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

Argyrides Argyrou, John S. Blanchard

Research output: Contribution to journalArticle

60 Citations (Scopus)

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 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 kcat/Km values, the maximum turnover numbers are similar, suggesting that the 2′-phosphate of NADPH contributes predominantly to binding and not to catalysis. While kcat was independent of pH in the region 6.0 ≤ pH ≤ 8.75, kcat/K actMn2+ decreased at low pH as two enzymatic groups with pKa 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.

Original languageEnglish (US)
Pages (from-to)4375-4384
Number of pages10
JournalBiochemistry
Volume43
Issue number14
DOIs
StatePublished - Apr 13 2004

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Ketol-Acid Reductoisomerase
Mycobacterium tuberculosis
Metal ions
Metals
Ions
Kinetics
NADP
Isotopes
NAD
Nucleotides
Enzymes
Catalysis
Complement C4
Column chromatography
Gene encoding
Deuterium
Escherichia coli Proteins
Biosynthesis
Terpenes
Isomerization

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and Chemical Mechanism of Mycobacterium tuberculosis 1-Deoxy-D-xylulose-5-phosphate Isomeroreductase. / Argyrou, Argyrides; Blanchard, John S.

In: Biochemistry, Vol. 43, No. 14, 13.04.2004, p. 4375-4384.

Research output: Contribution to journalArticle

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