8F5W

Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Dihydrothymine and NADPH Quasi-Anaerobically

Summary for 8F5W

• Entry DOI: 10.2210/pdb8f5w/pdb
• Descriptor: Dihydropyrimidine dehydrogenase [NADP(+)], IRON/SULFUR CLUSTER, FLAVIN-ADENINE DINUCLEOTIDE, ... (7 entities in total)
• Functional Keywords: dihydropyrimidine dehydrogenase, protein, mutant, dihyrdothymine, nadph, flavoprotein
• Biological source: Sus scrofa (pig)
• Total number of polymer chains: 4
• Total formula weight: 460429.08

Authors

Kaley, N.,Smith, M.,Forouzesh, D.,Liu, D.,Moran, G. (deposition date: 2022-11-15, release date: 2023-02-01, Last modification date: 2023-10-25)

Primary citation

Smith, M.M.,Forouzesh, D.C.,Kaley, N.E.,Liu, D.,Moran, G.R.
Mammalian dihydropyrimidine dehydrogenase: Added mechanistic details from transient-state analysis of charge transfer complexes.
Arch.Biochem.Biophys., 736:109517-109517, 2023

PubMed Abstract: Dihydropyrimidine dehydrogenase (DPD) is a flavin dependent enzyme that catalyzes the reduction of the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons from NADPH. DPD has two active sites that are separated by ∼60 Å. At one site NADPH binds adjacent to an FAD cofactor and at the other pyrimidine binds proximal to an FMN. Four FeS centers span the distance between these active sites. It has recently been established that the enzyme undergoes reductive activation prior to reducing the pyrimidine. In this initial process NADPH is oxidized at the FAD site and electrons are transmitted to the FMN via the FeS centers to yield the active state with a cofactor set of FAD•4(FeS)•FMNH. The catalytic chemistry of DPD can be studied in transient-state by observation of either NADPH consumption or charge transfer absorption associated with complexation of NADPH adjacent to the FAD. Here we have utilized both sets of absorption transitions to find evidence for specific additional aspects of the DPD mechanism. Competition for binding with NADP indicates that the two charge transfer species observed in activation/single turnover reactions arise from NADPH populating the FAD site before and after reductive activation. An additional charge transfer species is observed to accumulate at longer times when high NADPH concentrations are mixed with the enzyme•pyrimidine complex and this data can be modelled based on asymmetry in the homodimer. It was also shown that, like pyrimidines, dihydropyrimidines induce rapid reductive activation indicating that the reduced pyrimidine formed in turnover can stimulate the reinstatement of the active state of the enzyme. Investigation of the reverse reaction revealed that dihydropyrimidines alone can reductively activate the enzyme, albeit inefficiently. In the presence of dihydropyrimidine and NADP DPD will form NADPH but apparently without measurable reductive activation. Pyrimidines that have 5-substituent halogens were utilized to probe both reductive activation and turnover. The linearity of the Hammett plot based on the rate of hydride transfer to the pyrimidine establishes that, at least to the radius of an iodo-group, the 5-substituent volume does not have influence on the observed kinetics of pyrimidine reduction.

PubMed: 36681231
DOI: 10.1016/j.abb.2023.109517

Experimental method

X-RAY DIFFRACTION (1.97 Å)