combined to DXR prior to the NADPH reduction [4,15] and (d) after reduction, the final product MEP is released before the discharge of NADP + [9,16], we propose a catalytic cycle for the rearrangement/reduction reaction mediated by DXR (Fig. 5).…”

“…(a) The coordination of the hydroxyl groups of DXP with M 2+ increases their acidity and therefore facilitates deprotonation of the C4 hydroxy, which could be fundamental for the retro-aldol reaction. (b) The intermediates 6 and 7 both coordinate with M 2+ as monodentate ligands and could be strictly constrained [4,15]. (c) Although the enolate 6 is only partially stabilized in this binding mode, its C2 could be a better nucleophile because it has more carbanionic character [38] than it would as a bidentate ligand in the C2-C3 binding mode.…”

“…However, other studies have suggested that the C3 and C4 hydroxyl groups of DXP could be essential for the chelation of M 2+ (C3-C4 binding mode) [9,16]. Moreover, it has been demonstrated that the reaction intermediates 5, 6 and 7 remain strictly bound to the enzyme during DXP rearrangement [4,15], but how they bind is yet undetermined.…”

“…Exogenous 6 and 7, the two putative intermediates expected from the retro-aldol cleavage, were not recognized by DXR and converted to MEP [11,15,16], even in the presence of high concentrations of 6 (approximately 0.25 M) [15]. In addition, no fragment exchange was detected in the reverse reaction when a mixture of [1][2][3][4][5][6][7][8][9][10][11][12][13] C]-2 and [3-13 C]-2 was used as the substrate [15]. These results demonstrated that DXR does not have any aldolase activity and seemed to support the a-ketol mechanism; the explanation for this phenomenon in the context of the retroaldol/aldol mechanism remains unclear.…”