Crystal structure of ADP-dependent glucokinase from Methanocaldococcus jannaschii in complex with 5-iodotubercidin reveals phosphoryl transfer mechanism

Abstract

ADP-dependent glucokinase (ADPGK) is an alternative novel glucose phosphorylating enzyme in a modified glycolysis pathway of hyperthermophilic Archaea. In contrast to classical ATP-dependent hexokinases, ADPGK utilizes ADP as a phosphoryl group donor. Here, we present a crystal structure of archaeal ADPGK from Methanocaldococcus jannaschii in complex with an inhibitor, 5-iodotubercidin, d-glucose, inorganic phosphate, and a magnesium ion. Detailed analysis of the architecture of the active site allowed for confirmation of the previously proposed phosphorylation mechanism and the crucial role of the invariant arginine residue (Arg197). The crystal structure shows how the phosphate ion, while mimicking a β-phosphate group, is positioned in the proximity of the glucose moiety by arginine and the magnesium ion, thus providing novel insights into the mechanism of catalysis. In addition, we demonstrate that 5-iodotubercidin inhibits human ADPGK-dependent T cell activation-induced reactive oxygen species (ROS) release and downstream gene expression, and as such it may serve as a model compound for further screening for hADPGK-specific inhibitors.

Keywords: 5-iodotubercidin; ADP-dependent glucokinase; glycolysis; kinase inhibitor.

Figure 1

The overall structure of mjADPGK. (A) Stereo side view of protein architecture with indication of small and large domains. (B) 5‐iodotubercidin, glucose, and phosphate ion are located at the active site found between small and large domains. Protein is colored from N‐ (blue) to C‐terminus (red).

Figure 2

The architecture of mjADPGK active site. (A) Close‐up view on mjADPGK active site. 5‐iodotubercidin (blue), inorganic phosphate (orange), magnesium ion (red), and glucose (yellow) are indicated. Most prominent interactions of iodine atom and Ser195 and inorganic phosphate and Arg195 are shown by dashed lines. Water molecules are depicted as grey spheres, nucleotide binding loop is shown as green cartoon. 2F_o‐F_c electron density map contoured at 1σ is shown as grey mesh. (B) Major interactions guiding 5‐iodotubercidin binding. (C) Details of glucose, phosphate, and magnesium binding sites. (D) Superposition of mjADPGK and tlADPGK (PDB:4B8S) active sites. 5ITU, glucose, and side‐chains from M. jannashii are colored in blue, yellow, and wheat respectively. AMP, glucose and Arg205 from T. litoralis ADPGK are colored in teal. Inorganic phosphate from mjADPGK occupies the putative position of ADP β‐phosphate.

Figure 3

5‐iodotubercidin (5ITU) inhibits T cell activation‐induced ROS generation and subsequent NF‐κB‐dependent gene expression. (A) Jurkat T cells stained with H₂DCF‐DA and pre‐treated (30 min) with respective concentrations of 5ITU were activated by phorbol 12‐myristate 13‐acetate (PMA) treatment (1 hr) and the ‘oxidative signal’ was measured by fluorescence‐activated cell sorting (FACS) (mean values +/‐ SD). (B) 5ITU ‐pre‐treated (30 min) Jurkat T cells were activated by PMA/ionomycin for 1 hr. Next, IL‐2 and IκBα gene expression was assayed by RT‐PCR (mean values +/‐ SD). (C) Jurkat T cells were treated with +/‐ 5ITU for 4 hr and cell death was assayed by FACS measeurement of PI+ cells (mean values +/‐ SD).