ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.

In some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphory-late glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a divalent metal cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg 2+ concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phospho-fructokinase/glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein-ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADP-dependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases. Database Model data are available in the Protein Model Data Base under accession numbers PM0079106, PM0079107, PM0079108, PM0079109, PM0079110, PM0079111, PM0079112, PM0079113, PM0079114, PM0079115 and PM0079116 Abbreviations EM, Embden-Meyerhof; fructose 1,6-bisP, fructose 1,6-bisphosphate; fructose 6P, fructose 6-phosphate; GK, glucokinase; glucose 6P, glucose 6-phosphate; MmPFK/GK, bifunctional phosphofructokinase/glucokinase enzyme from Methanococcus maripaludis; PFK, phosphofructokinase.

Edited by Joseph Jez This work was supported in part by Fondo Nacional de Desarrollo Cientifico y Tecnológico from Chile Grants FONDECYT 1150460 (to V. G.) and FOND-ECYT Postdoctorado 3160332 (to V. C-F.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Figs. S1-S5 and Tables S1-S5. The atomic coordinates and structure factors (codes 5K27 and 5KKG) have been deposited in the Protein Data Bank (http://wwpdb.org/).

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