Department of Chemistry and Biochemistry, Department of Biomolecular Science and Engineering, University of California, Santa Barbara, California, United States.
Department of Chemistry and Biochemistry, Department of Biomolecular Science and Engineering, University of California, Santa Barbara, California, United States.
Biochim Biophys Acta Proteins Proteom. 2020 May;1868(5):140376. doi: 10.1016/j.bbapap.2020.140376. Epub 2020 Jan 22.
Two groups of metabolically related enzymes, the Group III family of Fe-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.
两组代谢相关的酶，即依赖铁的第三组家族醇脱氢酶（ADH）和独立的核苷二磷酸连接到 x（nudix）水解酶亚家族，它们激活第三组 ADH，尚未得到充分描述。在这里，我们报告了一种从铜绿假单胞菌克隆的第三组 ADH，即 γ-羟基丁酸脱氢酶（GHBDH，UniProt：Q59104）的稳态初始速度正向（醇→醛）反应。我们还报告了 nudix 水解酶对 GHBDH 反应的影响。在最佳 pH 9.0 下，两种不同的饱和纯化 nudix 水解酶，即巴氏甲烷激活酶（ACT，UniProt：I3EA59）和大肠杆菌 NudF（UniProt Q93K97）蛋白，使 GHBDH 反应激活约 2 倍。在生理 pH 值~7.0 下，ACT 激活超过 3.5 倍。在 pH 9.0 下对正向未激活和 ACT 激活反应的初始速率特征表明，对于两种情况，产物琥珀酸半醛对 GHB 的竞争抑制和另外三种底物-产物组合的非竞争性抑制。这种模式与 NAD 结合首先在单-Iso Theorell-Chance 动力学中一致。GHBDH 中一些可能重要残基的突变体也得到了表征。在所有第三组 ADH 中保守的 H265，以前被提议为关键的广义碱基，对 GHBDH 活性的帮助仅约为 4 倍，与 H265A 相关。以前提出的四个保守的 Fe 螯合剂（D193、H197、H261 和 H280）对 GHBDH 活性都是必不可少的。提出了一个两步解释，用于解释 ACT 在正向反应中的亚化学计量刺激以及在反向反应中缺乏 ACT 刺激的现象。
