The Puzzle of Ligand Binding to Corynebacterium ammoniagenes FAD Synthetase

Abstract

In bacteria, riboflavin phosphorylation and subsequent conversion of FMN into FAD are carried out by FAD synthetase, a single bifunctional enzyme. Both reactions require ATP and Mg 2؉ . The N-terminal domain of FAD synthetase appears to be responsible for the adenylyltransferase activity, whereas the C-terminal domain would be in charge of the kinase activity. Binding to Corynebacterium ammoniagenes FAD synthetase of its products and substrates, as well as of several analogues, is analyzed. Binding parameters for adenine nucleotides to each one of the two adenine nucleotide sites are reported. In addition, it is demonstrated for the first time that the enzyme presents two independent flavin sites, each one related with one of the enzymatic activities. The binding parameters of flavins to these sites are also provided. The presence of Mg 2؉ and of both adenine nucleotides and flavins cooperatively modulates the interaction parameters for the other ligands. Our data also suggest that during its double catalytic cycle FAD synthetase must suffer conformational changes induced by adenine nucleotide-Mg 2؉ or flavin binding. They might include not only rearrangement of the different protein loops but also alternative conformations between domains.Flavoproteins are involved in a large variety of biological processes (DNA repair, apoptosis, oxidative phosphorylation, photosynthesis, etc.) and require the riboflavin-derived redox cofactors FMN or FAD for their function. In vivo, riboflavin (RF) 3 is converted into FMN first and then into FAD via the sequential action of an ATP:riboflavin kinase (RFK) (EC 2.7.1.26) and an ATP:FMN adenylyltransferase (EC 2.7.7.2) (see Fig. 1A). Eukaryotes generally use two different enzymes for FMN and FAD production (1-7), whereas most prokaryotes depend on a single bifunctional enzyme, the FAD synthetase (FADS) (8, 9).The two catalytic cycles of FADS involve the binding of two ATP, one RF, and one FMN molecules as substrates and the production of one ADP, one PP i , one FMN, and one FAD. The proposed pathway for the phosphorylation reaction would be for RF to bind before ATP, whereas ADP releases prior FMN (10). In the adenylylation process, FMN is proposed to bind after ATP and the PP i to be released preceding FAD (10). The two enzymatic activities differ in their specificity for divalent cations, optimal pH, and temperature (8,11,12). The presence of Mg 2ϩ improves the turnover of both processes but, although low concentrations (Ͻ1 mM) enhance the kinase activity, much larger concentrations (ϳ10 mM) are required for maximal FAD production (8). These studies indicated the presence of two independent ATP-binding sites, one at the RF phosphorylation site and one at the FMN-adenylylation site, but a single pocket was proposed to allocate the isoalloxazine-ribityl moieties of both substrates, RF and FMN, in the two reactions (10).The only structure reported for an FADS is that of Thermotoga maritima (TmFADS), both free and in complex with several substrates (13,14). One of these structur...

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

“…This is in agreement with the strong affinity of the RFK module for FMN in the presence of ADP (Table 2). 9 These data suggest that once both ATP and FMN are bound to the FMNAT site, the catalytically competent complex active site will be different not only between the monomer and the trimer but also between trimers, depending on whether FMN enters from the external media or from the RFK module of the neighboring protomer. The similar maximal rates obtained in the overall riboflavin transformation exhibited by the monomer and the trimer (in contrast to the large difference obtained when direct entrance of exogenous FMN from the solution into the FMNAT module was performed) might also indicate that, in the quaternary ensemble, the interface between protomers might contribute to the direct transfer of the product of the first reaction (FMN) to the FMNAT catalytic site in the neighboring protomer, where it is a substrate.…”

“…[8][9][10][11][12] Crystal structures of HsRFK and SpRFK revealed large ligand-induced structural rearrangements during catalysis. [8][9][10][11][12] These changes mainly involved two regions, flap I and flap II (L1c and L4c in CaFADS), which, on one hand, close the flavin site after riboflavin binding and, on the other hand, open the adenine nucleotide site (inaccessible in apo form). This catalytically active conformation approaches from the O5′-hydroxyl of riboflavin to the catalytic base (Glu268 in CaFADS) and promotes the coordination of Mg 2+ with oxygens from ATP phosphates and Asn from the consensus PTAN motive (Asn210 in CaFADS).…”

“…It has been reported that, first, one of the modules influences nucleotide binding in the other one; second, the isolated FMNAT module is unable to transform FMN into FAD; and, third, a strong decrease in ligand affinity is observed at the C-terminal module when it is independently expressed. 8,9 Biophysical characterization of the CaFADS oligomeric state…”

“…7 This protein, referred to as FAD synthetase (FADS), presents a C-terminal region showing a high level of homology with RFKs. [8][9][10][11][12] The N-terminal region does not show similarity with eukaryotic FMNATs and rather belongs to the nucleotidyl transferase (NT) superfamily. 8,13,14 Most pathogenic bacteria and parasites rely on a prokaryotic-type FADS to provide flavoproteins with the required FAD and FMN cofactors.…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…In eukaryotic organisms, only monofunctional FAD synthetases are known, whereas in bacteria, FAD synthetases that act as part of bifunctional RF kinase/FAD synthetase were found (133,134). Genome sequence analysis of nearly 800 prokaryotes revealed a bifunctional RF kinase/FAD synthetase with conservation of several consensus regions and highly conserved residues.…”