Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu, 241000, Anhui, China; College of Biological and Food Engineering, Anhui Polytechnic University, Wuhu, 241000, Anhui, China.
Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu, 241000, Anhui, China.
Biochimie. 2021 Feb;181:77-85. doi: 10.1016/j.biochi.2020.12.001. Epub 2020 Dec 5.
Acinetobacter baumannii encodes all enzymes required in the tricarboxylic acid (TCA) cycle and glyoxylate bypass except for isocitrate dehydrogenase kinase/phosphatase (IDHKP), which can phosphorylate isocitrate dehydrogenase (IDH) at a substrate-binding Ser site and control the carbon flux in enterobacteria, such as Escherichia coli. The potential kinase was not successfully pulled down from A. baumannii cell lyase; therefore, whether the IDH 1 from A. baumannii (AbIDH1) can be phosphorylated to regulate intracellular carbon flux has not been clarified. Herein, the AbIDH1 gene was cloned, the encoded protein was expressed and purified to homogeneity, and phosphorylation and enzyme kinetics were evaluated in vitro. Gel filtration and SDS-PAGE analyses showed that AbIDH1 is an 83.5 kDa homodimer in solution. The kinetics showed that AbIDH1 is a fully active NADP-dependent enzyme. The Michaelis constant K is 46.6 (Mn) and 18.1 μM (Mg) for NADP and 50.5 (Mn) and 65.4 μM (Mg) for the substrate isocitrate. Phosphorylation experiments in vitro indicated that AbIDH1 is a substrate for E. coli IDHKP. The activity of AbIDH1 treated with E. coli IDHKP immediately decreased by 80% within 9 min. Mass spectrometry indicated that the conserved Ser113 of AbIDH1 is phosphorylated. Continuous phosphorylation-mimicking mutants (Ser113Glu and Ser113Asp) lack almost all enzymatic activity. Side-chain mutations at Ser113 (Ser113Thr, Ser113Ala, Ser113Gly and Ser113Tyr) remarkably reduce the enzymatic activity. Understanding the potential of AbIDH1 phosphorylation enables further investigations of the AbIDH1 physiological functions in A. baumannii.
鲍曼不动杆菌编码三羧酸 (TCA) 循环和乙醛酸旁路所需的所有酶，除了异柠檬酸脱氢酶激酶/磷酸酶 (IDHKP) 外，后者可以在底物结合 Ser 位点磷酸化异柠檬酸脱氢酶 (IDH)，并控制大肠杆菌等肠杆菌中的碳通量。未能从鲍曼不动杆菌细胞裂解物中成功提取潜在的激酶；因此，鲍曼不动杆菌的 IDH1（AbIDH1）是否可以被磷酸化以调节细胞内碳通量尚不清楚。在此，克隆了 AbIDH1 基因，表达并纯化了编码的蛋白质至均相，并在体外评估了磷酸化和酶动力学。凝胶过滤和 SDS-PAGE 分析表明，AbIDH1 在溶液中是 83.5 kDa 的同二聚体。动力学表明，AbIDH1 是一种完全活性的 NADP 依赖性酶。对于 NADP 的米氏常数 K 为 46.6（Mn）和 18.1 μM（Mg），对于底物异柠檬酸的米氏常数 K 为 50.5（Mn）和 65.4 μM（Mg）。体外磷酸化实验表明，AbIDH1 是大肠杆菌 IDHKP 的底物。用大肠杆菌 IDHKP 处理的 AbIDH1 的活性在 9 分钟内立即下降了 80%。质谱分析表明，AbIDH1 的保守 Ser113 被磷酸化。连续磷酸化模拟突变体（Ser113Glu 和 Ser113Asp）几乎丧失了所有酶活性。Ser113 侧链突变（Ser113Thr、Ser113Ala、Ser113Gly 和 Ser113Tyr）显著降低了酶活性。了解 AbIDH1 磷酸化的潜力可以进一步研究 AbIDH1 在鲍曼不动杆菌中的生理功能。
