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Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes
Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among spec...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829536/ https://www.ncbi.nlm.nih.gov/pubmed/31614972 http://dx.doi.org/10.3390/ijms20205083 |
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author | Arilla-Luna, Sonia Serrano, Ana Medina, Milagros |
author_facet | Arilla-Luna, Sonia Serrano, Ana Medina, Milagros |
author_sort | Arilla-Luna, Sonia |
collection | PubMed |
description | Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis. |
format | Online Article Text |
id | pubmed-6829536 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-68295362019-11-18 Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes Arilla-Luna, Sonia Serrano, Ana Medina, Milagros Int J Mol Sci Article Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis. MDPI 2019-10-14 /pmc/articles/PMC6829536/ /pubmed/31614972 http://dx.doi.org/10.3390/ijms20205083 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Arilla-Luna, Sonia Serrano, Ana Medina, Milagros Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes |
title | Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes |
title_full | Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes |
title_fullStr | Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes |
title_full_unstemmed | Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes |
title_short | Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes |
title_sort | specific features for the competent binding of substrates at the fmn adenylyltransferase site of fad synthase from corynebacterium ammoniagenes |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829536/ https://www.ncbi.nlm.nih.gov/pubmed/31614972 http://dx.doi.org/10.3390/ijms20205083 |
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