Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5-iodouracil. Implications for mechanism of inhibition and electron transfer.
Dihydroprymidine dehydrogenase catalyzes the first and rate-limiting step in pyrimidine degradation by converting pyrimidines to the corresponding 5,6- dihydro compounds. The three-dimensional structures of a binary complex with the inhibitor 5-iodouracil and two ternary complexes with NADPH and the inhibitors 5-iodouracil and uracil-4-acetic acid were determined by x-ray crystallography. In the ternary complexes, NADPH is bound in a catalytically competent fashion, with the nicotinamide ring in a position suitable for hydride transfer to FAD. The structures provide a complete picture of the electron transfer chain from NADPH to the substrate, 5-iodouracil, spanning a distance of 56 A and involving FAD, four [Fe-S] clusters, and FMN as cofactors. The crystallographic analysis further reveals that pyrimidine binding triggers a conformational change of a flexible active-site loop in the alpha/beta-barrel domain, resulting in placement of a catalytically crucial cysteine close to the bound substrate. Loop closure requires physiological pH, which is also necessary for correct binding of NADPH. Binding of the voluminous competitive inhibitor uracil-4-acetic acid prevents loop closure due to steric hindrance. The three-dimensional structure of the ternary complex enzyme-NADPH-5-iodouracil supports the proposal that this compound acts as a mechanism-based inhibitor, covalently modifying the active-site residue Cys-671, resulting in S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteine.
Dobritzsch
Doreen
D
Division of Molecular Structural Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden.
Ricagno
Stefano
S
Schneider
Gunter
G
Schnackerz
Klaus D
KD
Lindqvist
Ylva
Y
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D042943:Dihydrouracil Dehydrogenase (NADP); D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014498:Uracil / Q000031:analogs & derivatives* / Q000378:metabolism*
D008024:Ligands; D011994:Recombinant Proteins; D009249:NADP; D014498:Uracil; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); C048747:5-iodouracil