Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis
The last step in the biosynthesis of flavin adenine dinucleotide (FAD) is considered a target for the design of antimicrobial drugs because it is carried out by two non-homologous proteins in eukaryotic and prokaryotic organisms. Monofunctional FMN: adenylyltransferases (FMNAT) in Eukarya and FMNAT...
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2020-05-01
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author | Ana Serrano Sonia Arilla-Luna Milagros Medina |
author_facet | Ana Serrano Sonia Arilla-Luna Milagros Medina |
author_sort | Ana Serrano |
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description | The last step in the biosynthesis of flavin adenine dinucleotide (FAD) is considered a target for the design of antimicrobial drugs because it is carried out by two non-homologous proteins in eukaryotic and prokaryotic organisms. Monofunctional FMN: adenylyltransferases (FMNAT) in Eukarya and FMNAT modules of bifunctional FAD synthases (FADS) in Prokarya belong to different structural families with dissimilar chemistry and binding modes for the substrates. In this study, we analyzed the relevance of the hydrophobic environment of the flavin isoalloxazine in the FMNAT active site of <i>Corynebacterium ammoniagenes</i> FADS (<i>Ca</i>FADS) through the mutational analysis of its F62, Y106, and F128 residues. They form the isoalloxazine binding cavity and are highly conserved in the prokaryotic FADS family. The spectroscopic, steady-state kinetics and thermodynamic data presented indicate that distortion of aromaticity at the FMNAT isoalloxazine binding cavity prevents FMN and FAD from correct accommodation in their binding cavity and, as a consequence, decreases the efficiency of the FMNAT activity. Therefore, the side-chains of F62, Y106 and F128 are relevant in the formation of the catalytic competent complex during FMNAT catalysis in <i>Ca</i>FADS. The introduced mutations also modulate the activity occurring at the riboflavin kinase (RFK) module of <i>Ca</i>FADS, further evidencing the formation of quaternary assemblies during catalysis. |
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spelling | doaj.art-a413084b77cc41a5857c22086fd1a7d32023-11-20T01:43:10ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-05-012110373810.3390/ijms21103738Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and CatalysisAna Serrano0Sonia Arilla-Luna1Milagros Medina2Department of Biochemistry and Molecular and Cellular Biology, Faculty of Sciences, and Institute of Biocomputation and Physics of Complex Systems (Joint Units: BIFI-IQFR and GBsC-CSIC), University of Zaragoza, E-50009 Zaragoza, SpainDepartment of Biochemistry and Molecular and Cellular Biology, Faculty of Sciences, and Institute of Biocomputation and Physics of Complex Systems (Joint Units: BIFI-IQFR and GBsC-CSIC), University of Zaragoza, E-50009 Zaragoza, SpainCentro de Investigaciones Biológicas Margarita Salas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, SpainThe last step in the biosynthesis of flavin adenine dinucleotide (FAD) is considered a target for the design of antimicrobial drugs because it is carried out by two non-homologous proteins in eukaryotic and prokaryotic organisms. Monofunctional FMN: adenylyltransferases (FMNAT) in Eukarya and FMNAT modules of bifunctional FAD synthases (FADS) in Prokarya belong to different structural families with dissimilar chemistry and binding modes for the substrates. In this study, we analyzed the relevance of the hydrophobic environment of the flavin isoalloxazine in the FMNAT active site of <i>Corynebacterium ammoniagenes</i> FADS (<i>Ca</i>FADS) through the mutational analysis of its F62, Y106, and F128 residues. They form the isoalloxazine binding cavity and are highly conserved in the prokaryotic FADS family. The spectroscopic, steady-state kinetics and thermodynamic data presented indicate that distortion of aromaticity at the FMNAT isoalloxazine binding cavity prevents FMN and FAD from correct accommodation in their binding cavity and, as a consequence, decreases the efficiency of the FMNAT activity. Therefore, the side-chains of F62, Y106 and F128 are relevant in the formation of the catalytic competent complex during FMNAT catalysis in <i>Ca</i>FADS. The introduced mutations also modulate the activity occurring at the riboflavin kinase (RFK) module of <i>Ca</i>FADS, further evidencing the formation of quaternary assemblies during catalysis.https://www.mdpi.com/1422-0067/21/10/3738flavin biosynthesisprokaryotic FAD synthaseeukaryotic ATP:FMN:adenylyltransferasearomatic residuesisoalloxazinebinding |
spellingShingle | Ana Serrano Sonia Arilla-Luna Milagros Medina Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis International Journal of Molecular Sciences flavin biosynthesis prokaryotic FAD synthase eukaryotic ATP:FMN:adenylyltransferase aromatic residues isoalloxazine binding |
title | Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis |
title_full | Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis |
title_fullStr | Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis |
title_full_unstemmed | Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis |
title_short | Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis |
title_sort | insights into the fmnat active site of fad synthase aromaticity is essential for flavin binding and catalysis |
topic | flavin biosynthesis prokaryotic FAD synthase eukaryotic ATP:FMN:adenylyltransferase aromatic residues isoalloxazine binding |
url | https://www.mdpi.com/1422-0067/21/10/3738 |
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