Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity

Serrano, Ana; Sebastián, María; Arilla-Luna, Sonia; Baquedano, Silvia; Pallarés, María Carmen; Lostao, Anabel; Herguedas, B.; Velázquez‐Campoy, Adrián; Martínez‐Júlvez, Marta; Medina, Milagros

doi:10.1016/j.bbapap.2015.03.005

Cited by 19 publications

(36 citation statements)

References 33 publications

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“…Most mutants primarily purified as monomers, with a small population of oligomeric species (Figure SP1), similar to WT Ca FADS 8, 19 . Far-ultraviolet (UV) circular dichroism (CD) spectra were similar to those of the WT 20 , indicating minor impact of the mutations on the enzyme’s secondary structure (Figures SP2A and SP2B).…”

Section: Resultsmentioning

confidence: 86%

“…The most significant changes of peak shapes in the spectra were observed when titrating with FAD and FMN, particularly for the F206W, D298E and E301K variants (Figures SP3C–SP3F), suggesting different environments of the isoalloxazine ring in the flavin binding site at the FMNAT module. The increase in the magnitude of the difference spectra upon titration of the preformed WT Ca FADS:ADP:Mg 2+ complex with FMN (~8-times higher than in the absence of ADP) is related to the formation of the FMN binding site at the RFK module 16, 19, 20 . Noticeably, this effect was smaller for most of our variants and undetectable for E301K (Figures SP3G and SP3H).…”

Section: Resultsmentioning

confidence: 97%

“…Previous studies showed that monomeric WT Ca FADS, incubated with the products of the RFK activity (FMN and ADP in the presence of Mg 2+ ), stabilizes a transient dimer of trimers, which mostly dissociates into the monomer form upon the ligands’ removal by gel filtration chromatography 8, 19 . This is due to the protomers of Ca FADS rapidly assembling in response to ligand binding, and disassembling when ligands are eliminated.…”

Section: Resultsmentioning

confidence: 99%

“…However, a small fraction of oligomers always remains after gel filtration, which probably reflects the presence of poorly fit assemblies with a large kinetic barrier to dissociation. This might be a consequence of the thermodynamic complexity of the system (see text and data below), which forms other assemblies than the transient dimer of trimers 8, 19 . In practice, these observations allow the evaluation of whether the introduced mutations modulate assembly and disassembly profiles of WT Ca FADS 8 .…”

Section: Resultsmentioning

confidence: 99%

“…L1c-FlapI, L6c and α1c, together with loop L4c-FlapII, form the closed conformation of the RFK flavin-binding site that is reached after being triggered by substrate/product binding (Figure 1B) 9 . In addition, a salt-bridge between the catalytic base at the RFK module, E268, and R66 at loop L4n stabilizes each trimer within the dimer of trimers 16, 19 , while residues at the RFK catalytic site modulate binding parameters and catalytic efficiency at the FMNAT active site 16 .…”

Section: Introductionmentioning

confidence: 99%

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The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes

Serrano

Sebastián

Arilla-Luna

et al. 2017

Sci Rep

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Bifunctional FAD synthetases (FADSs) fold in two independent modules; The C-terminal riboflavin kinase (RFK) catalyzes the RFK activity, while the N-terminal FMN-adenylyltransferase (FMNAT) exhibits the FMNAT activity. The search for macromolecular interfaces in the Corynebacterium ammoniagenes FADS (CaFADS) crystal structure predicts a dimer of trimers organization. Within each trimer, a head-to-tail arrangement causes the RFK and FMNAT catalytic sites of the two neighboring protomers to approach, in agreement with active site residues of one module influencing the activity at the other. We analyze the relevance of the CaFADS head-to-tail macromolecular interfaces to stabilization of assemblies, catalysis and ligand binding. With this aim, we evaluate the effect of point mutations in loop L1c-FlapI, loop L6c, and helix α1c of the RFK module (positions K202, E203, F206, D298, V300, E301 and L304), regions at the macromolecular interface between two protomers within the trimer. Although none of the studied residues is critical in the formation and dissociation of assemblies, residues at L1c-FlapI and helix α1c particularly modulate quaternary architecture, as well as ligand binding and kinetic parameters involved with RFK and FMNAT activities. These data support the influence of transient oligomeric structures on substrate accommodation and catalysis at both CaFADS active sites.

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes

Serrano

Sebastián

Arilla-Luna

et al. 2017

Sci Rep

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Cofactors and pathogens: Flavin mononucleotide and flavin adenine dinucleotide (FAD) biosynthesis by the FAD synthase from Brucella ovis

et al. 2021

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The biosynthesis of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), cofactors used by 2% of proteins, occurs through the sequential action of two ubiquitous activities: a riboflavinkinase (RFK) that phosphorylates the riboflavin (RF) precursor to FMN, and a FMN:adenylyltransferase (FMNAT) that transforms FMN into FAD. In most mammals two different monofunctional enzymes have each of these activities, but in prokaryotes a single bifunctional enzyme, FAD synthase (FADS), holds them. Differential structural and functional traits for RFK and FMNAT catalysis between bacteria and mammals, as well as within the few bacterial FADSs so far characterized, has envisaged the potentiality of FADSs from pathogens as targets for the development of species-specific inhibitors. Here, we particularly characterize the FADS from the ovine pathogen Brucella ovis (BoFADS), causative agent of brucellosis. We show that BoFADS has RFK activity independently of the media redox status, but its FMNAT activity (in both forward and reverse senses) only occurs under strong reducing conditions. Moreover, kinetics for flavin and adenine nucleotides binding to the RFK site show that BoFADS binds preferentially the substrates of the RFK reaction over the products and that the adenine nucleotide must bind prior to flavin entrapment. These results, together with multiple

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Atomic Force Microscopy: Single-Molecule Imaging and Force Spectroscopy in the Study of Flavoproteins Ligand Binding and Reaction Mechanisms

Lostao

Medina

2021

Methods in Molecular Biology

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Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity

Cited by 19 publications

References 33 publications

The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes

The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes

Cofactors and pathogens: Flavin mononucleotide and flavin adenine dinucleotide (FAD) biosynthesis by the FAD synthase from Brucella ovis

Atomic Force Microscopy: Single-Molecule Imaging and Force Spectroscopy in the Study of Flavoproteins Ligand Binding and Reaction Mechanisms

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