The Differences are in the Details: The Mechanism of Flavin Reduction inClass 1A and Class 2 Dihydroorotate Dehydrogenases.

[摘要] Several flavoproteins catalyze very similar oxidation/reduction reactions in pyrimidine metabolism.One such enzyme is dihydroorotate dehydrogenase (DHOD), which catalyzes the only redox step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate (DHO) to orotate.Due to their important biological role, DHODs are interesting drug targets.A better understanding of the detailed chemical mechanism of DHODs from different phylogenetic classes could aid in class-specific inhibitor development. During DHO oxidation, an active site base deprotonates C5 of DHO and a hydride is transferred from C6 to the isoalloxazine ring of the FMN prosthetic group.The active site bases differ between classes of DHODs – Class 1A enzymes have cysteine while Class 2 enzymes have serine – but the rest of the active site is nearly identical.The fundamental question of whether the scission of the two substrate C-H bonds is stepwise or concerted was addressed by determining deuterium kinetic isotope effects on flavin reduction under anaerobic conditions.Interestingly, different mechanisms were observed; Class 1A enzymes utilize a concerted mechanism while Class 2 enzymes use a stepwise mechanism.When the active site base of the Class 1A enzyme from L. lactis was switched to that of Class 2 enzymes, the rate of flavin reduction was significantly decreased, but the mechanism of flavin reduction was not changed, indicating that the identity of the active site base is not responsible for determining the mechanism of DHO oxidation. The active sites of all DHODs contain many strictly conserved residues that hydrogen-bond to pyrimidines.Site-directed mutagenesis was used to investigate their roles in DHO oxidation.Mutation of several analogous residues in the two classes caused different changes in the behavior of the enzymes.Some residues have been shown to be important for DHO binding, while others were critical for DHO oxidation.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] Dihydroorotate Dehydrogenase;Biological Chemistry;Science;Biological Chemistry