Porcine Recombinant Dihydropyrimidine Dehydrogenase: Comparison of the Spectroscopic and Catalytic Properties of the Wild-Type and C671A Mutant Enzymes
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- 作者：Katrin Rosenbaum ; Karin Jahnke ; Bruno Curti ; Wilfred R. Hagen ; Klaus D. Schnackerz ; and Maria A. Vanoni
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：December 15, 1998
- 年：1998
- 卷：37
- 期：50
- 页码：17598 - 17609
- 全文大小：175K
- 年卷期：v.37,no.50(December 15, 1998)
- ISSN：1520-4995
文摘
Dihydropyrimidine dehydrogenase catalyzes, in the rate-limiting step of the pyrimidinedegradation pathway, the NADPH-dependent reduction of uracil and thymine to dihydrouracil anddihydrothymine, respectively. The porcine enzyme is a homodimeric iron-sulfur flavoprotein (2 × 111kDa). C671, the residue postulated to be in the uracil binding site and to act as the catalytically essentialacidic residue of the enzyme oxidative half-reaction, was replaced by an alanyl residue. The mutantenzyme was overproduced in Escherichia coli DH5
cells, purified to homogeneity, and characterized incomparison with the wild-type species. An extinction coefficient of 74 mM-1 cm-1 was determined at450 nm for the wild-type and mutant enzymes. Chemical analyses of the flavin, iron, and acid-labilesulfur content of the enzyme subunits revealed similar stoichiometries for wild-type and C671Adihydropyrimidine dehydrogenases. One FAD and one FMN per enzyme subunit were found.Approximately 16 iron atoms and 16 acid-labile sulfur atoms were found per wild-type and mutant enzymesubunit. The C671A dihydropyrimidine dehydrogenase mutant exhibited approximately 1% of the activityof the wild-type enzyme, thus preventing its steady-state kinetic analysis. Therefore, the ability of theC671A mutant and, for comparison, of the wild-type enzyme species to interact with reaction substrates,products, or their analogues were studied by absorption spectroscopy. Both enzyme forms did not reactwith sulfite. The wild-type and mutant enzymes were very similar to each other with respect to thespectral changes induced by binding of the reaction product NADP+ or of its nonreducible analogue3-aminopyridine dinucleotide phosphate. Uracil also induced qualitatively and quantitatively similarabsorbance changes in the visible region of the absorbance spectrum of the two enzyme forms. However,the calculated Kd of the enzyme-uracil complex was significantly higher for the C671A mutant (9.1 ±0.7 
M) than for the wild-type dihydropyrimidine dehydrogenase (0.7 ± 0.09 M). In line with theseobservations, the two enzyme forms behaved in a similar way when titrated anaerobically with a NADPHsolution. Addition of an up to 10-fold excess of NADPH to both dihydropyrimidine dehydrogenase formsled to absorbance changes consistent with reduction of approximately 0.5 flavin per subunit, with noindication of reduction of the enzyme iron-sulfur clusters. Absorbance changes consistent with reductionof both enzyme flavins were obtained by removing NADP+ with a NADPH-regenerating system. On thecontrary, the two enzyme species differed significantly with respect to their reactivity with dihydrouracil.Addition of dihydrouracil to the wild-type enzyme species, under anaerobic conditions, led to absorbancechanges that could be interpreted to result from both partial flavin reduction and the formation of a complexbetween the enzyme and (dihydro)uracil. In contrast, only spectral changes consistent with formation ofa complex between the oxidized enzyme and dihydrouracil were observed when a C671A mutant enzymesolution was titrated with this compound. Furthermore, enzyme-monitored turnover experiments werecarried out anaerobically in the presence of a limiting amount of NADPH and excess uracil with the twoenzyme forms in a stopped-flow apparatus. These experiments directly demonstrated that the substitutionof an alanyl residue for C671 in dihydropyrimidine dehydrogenase specifically prevents enzyme-catalyzedreduction of uracil. Finally, sequence analysis of dihydropyrimidine dehydrogenase revealed that it exhibitsa modular structure; the N-terminal region, similar to the 
subunit of bacterial glutamate synthases, isproposed to be responsible for NADPH binding and oxidation with reduction of the FAD cofactor ofdihydropyrimidine dehydrogenase. The central region, similar to the FMN subunit of dihydroorotatedehydrogenases, is likely to harbor the site of (dihydro)pyrimidine binding and the FMN cofactor of theenzyme. Two regions containing cysteine residues, which conform to the consensus sequence for theformation of 4Fe-4S clusters, are within the enzyme C-terminal region, while two cysteine-rich regions,conserved in all dihydropyrimidine dehydrogenases and in glutamate synthase subunits, have been foundand may play a role in formation of additional iron-sulfur clusters of dihydropyrimidine dehydrogenases.
cells, purified to homogeneity, and characterized incomparison with the wild-type species. An extinction coefficient of 74 mM-1 cm-1 was determined at450 nm for the wild-type and mutant enzymes. Chemical analyses of the flavin, iron, and acid-labilesulfur content of the enzyme subunits revealed similar stoichiometries for wild-type and C671Adihydropyrimidine dehydrogenases. One FAD and one FMN per enzyme subunit were found.Approximately 16 iron atoms and 16 acid-labile sulfur atoms were found per wild-type and mutant enzymesubunit. The C671A dihydropyrimidine dehydrogenase mutant exhibited approximately 1% of the activityof the wild-type enzyme, thus preventing its steady-state kinetic analysis. Therefore, the ability of theC671A mutant and, for comparison, of the wild-type enzyme species to interact with reaction substrates,products, or their analogues were studied by absorption spectroscopy. Both enzyme forms did not reactwith sulfite. The wild-type and mutant enzymes were very similar to each other with respect to thespectral changes induced by binding of the reaction product NADP+ or of its nonreducible analogue3-aminopyridine dinucleotide phosphate. Uracil also induced qualitatively and quantitatively similarabsorbance changes in the visible region of the absorbance spectrum of the two enzyme forms. However,the calculated Kd of the enzyme-uracil complex was significantly higher for the C671A mutant (9.1 ±0.7 M) than for the wild-type dihydropyrimidine dehydrogenase (0.7 ± 0.09 M). In line with theseobservations, the two enzyme forms behaved in a similar way when titrated anaerobically with a NADPHsolution. Addition of an up to 10-fold excess of NADPH to both dihydropyrimidine dehydrogenase formsled to absorbance changes consistent with reduction of approximately 0.5 flavin per subunit, with noindication of reduction of the enzyme iron-sulfur clusters. Absorbance changes consistent with reductionof both enzyme flavins were obtained by removing NADP+ with a NADPH-regenerating system. On thecontrary, the two enzyme species differed significantly with respect to their reactivity with dihydrouracil.Addition of dihydrouracil to the wild-type enzyme species, under anaerobic conditions, led to absorbancechanges that could be interpreted to result from both partial flavin reduction and the formation of a complexbetween the enzyme and (dihydro)uracil. In contrast, only spectral changes consistent with formation ofa complex between the oxidized enzyme and dihydrouracil were observed when a C671A mutant enzymesolution was titrated with this compound. Furthermore, enzyme-monitored turnover experiments werecarried out anaerobically in the presence of a limiting amount of NADPH and excess uracil with the twoenzyme forms in a stopped-flow apparatus. These experiments directly demonstrated that the substitutionof an alanyl residue for C671 in dihydropyrimidine dehydrogenase specifically prevents enzyme-catalyzedreduction of uracil. Finally, sequence analysis of dihydropyrimidine dehydrogenase revealed that it exhibitsa modular structure; the N-terminal region, similar to the subunit of bacterial glutamate synthases, isproposed to be responsible for NADPH binding and oxidation with reduction of the FAD cofactor ofdihydropyrimidine dehydrogenase. The central region, similar to the FMN subunit of dihydroorotatedehydrogenases, is likely to harbor the site of (dihydro)pyrimidine binding and the FMN cofactor of theenzyme. Two regions containing cysteine residues, which conform to the consensus sequence for theformation of 4Fe-4S clusters, are within the enzyme C-terminal region, while two cysteine-rich regions,conserved in all dihydropyrimidine dehydrogenases and in glutamate synthase subunits, have been foundand may play a role in formation of additional iron-sulfur clusters of dihydropyrimidine dehydrogenases.