Isocitrate dehydrogenase 1 from Acinetobacter baummanii (AbIDH1) enzymatic characterization and its regulation by phosphorylation.

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.