At least three families of enzymes catalyze the interconversion of pyrimidine and dihydropyrimidine rings using a flavin prosthetic group (always FMN): dihydroorotate dehydrogenases (DHOD), dihydrouridine synthases (DUS), and dihydropyrimidine dehydrogenases (DHPDH). Flavoenzyme catalytic cycles can generally be divided into half-reactions. In the pyrimidine-converting half-reaction, all these enzymes transfer a hydride and a proton. Structures have been determined of examples of all these enzymes. In keeping with their common chemical task, the sites where pyrimidine substrates react with FMN are all very similar (some extremely so). Nonetheless, careful examination of reaction mechanisms, using isotope effects and site-directed mutants, detect important differences in mechanisms.

In the other half-reaction, FMN can react very differently from enzyme to enzyme. Different DHODs react with fumarate, NAD, or ubiquinone; DUSs react with pyridine nucleotides; DHPDHs react with NAD. These enzymes manage to do this with biologically acceptable specificity despite the need to also accomplish the same pyrimidine chemistry during the other half-reaction. This highlights the exquisite control protein exert over the reactivity of flavins. It is becoming increasingly clear that one important task enzymes accomplish is to prevent some reactions – so-called negative catalysis – while accelerating biologically desirable reactions. We are investigating the strategies employed by several flavoenzymes from pyrimidine metabolism.