7M32

Dihydropyrimidine Dehydrogenase (DPD) C671A Mutant Soaked with Uracil and NADPH Anaerobically

7M32 の概要

• エントリーDOI: 10.2210/pdb7m32/pdb
• 分子名称: Dihydropyrimidine dehydrogenase [NADP(+)], IRON/SULFUR CLUSTER, FLAVIN-ADENINE DINUCLEOTIDE, ... (9 entities in total)
• 機能のキーワード: dihydropyrimidine dehydrogenase, dpd, mutant, uracil, flavin, soaking, ligand, fmn, fad, flavoprotein, oxidoreductase
• 由来する生物種: Sus scrofa (Pig)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 457891.01

構造登録者

Butrin, A.,Beaupre, B.,Forouzesh, D.,Liu, D.,Moran, G. (登録日: 2021-03-18, 公開日: 2021-06-09, 最終更新日: 2023-10-18)

主引用文献

Beaupre, B.A.,Forouzesh, D.C.,Butrin, A.,Liu, D.,Moran, G.R.
Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase.
Biochemistry, 60:1764-1775, 2021

PubMed Abstract: The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by ∼60 Å that are bridged by four FeS centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) , 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or FeS centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.

PubMed: 34032117
DOI: 10.1021/acs.biochem.1c00243

実験手法

X-RAY DIFFRACTION (1.82 Å)