Overexpression and characterization of Escherichia coli dihydropyrimidine dehydrogenase: a four iron-sulphur cluster containing flavoprotein

Escherichia coli dihydropyrimidine dehydrogenase (EcDPD) catalyses 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 His(6)-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 mu mol min(-1) mg(-1). The purified EcDPD was a heterotetr amer and contained 0.81 FAD, 0.99 FMN, 14 acid-labile sulphur 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 5.5-10. For dihydrouracil (DM) dehydrogenation, the pH-dependence of the k(cat) and k(cat)/KNAD+ 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.