Kinetic and Chemical Mechanism of Mycobacterium tuberculosis 1-Deoxy-D-xylulose-5-phosphate Isomeroreductase

1-Deoxy-D-xylulose-5-phosphate (DXP) isomeroreductase catalyzes the isomerization and reduced nicotinamide adenine dinucleotide phosphate- (NADPH-) dependent reduction of DXP to generate 2-C-methylerythritol 4-phosphate (MEP) in the first committed step of the MEP pathway of isoprenoid biosynthesis. We have cloned the gene encoding the Mycobacterium tuberculosis DXP isomeroreductase, expressed the protein in Escherichia coli, and purified the enzyme to homogeneity using conventional column chromatography methods. DXP isomeroreductase is a metal ion-activated enzyme displaying superior specificity for Co²⁺, good specificity for Mn²⁺, and poor specificity for Mg²⁺. Although NADPH is preferred over reduced nicotinamide adenine dinucleotide (NADH) about 100-fold as evaluated by the relative k_cat/K_m values, the maximum turnover numbers are similar, suggesting that the 2′-phosphate of NADPH contributes predominantly to binding and not to catalysis. While k_cat was independent of pH in the region 6.0 ≤ pH ≤ 8.75, k_cat/K _act^Mn2+ decreased at low pH as two enzymatic groups with pK_a values of 7.4 are protonated. These groups likely represent carboxylate groups that coordinate the divalent metal ion in the active site. The results also support an electrostatic role for the divalent metal ion in catalysis. The results of product inhibition studies and isotope effects suggest that the enzyme utilizes a steady-state random mechanism. Significant isotope effects were observed with [4S-²H]NAD(P)H, establishing that the enzyme promotes transfer of the C₄-proS hydride of the reduced pyridine nucleotide. The magnitude of these primary deuterium kinetic isotope effects varied with metal ion and reduced pyridine nucleotide identities. The results are discussed in terms of significant differences in the commitment factors for the various metal ions and pyridine nucleotides.