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.

Structural Characterization of Glycerol Kinase from the Thermophilic Fungus Chaetomium thermophilum

“…Using a well-established pyruvate kinase/lactate dehydrogenase-coupled activity assay ( Figure 3 ), we measured the apparent Km value for ATP (0.052 ± 0.009 mM) ( Figure 4 A). Then, encouraged by our previous work on ADP-dependent glucokinases (ADPGKs) [ 18 ] and further studies by Ueda and Sakasegawa employing ADPGK to measure creatine kinase activity [ 19 ], we decided to test whether ADPGK-coupled enzymatic assay could be used to assess the CtGK activity. ADPGK was first described in hyperthermophilic archaea, where, together with ADP-dependent phosphofructokinase (ADP-PFK), it takes part in a modified glycolysis pathway [ 20 , 21 ].…”

“…To uncover the structural determinants of nucleotide-binding by CtGK, we attempted to crystalize either binary CtGK–ATP or ternary CtGK–ATP–glycerol complexes. Despite significant efforts, including co-crystallization or ligand soaking procedures, and testing the known nucleotide-mimicking compounds such as 8-Br-AMP, 5-ITU, or AMPNP [ 18 , 28 , 29 ], no electron density allowing for reliable nucleotide modeling was identified in any of the analyzed crystals. However, taking into account the high structural similarity between CtGK and PfGK, especially in the active site, we can infer the most likely position of the ATP by comparison of these two structures.…”

“…MjADPGK and phADPGK/PFK were obtained as previously described [ 18 , 28 ]. Briefly, genes encoding full-length mjADPGK and phADPGK were synthesized (GenScript, Leiden, Netherlands) and cloned into pET24d and pET15b plasmid, respectively.…”

Crystal Structure of Kluyveromyces lactis Glucokinase (KlGlk1)

“…Kl Glk1 activity was assessed by monitoring the level of one of the reaction products (glucose-6-phosphate) in a coupled glucose-6-phosphate dehydrogenase (G6PD) reaction essentially as described previously [5,6]. Unless stated otherwise, the assays were performed at 37 °C in reaction buffer (20 mM Tris-HCl pH 7.4, 250 mM sucrose, 50 mM KCl, 5 mM MgCl 2 ) supplemented with glucose-6-phosphate dehydrogenase (1 unit) and nicotinamide adenine dinucleotide phosphate (NADP) (0.5 mM).…”

“…Glucose phosphorylation is also considered as drug target against parasitic protists [1,2,3]. Additionally, some ADP-dependent sugar kinases were also characterized, but their role remains more elusive [4,5,6].…”