title: Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.
abstract: Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain; Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Leiria, P-2411-901 Leiria, Portugal.
authors.forename: Joaquim Rui
authors.initials: JR
authors.lastname: Rodrigues
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Couto
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Cabezas
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain.
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Pinto
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain.
authors.forename: João Meireles
authors.initials: JM
authors.lastname: Ribeiro
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Canales
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Costas
authors.affiliation: Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain. Electronic address: camselle@unex.es.
authors.forename: José Carlos
authors.initials: JC
authors.lastname: Cameselle
mesh_terms: D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005632:Fructose / Q000737:chemistry; D005985:Glyceraldehyde / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry* / Q000502:physiology*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013552:Swine
chemical_list: D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005632:Fructose; D005985:Glyceraldehyde; D017853:Phosphotransferases (Alcohol Group Acceptor); C023190:triokinase; D019761:Phosphorus-Oxygen Lyases; C111330:FMN cyclase
keywords: Bifunctional Kinase/Cyclase; Cyclic FMN; DAK Gene; Dihydroxyacetone Phosphorylation; FAD; Fructose Metabolism; Glyceraldehyde Phosphorylation; Molecular Docking; Molecular Dynamics; Mutagenesis, Site-specific