Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes

Abstract Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-terminal and N-terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine di...

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Autores principales:	María Sebastián, Ana Serrano, Adrián Velázquez-Campoy, Milagros Medina
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/47982cd4148545978d3dd530479c520b
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spelling	oai:doaj.org-article:47982cd4148545978d3dd530479c520b2021-12-02T15:05:04ZKinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes10.1038/s41598-017-07875-52045-2322https://doaj.org/article/47982cd4148545978d3dd530479c520b2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07875-5https://doaj.org/toc/2045-2322Abstract Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-terminal and N-terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These act as cofactors of a plethora of flavoproteins in all organisms. Therefore, regulation of their production maintains the cellular flavoproteome homeostasis. Here, we focus on regulation of the FMN synthesis in Corynebacterium ammoniagenes (Ca) by the inhibition of its RFK activity by substrates and products of the reaction. We use a truncated CaFADS variant consisting in the isolated C-terminal RFK module, whose RFK activity is similar to that of the full-length enzyme. Inhibition of the RFK activity by the RF substrate is independent of the FMNAT module, and FMN production, in addition to being inhibited by an excess of RF, is also inhibited by both of the reaction products. Pre-steady-state kinetic and thermodynamic studies reveal key aspects to the substrates induced fit to produce the catalytically competent complex. Among them, the role of Mg2+ in the concerted allocation of substrates for catalysis and the ensemble of non-competent complexes that contribute to the regulated inhibition of the RFK activity are particularly relevant.María SebastiánAna SerranoAdrián Velázquez-CampoyMilagros MedinaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q María Sebastián Ana Serrano Adrián Velázquez-Campoy Milagros Medina Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes
description	Abstract Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-terminal and N-terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These act as cofactors of a plethora of flavoproteins in all organisms. Therefore, regulation of their production maintains the cellular flavoproteome homeostasis. Here, we focus on regulation of the FMN synthesis in Corynebacterium ammoniagenes (Ca) by the inhibition of its RFK activity by substrates and products of the reaction. We use a truncated CaFADS variant consisting in the isolated C-terminal RFK module, whose RFK activity is similar to that of the full-length enzyme. Inhibition of the RFK activity by the RF substrate is independent of the FMNAT module, and FMN production, in addition to being inhibited by an excess of RF, is also inhibited by both of the reaction products. Pre-steady-state kinetic and thermodynamic studies reveal key aspects to the substrates induced fit to produce the catalytically competent complex. Among them, the role of Mg2+ in the concerted allocation of substrates for catalysis and the ensemble of non-competent complexes that contribute to the regulated inhibition of the RFK activity are particularly relevant.
format	article
author	María Sebastián Ana Serrano Adrián Velázquez-Campoy Milagros Medina
author_facet	María Sebastián Ana Serrano Adrián Velázquez-Campoy Milagros Medina
author_sort	María Sebastián
title	Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes
title_short	Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes
title_full	Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes
title_fullStr	Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes
title_full_unstemmed	Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes
title_sort	kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the fad synthetase from corynebacterium ammoniagenes
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/47982cd4148545978d3dd530479c520b
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Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes

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