title: Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension.
abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.
authors: Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense 400, C.P. 369, 13560-970, São Carlos, SP, Brazil.
authors: Sheila G
authors: SG
authors: Couto
authors: M
authors: M
authors: Cristina Nonato
authors: Antonio J
authors: AJ
authors: Costa-Filho
mesh_terms: D002384:Catalysis; D000090722:Dihydroorotate Dehydrogenase; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D060526:Magnetic Fields; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013113:Spin Labels
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D013113:Spin Labels; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors