Structural Basis of Redox-Dependent Affinities of Dihydroorotate Dehydrogenase for Its Substrates and Products.

Dihydroorotate dehydrogenase (DHODH), containing a cofactor, flavin mononucleotide (FMN), catalyzes the oxidation of dihydroorotate to orotate. In Trypanosoma brucei, the causative parasite of African trypanosomiasis, the DHODH enzyme (TbDHODH) couples this reaction with the reduction of fumarate to succinate. These reactions occur successively in the same catalytic site through a ping-pong bi-bi mechanism. FMN is reduced in the first step and re-oxidized in the second one. Here, we investigated how the redox state of FMN influences the binding of substrates and products to TbDHODH by NMR analyses of a catalytically impaired mutant. The affinities for the substrates, dihydroorotate and fumarate, were redox-dependent: dihydroorotate preferred the oxidized state, whereas fumarate preferred the reduced state. One of the products, succinate, exhibited no detectable binding to either of the redox states, suggesting its efficient product release. In contrast, the product of the first-half reaction, orotate, showed stronger binding to the oxidized state, suggesting that product inhibition may occur unless orotate is rapidly consumed in the subsequent biosynthetic step. The structural basis of these findings was elucidated by crystallography of the complexes with the above ligands in both redox states, along with quantitative analyses of the interactions using quantum chemical calculations. Upon reduction, the FMN isoalloxazine ring gains two additional hydrogens and becomes more bent at its center. These changes perturb the interactions among the ligands, flavin, and surrounding residues (notably Lys44, Asn68, and Asn195), primarily in the electrostatic term, which quantitatively explains the difference in binding affinities.