title: Bifunctional ADP-dependent phosphofructokinase/glucokinase activity in the order Methanococcales--biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis.
abstract: 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 phosphorylate 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 phosphofructokinase/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.
authors.forename: Victor
authors.initials: V
authors.lastname: Castro-Fernandez
authors.forename: Felipe
authors.initials: F
authors.lastname: Bravo-Moraga
authors.forename: Alejandra
authors.initials: A
authors.lastname: Herrera-Morande
authors.forename: Victoria
authors.initials: V
authors.lastname: Guixe
mesh_terms: D005941:Glucokinase / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D005943:Gluconeogenesis; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology*; D025221:Phosphofructokinases / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D010802:Phylogeny
chemical_list: D025221:Phosphofructokinases; D005941:Glucokinase; D005947:Glucose