Analysis of Flavin Oxidation and Electron-Transfer Inhibition in Plasmodium falciparum Dihydroorotate Dehydrogenase

• Published in: Biochemistry (Easton) 2008-01, Vol.47 (8), p.2466-2475
• Main Authors: Malmquist, Nicholas A, Gujjar, Ramesh, Rathod, Pradipsinh K, Phillips, Margaret A
• Format: Article
• Language: English
• Subjects: Plasmodium falciparum
• ISSN: 0006-2960
• DOI: 10.1021/bi702218cPII:S0006-2960(70)02218-3

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 K sub(m) super(app) for CoQ was observed, although mutation of Y528 in particular caused a substantial reduction in k sub(cat) (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 K sub(d) super(ox) 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.