title: Analysis of flavin oxidation and electron-transfer inhibition in Plasmodium falciparum dihydroorotate dehydrogenase.
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.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Boulevard, Dallas, Texas 75390-9041, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Malmquist
authors.forename: Ramesh
authors.initials: R
authors.lastname: Gujjar
authors.forename: Pradipsinh K
authors.initials: PK
authors.lastname: Rathod
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Phillips
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D003437:Crotonates; D018360:Crystallography, X-Ray; D000090722:Dihydroorotate Dehydrogenase; D004579:Electron Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D006885:Hydroxybutyrates / Q000494:pharmacology; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009570:Nitriles; D010084:Oxidation-Reduction / Q000187:drug effects; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D014052:Toluidines
chemical_list: D000814:Aniline Compounds; D001549:Benzamides; D003437:Crotonates; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D006885:Hydroxybutyrates; D050505:Mutant Proteins; C528506:N-(3,5-dichlorophenyl)-2-methyl-3-nitrobenzamide; D009570:Nitriles; D014052:Toluidines; C527525:teriflunomide; D005486:Flavin Mononucleotide; D044925:Oxidoreductases Acting on CH-CH Group Donors