Bifunctional Homodimeric Triokinase/FMN Cyclase: CONTRIBUTION OF PROTEIN DOMAINS TO THE ACTIVITIES OF THE HUMAN ENZYME AND MOLECULAR DYNAMICS SIMULATION OF DOMAIN MOVEMENTS* |
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Authors: | Joaquim Rui Rodrigues Ana Couto Alicia Cabezas Rosa María Pinto Jo?o Meireles Ribeiro José Canales María Jesús Costas José Carlos Cameselle |
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Affiliation: | From the ‡Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina, Universidad de Extremadura, E-06006 Badajoz, Spain and ;the §Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Leiria, P-2411-901 Leiria, Portugal |
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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 Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. |
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Keywords: | FAD Fructose Metabolism Molecular Docking Molecular Dynamics Mutagenesis Site-specific Bifunctional Kinase/Cyclase Cyclic FMN DAK Gene Dihydroxyacetone Phosphorylation Glyceraldehyde Phosphorylation |
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