Yoshioka Hiroshi, Ishida Tetsuo, Mihara Hisaaki
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan.
Department of Chemistry, Biology and Marine Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-2013, Japan.
J Biochem. 2021 Dec 4;170(4):511-520. doi: 10.1093/jb/mvab067.
Escherichia coli dihydropyrimidine dehydrogenase (EcDPD) catalyses the NADH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines to control their metabolite pools. EcDPD consists of two subunits, PreT and PreA, and requires FAD, FMN and Fe-S clusters for activity. Recombinant EcDPD with a C-terminal His6-tagged-PreA subunit was overproduced in a DPD-lacking E. coli cells with augmented Fe-S cluster synthesis. Anaerobic purification resulted in purified enzyme with a specific activity of 13 μmol min-1 mg-1. The purified EcDPD was a heterotetramer and contained 0.81 FAD, 0.99 FMN, 14 acid-labile sulphur and 15 iron per PreT-PreA dimer. The enzyme exhibited Michaelis-Menten kinetics for both the forward and reverse reactions, which is distinct from mammalian DPDs showing substrate inhibition kinetics. For uracil reduction, the kcat, kcat/KNADH and kcat/Kuracil values were constant over the pH range of 5.5-10. For dihydrouracil (DHU) dehydrogenation, the pH-dependence of the kcat and kcat/KNAD+ values indicated that a residue with a pKa of 6.6 must be deprotonated for activity. Biochemical and kinetic comparisons with pig DPD revealed that protonation sates of the catalytically competent forms of EcDPD are distinct from those of pig enzyme.
大肠杆菌二氢嘧啶脱氢酶（EcDPD）催化尿嘧啶和胸腺嘧啶依赖NADH还原为相应的5,6 - 二氢嘧啶，以控制它们的代谢物库。EcDPD由两个亚基PreT和PreA组成，并且其活性需要FAD、FMN和铁硫簇。带有C末端His6标签 - PreA亚基的重组EcDPD在缺乏DPD且铁硫簇合成增强的大肠杆菌细胞中过量表达。厌氧纯化得到了比活性为13 μmol min-1 mg-1的纯化酶。纯化后的EcDPD是一种异源四聚体，每个PreT - PreA二聚体含有0.81个FAD、0.99个FMN、14个酸不稳定硫和15个铁。该酶在正向和反向反应中均表现出米氏动力学，这与表现出底物抑制动力学的哺乳动物DPD不同。对于尿嘧啶还原，在pH 5.5 - 10范围内，kcat、kcat/KNADH和kcat/Kuracil值保持恒定。对于二氢尿嘧啶（DHU）脱氢反应，kcat和kcat/KNAD+值对pH的依赖性表明，一个pKa为6.6的残基必须去质子化才能具有活性。与猪DPD的生化和动力学比较表明，EcDPD催化活性形式的质子化状态与猪酶不同。
