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

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 meta...

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Autores principales: Rivero, Maribel, Boneta, Sergio, Novo, Nerea, Velázquez-Campoy, Adrián, Polo, Victor, Medina, Milagros
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10086132/
https://www.ncbi.nlm.nih.gov/pubmed/37056721
http://dx.doi.org/10.3389/fmolb.2023.1167348
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author Rivero, Maribel
Boneta, Sergio
Novo, Nerea
Velázquez-Campoy, Adrián
Polo, Victor
Medina, Milagros
author_facet Rivero, Maribel
Boneta, Sergio
Novo, Nerea
Velázquez-Campoy, Adrián
Polo, Victor
Medina, Milagros
author_sort Rivero, Maribel
collection PubMed
description 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.
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spelling pubmed-100861322023-04-12 Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery Rivero, Maribel Boneta, Sergio Novo, Nerea Velázquez-Campoy, Adrián Polo, Victor Medina, Milagros Front Mol Biosci Molecular Biosciences 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. Frontiers Media S.A. 2023-03-28 /pmc/articles/PMC10086132/ /pubmed/37056721 http://dx.doi.org/10.3389/fmolb.2023.1167348 Text en Copyright © 2023 Rivero, Boneta, Novo, Velázquez-Campoy, Polo and Medina. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Rivero, Maribel
Boneta, Sergio
Novo, Nerea
Velázquez-Campoy, Adrián
Polo, Victor
Medina, Milagros
Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery
title Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery
title_full Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery
title_fullStr Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery
title_full_unstemmed Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery
title_short Riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery
title_sort riboflavin kinase and pyridoxine 5′-phosphate oxidase complex formation envisages transient interactions for fmn cofactor delivery
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10086132/
https://www.ncbi.nlm.nih.gov/pubmed/37056721
http://dx.doi.org/10.3389/fmolb.2023.1167348
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