Analysis of flavin oxidation and electron-transfer inhibition in Plasmodium falciparum dihydroorotate dehydrogenase

Biochemistry. 2008 Feb 26;47(8):2466-75. doi: 10.1021/bi702218c. Epub 2008 Jan 29.

Abstract

Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) is a flavin-dependent mitochondrial enzyme that provides the only route to pyrimidine biosynthesis in the parasite. Clinically significant inhibitors of human DHODH (e.g., A77 1726) bind to a pocket on the opposite face of the flavin cofactor from dihydroorotate (DHO). This pocket demonstrates considerable sequence variability, which has allowed species-specific inhibitors of the malarial enzyme to be identified. Ubiquinone (CoQ), the physiological oxidant in the reaction, has been postulated to bind this site despite a lack of structural evidence. To more clearly define the residues involved in CoQ binding and catalysis, we undertook site-directed mutagenesis of seven residues in the structurally defined A77 1726 binding site, which we term the species-selective inhibitor site. Mutation of several of these residues (H185, F188, and F227) to Ala substantially decreased the affinity of pfDHODH-specific inhibitors (40-240-fold). In contrast, only a modest increase in the Kmapp for CoQ was observed, although mutation of Y528 in particular caused a substantial reduction in kcat (40-100-fold decrease). Pre-steady-state kinetic analysis by single wavelength stopped-flow spectroscopy showed that the mutations had no effect on the rate of the DHO-dependent reductive half-reaction, but most reduced the rate of the CoQ-dependent flavin oxidation step (3-20-fold decrease), while not significantly altering the Kdox for CoQ. As with the mutants, inhibitors that bind this site block the CoQ-dependent oxidative half-reaction without affecting the DHO-dependent step. These results identify residues involved in inhibitor binding and electron transfer to CoQ. Importantly, the data provide compelling evidence that the binding sites for CoQ and species-selective site inhibitors do not overlap, and they suggest instead that inhibitors act either by blocking the electron path between flavin and CoQ or by stabilizing a conformation that excludes CoQ binding.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds / pharmacology
  • Animals
  • Base Sequence
  • Benzamides / pharmacology
  • Binding Sites / genetics
  • Crystallography, X-Ray
  • Electron Transport / drug effects
  • Enzyme Inhibitors / pharmacology
  • Flavin Mononucleotide / analysis*
  • Flavin Mononucleotide / metabolism*
  • Humans
  • Hydroxybutyrates / pharmacology
  • Models, Biological
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Oxidation-Reduction / drug effects
  • Oxidoreductases Acting on CH-CH Group Donors / antagonists & inhibitors*
  • Oxidoreductases Acting on CH-CH Group Donors / chemistry*
  • Oxidoreductases Acting on CH-CH Group Donors / genetics
  • Oxidoreductases Acting on CH-CH Group Donors / metabolism
  • Plasmodium falciparum / drug effects
  • Plasmodium falciparum / enzymology*
  • Plasmodium falciparum / genetics
  • Species Specificity

Substances

  • Aniline Compounds
  • Benzamides
  • Enzyme Inhibitors
  • Hydroxybutyrates
  • Mutant Proteins
  • N-(3,5-dichlorophenyl)-2-methyl-3-nitrobenzamide
  • A 771726
  • Flavin Mononucleotide
  • Oxidoreductases Acting on CH-CH Group Donors
  • dihydroorotate dehydrogenase