Kinetic and chemical mechanism of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate isomeroreductase

Biochemistry. 2004 Apr 13;43(14):4375-84. doi: 10.1021/bi049974k.

Abstract

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 Co(2+), good specificity for Mn(2+), and poor specificity for Mg(2+). 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 <or= pH <or= 8.75, k(cat)/K(act)(Mn)2+ 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-(2)H]NAD(P)H, establishing that the enzyme promotes transfer of the C(4)-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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aldose-Ketose Isomerases / antagonists & inhibitors
  • Aldose-Ketose Isomerases / chemistry*
  • Aldose-Ketose Isomerases / genetics
  • Aldose-Ketose Isomerases / isolation & purification
  • Bacterial Proteins / antagonists & inhibitors
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Catalysis
  • Cations, Divalent / chemistry
  • Cloning, Molecular / methods
  • Deuterium Exchange Measurement
  • Enzyme Stability
  • Erythritol / analogs & derivatives*
  • Erythritol / chemistry
  • Hydrogen-Ion Concentration
  • Kinetics
  • Metals, Heavy / chemistry
  • Multienzyme Complexes / antagonists & inhibitors
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / isolation & purification
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / genetics
  • NADP / chemistry
  • Oxidoreductases / antagonists & inhibitors
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics
  • Oxidoreductases / isolation & purification
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Substrate Specificity
  • Sugar Phosphates / chemistry

Substances

  • 2-C-methylerythritol 4-phosphate
  • Bacterial Proteins
  • Cations, Divalent
  • Metals, Heavy
  • Multienzyme Complexes
  • Recombinant Proteins
  • Sugar Phosphates
  • NADP
  • Oxidoreductases
  • 1-deoxy-D-xylulose 5-phosphate reductoisomerase
  • Aldose-Ketose Isomerases
  • Erythritol