Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery

Abstract

Enzymes catalysing sequential reactions have developed different mechanisms to control the transport and flux of reactants and intermediates along metabolic pathways, which usually involve direct transfer of metabolites from an enzyme to the next one in a cascade reaction. Despite the fact that metabolite or substrate channelling has been widely studied for reactant molecules, such information is seldom available for cofactors in general, and for flavins in particular. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) act as cofactors in flavoproteins and flavoenzymes involved in a wide range of physiologically relevant processes in all type of organisms. Homo sapiens riboflavin kinase (RFK) catalyses the biosynthesis of the flavin mononucleotide cofactor, and might directly interplay with its flavin client apo-proteins prior to the cofactor transfer. Non-etheless, none of such complexes has been characterized at molecular or atomic level so far. Here, we particularly evaluate the interaction of riboflavin kinase with one of its potential FMN clients, pyridoxine-5′-phosphate oxidase (PNPOx). The interaction capacity of both proteins is assessed by using isothermal titration calorimetry, a methodology that allows to determine dissociation constants for interaction in the micromolar range (in agreement with the expected transient nature of the interaction). Moreover, we show that; i) both proteins become thermally stabilized upon mutual interaction, ii) the tightly bound FMN product can be transferred from RFK to the apo-form of PNPOx producing an efficient enzyme, and iii) the presence of the apo-form of PNPOx slightly enhances RFK catalytic efficiency. Finally, we also show a computational study to predict likely RFK-PNPOx binding modes that can envisage coupling between the FMN binding cavities of both proteins for the potential transfer of FMN.

Pyridoxal 5′-Phosphate Biosynthesis by Pyridox-(am)-ine 5′-Phosphate Oxidase: Species-Specific Features

“…In HsPNPOx, such a patch might be formed by the highly conserved Arg258 of one protomer together with Arg181 and Arg185 situated in the other protomer in one of the helices that undergo disorder upon PLP binding at the allosteric site in EcPNPOx ( Figure 6 ). Notably, molecular dynamics simulations in HsPNPOx identified the 181–195 α6 helix and the 195–198 β-turn at the interdomain cap folding out of the PNPOx and PNP_phzG_C domains as the most flexible region of the protein [ 31 ]. Arg181 at the N-terminus of the longest helix is conserved in ScPNPOx and MtPNPOx (as Arg160 and Arg162, respectively) but not Arg185.…”

“…Similarly to PLP, the FMN cofactor plays a crucial role in assisting the function of numerous flavoproteins and flavoenzymes within the cell (around 25% of flavoproteins use FMN in Homo sapiens ) while maintaining its free concentration at a low level. FMN synthesis is catalysed by riboflavin kinase (RFK), and the transient interaction between RFK and PNPOx has been confirmed in the case of H. sapiens enzymes [ 31 , 68 ]. Furthermore, these studies have demonstrated the transfer of tightly bound FMN from HsRFK to apo-HsPNPOx, as well as the mutual influence between the two proteins in catalysis and ligand binding steps.…”

“…Various complex arrangements are envisaged to result from diverse ligation and conformational states expected for HsRFK during catalysis, particularly influenced by changes in the conformation of the loops forming the FMN binding site and its exit channel [ 68 ]. Importantly, predictions suggest that loops in HsPNPOx also contribute to protein–protein fitting, hinting again at transient “loop-mediated” interactions whose reorganization might mediate cofactor transfer [ 31 ]. Altogether, an intricate thermodynamic landscape governing binding and transference, and highly sensitive to particular HsRFK and HsPNPOx ligation states, was proposed to regulate coupling and overall channelling processes between these two proteins [ 31 ].…”

“…Importantly, predictions suggest that loops in HsPNPOx also contribute to protein–protein fitting, hinting again at transient “loop-mediated” interactions whose reorganization might mediate cofactor transfer [ 31 ]. Altogether, an intricate thermodynamic landscape governing binding and transference, and highly sensitive to particular HsRFK and HsPNPOx ligation states, was proposed to regulate coupling and overall channelling processes between these two proteins [ 31 ]. Residues predicted to be involved in this interaction in HsPNPOx were located in α1 (Phe66 and Glu67) and α4-tun (Glu152-Glu153, Glu155, Tyr157-Phe158 and Arg161-Pro162), and to a lesser extent in the β3-β4-hairpin (Phe102 and Arg108).…”

From Metabolism to Vitality: Uncovering Riboflavin’s Importance in Poultry Nutrition

“…FMN is synthesized through the phosphorylation of riboflavin catalyzed by riboflavin kinase in the presence of ATP:Mg 2+ . This conversion represents a major rate-limiting step in FAD biosynthesis [39]. FAD formation occurs as FMN:ATP adenylyl transferase catalytically adenylylates FMN to produce FAD [40].…”