Bifunctional Homodimeric Triokinase/FMN Cyclase

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.

Closure of the Human TKFC Active Site: Comparison of the Apoenzyme and the Complexes Formed with Either Triokinase or FMN Cyclase Substrates

“…Citrobacter DHA kinase has a known crystal structure with a unique fold [ 16 ] that defines Group 9 in the classification of kinases [ 17 , 18 ]. This enzyme, and the hTKFC model constructed by homology [ 2 ], show up a homodimer (in agreement with size exclusion chromatography data for hTKFC [ 2 ]), each subunit with two domains, K and L, linked by a long spacer ( Figure 1 ). The intertwined subunits form an elongated L2-K1-K2-L1 arrangement.…”

“…It has been suggested that K- and L-domains, connected just by a long spacer, could move with respect to each other, and that domain mobility would be required for kinase activity [10,16]. The relative mobility of both domains has been actually shown by us in a previous work with the homology model of hTKFC, where, in the course of a 75-ns trajectory of molecular dynamics, transient ATP-to-DHA approximations are seen in the L2-K1 site, displaying apparent near-attack conformations that, however, last very shortly [2].…”

“…Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) is a bifunctional enzyme which catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde (GA) and dihydroxyacetone (DHA), and also the Mn 2+ -dependent splitting of flavin adenine dinucleotide (FAD) by an internal cyclizing reaction that forms adenosine monophosphate (AMP) and riboflavin 4′,5′-phosphate or cyclic FMN [ 1 , 2 ]. GA kinase represents the third step of fructose metabolism [ 3 , 4 , 5 ] and DHA kinase is necessary for the metabolism of exogenous DHA [ 6 , 7 , 8 , 9 ] and the eventually generated endogenous DHA [ 10 ].…”

“…GA kinase represents the third step of fructose metabolism [ 3 , 4 , 5 ] and DHA kinase is necessary for the metabolism of exogenous DHA [ 6 , 7 , 8 , 9 ] and the eventually generated endogenous DHA [ 10 ]. On the other hand, the biological role of the cyclase activity is unknown [ 2 , 11 , 12 ]. hTKFC is functionally and structurally similar to Citrobacter sp.…”

Phosphorylation Catalyzed by Dihydroxyacetone Kinase

“…The recent discovery of a glyceraldehyde‐phosphorylating function to a human triokinase shows the importance of this enzyme function in d ‐fructose utilization for human health and is of much interest to the molecular understanding of the Hers metabolic pathway …”