Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination.
In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.
Lohkamp
Bernhard
B
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden.
Voevodskaya
Nina
N
Lindqvist
Ylva
Y
Dobritzsch
Doreen
D
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013552:Swine
D005415:Flavins; D011743:Pyrimidines; D005973:Glutamine; D005182:Flavin-Adenine Dinucleotide; D012694:Serine; D006639:Histidine; D001120:Arginine; D007501:Iron; D042943:Dihydrouracil Dehydrogenase (NADP); D003545:Cysteine