Bifunctional Homodimeric Triokinase/FMN Cyclase

• Published in: The Journal of biological chemistry Vol. 289; no. 15; pp. 10620 - 10636
• Main Authors: Rodrigues, Joaquim Rui, Couto, Ana, Cabezas, Alicia, Pinto, Rosa María, Ribeiro, João Meireles, Canales, José, Costas, María Jesús, Cameselle, José Carlos
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 11.04.2014
• Subjects: Bifunctional Kinase/Cyclase; Cyclic FMN; DAK Gene; Dihydroxyacetone Phosphorylation; FAD; Fructose Metabolism; Glyceraldehyde Phosphorylation; Molecular Docking; Molecular Dynamics; Mutagenesis, Site-specific; Glyceraldehyde Phosphorylation; Molecular Dynamics; DAK Gene; Cyclic FMN; Molecular Docking; FAD; Bifunctional Kinase/Cyclase; Dihydroxyacetone Phosphorylation; Fructose Metabolism; Mutagenesis, Site-specific
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.525626

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. Background: Triokinase, which phosphorylates dihydroxyacetone and fructose-derived glyceraldehyde, remains molecularly unidentified. Results: Human DAK gene encodes homodimeric triokinase/FMN cyclase formed by two-domain subunits. Although kinase activity requires intact homodimers, cyclase requires only a truncated, single domain subunit. Conclusion: Triokinase/FMN cyclase identity and bifunctionality are established. Significance: This study molecularly dissects a bifunctional enzyme of unusual specificity and finishes the molecular identification of fructose pathway enzymes.