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.

中文翻译：

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大肠杆菌二氢嘧啶脱氢酶的过表达和表征：一种含有黄素蛋白的四铁硫簇

大肠杆菌二氢嘧啶脱氢酶 (EcDPD) 催化尿嘧啶和胸腺嘧啶的 NADH 依赖性还原为相应的 5,6-二氢嘧啶，以控制其代谢物库。EcDPD 由两个亚基 PreT 和 PreA 组成，并且需要 FAD、FMN 和 Fe-S 簇进行活动。具有 C 末端 His6 标记 PreA 亚基的重组 EcDPD 在缺乏 DPD 的大肠杆菌细胞中过量产生，具有增强的 Fe-S 簇合成。厌氧纯化产生比活性为 13 μmol min-1 mg-1 的纯化酶。纯化的 EcDPD 是异四聚体，每个 PreT-PreA 二聚体含有 0.81 FAD、0.99 FMN、14 个酸不稳定硫和 15 个铁。该酶在正向和反向反应中都表现出 Michaelis-Menten 动力学，这与哺乳动物 DPD 显示底物抑制动力学不同。对于尿嘧啶还原，kcat、kcat/KNADH 和 kcat/Kuracil 值在 5.5-10 的 pH 范围内是恒定的。对于二氢尿嘧啶 (DHU) 脱氢，kcat 和 kcat/KNAD+ 值的 pH 依赖性表明 pKa 为 6.6 的残基必须去质子化才能发挥活性。与猪 DPD 的生化和动力学比较表明，催化活性形式的 EcDPD 的质子化状态与猪酶的质子化状态不同。