Porcine recombinant dihydropyrimidine dehydrogenase: Comparison of the spectroscopic and catalytic properties of the wild-type and C671A mutant enzymes

Dihydropyrimidine dehydrogenase catalyzes, in the rate-limiting step of the pyrimidine degradation pathway, the NADPH-dependent reduction of uracil an d thymine to dihydrouracil and dihydrothymine, respectively. The porcine en zyme is a homodimeric iron-sulfur flavoprotein (2 x 111 kDa). C671, the res idue postulated to be in the uracil binding site and to act as the catalyti cally essential acidic residue of the enzyme oxidative half-reaction, was r eplaced by an alanyl residue. The mutant enzyme was overproduced in Escheri chia coli DH5 alpha cells, purified to homogeneity, and characterized in co mparison with the wild-type species. An extinction coefficient of 74 mM(-1) cm(-1) was determined at 450 nm for the wild-type and mutant enzymes. Chem ical analyses of the flavin, iron, and acid-labile sulfur content of the en zyme subunits revealed similar stoichiometries for wild-type and C671A dihy dropyrimidine dehydrogenases. One FAD and one FMN per enzyme subunit were f ound. Approximately 16 iron atoms and 16 acid-labile sulfur atoms were foun d per wild-type and mutant enzyme subunit. The C671A dihydropyrimidine dehy drogenase mutant exhibited approximately 1% of the activity of the wild-typ e enzyme, thus preventing its steady-state kinetic analysis. Therefore, the ability of the C671A mutant and, for comparison, of the wild-type enzyme s pecies to interact with reaction substrates, products, or their analogues w ere studied by absorption spectroscopy. Both enzyme forms did not react wit h sulfite. The wild-type and mutant enzymes were very similar to each other with respect to the spectral changes induced by binding of the reaction pr oduct NADP(+) or of its nonreducible analogue 3-aminopyridine dinucleotide phosphate. Uracil also induced qualitatively and quantitatively similar abs orbance changes in the visible region of the absorbance spectrum of the two enzyme forms. However, the calculated K-d of the enzyme-uracil complex was significantly higher for the C671A mutant (9.1 +/- 0.7 mu M) than for the wild-type dihydropyrimidine dehydrogenase (0.7 +/- 0.09 mu M). In line with these observations, the two enzyme forms behaved in a similar way when tit rated anaerobically with a NADPH solution. Addition of an up to 10-fold exc ess of NADPH to both dihydropyrimidine dehydrogenase forms led to absorbanc e changes consistent with reduction of approximately 0.5 flavin per subunit , with no indication of reduction of the enzyme iron-sulfur clusters. Absor bance changes consistent with reduction of both enzyme flavins were obtaine d by removing NADP(+) with a NADPH-regenerating system. On the contrary, th e two enzyme species differed significantly with respect to their reactivit y with dihydrouracil. Addition of dihydrouracil to the wild-type enzyme spe cies, under anaerobic conditions, led to absorbance changes that could be i nterpreted to result from both partial flavin reduction and the formation o f a complex between the enzyme and (dihydro)uracil. In contrast, only spect ral changes consistent with formation of a complex between the oxidized enz yme and dihydrouracil were observed when a C671A mutant enzyme solution was titrated with this compound. Furthermore, enzyme-monitored turnover experi ments were carried out anaerobically in the presence of a limiting amount o f NADPH and excess uracil with the two enzyme forms in a stopped-flow appar atus. These experiments directly demonstrated that the substitution of an a lanyl residue for C671 in dihydropyrimidine dehydrogenase specifically prev ents enzyme-catalyzed reduction of uracil. Finally, sequence analysis of dihydropyrimidine dehydrogenase revealed that it exhibits a modular structure; the N-terminal region, similar to the bet a subunit of bacterial glutamate synthases, is proposed to be responsible f or NADPH binding and oxidation with reduction of the FAD cofactor of dihydr opyrimidine dehydrogenase. The central region, similar to the FMN subunit o f dihydroorotate dehydrogenases, is likely to harbor the site of (dihydro)p yrimidine binding and the FMN cofactor of the enzyme. Two regions containin g cysteine residues, which conform to the consensus sequence for the format ion of 4Fe-4S clusters, are within the enzyme C-terminal region, while two cysteine-rich regions, conserved in all dihydropyrimidine dehydrogenases an d in glutamate synthase beta subunits, have been found and may play a role in formation of additional iron-sulfur clusters of dihydropyrimidine dehydr ogenases.