Overexpression and characterization of Escherichia coli dihydropyrimidine dehydrogenase, a four iron-sulfur cluster containing flavoprotein.

Escherichia coli dihydropyrimidine dehydrogenase (EcDPD) catalyzes the NADH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines to control their metabolite pools. EcDPD consists of two subunits, PreT and PreA, and requires FAD, FMN, and Fe-S clusters for activity. Recombinant EcDPD with a C-terminal His6-tagged-PreA subunit was overproduced in a DPD-lacking E. coli cells with augmented Fe-S cluster synthesis. Anaerobic purification resulted in purified enzyme with a specific activity of 13 μmol min-1 mg-1. The purified EcDPD was a heterotetramer and contained 0.81 FAD, 0.99 FMN, 14 acid-labile sulfur, and 15 iron per PreT-PreA dimer. The enzyme exhibited Michaelis-Menten kinetics for both the forward and reverse reactions, which is distinct from mammalian DPDs showing substrate inhibition kinetics. For uracil reduction, the k cat, k cat/K NADH, and k cat/K uracil values were constant over the pH range of 5.5 to 10. For dihydrouracil dehydrogenation, the pH-dependence of the k cat and k cat/K NAD+ values indicated that a residue with a pK a of 6.6 must be deprotonated for activity. Biochemical and kinetic comparisons with pig DPD revealed that protonation sates of the catalytically competent forms of EcDPD are distinct from those of pig enzyme.