title: Defects in vesicle core induced by escherichia coli dihydroorotate dehydrogenase.
abstract: Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 10-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.
authors.affiliation: Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970, São Carlos, SP, Brazil.
authors.forename: Sheila G
authors.initials: SG
authors.lastname: Couto
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Nonato
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Costa-Filho
mesh_terms: D002384:Catalysis; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology* / Q000473:pathology; D003600:Cytosol / Q000201:enzymology* / Q000473:pathology; D000090722:Dihydroorotate Dehydrogenase; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005997:Glycerylphosphorylcholine / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors / Q000378:metabolism; D011743:Pyrimidines / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D013194:Staining and Labeling; D013379:Substrate Specificity
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D005997:Glycerylphosphorylcholine; D044925:Oxidoreductases Acting on CH-CH Group Donors