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• W2022784171 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 Mg2+. 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 Mg2+ 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-Mg2+ or flavin binding. They might include not only rearrangement of the different protein loops but also alternative conformations between domains. 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 Mg2+. 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 Mg2+ 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-Mg2+ 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) 3The abbreviations used are: RF, riboflavin; FADS, FAD synthetase; LF, lumiflavin; RFK, riboflavin kinase; AMPPNP, 5′-adenosine 5′-(β,γ-imido) triphosphate; ITC, isothermal titration calorimetry; Tm, Thermotoga maritima; Ca, Corynebacterium ammoniagenes; WT, wild type. 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 (1Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (78) Google Scholar, 2McCormick D.B. Oka M. Bowers-Komro D.M. Yamada Y. Hartman H.A. Methods Enzymol. 1997; 280: 407-413Crossref PubMed Scopus (17) Google Scholar, 3Oka M. McCormick D.B. J. Biol. Chem. 1987; 262: 7418-7422Abstract Full Text PDF PubMed Google Scholar, 4Merrill Jr., A.H. McCormick D.B. J. Biol. Chem. 1980; 255: 1335-1338Abstract Full Text PDF PubMed Google Scholar, 5Galluccio M. Brizio C. Torchetti E.M. Ferranti P. Gianazza E. Indiveri C. Barile M. Protein Expr. Purif. 2007; 52: 175-181Crossref PubMed Scopus (36) Google Scholar, 6Santos M.A. Jimenez A. Revuelta J.L. J. Biol. Chem. 2000; 275: 28618-28624Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Giancaspero T.A. Locato V. de Pinto M.C. De Gara L. Barile M. FEBS J. 2009; 276: 219-231Crossref PubMed Scopus (47) Google Scholar), whereas most prokaryotes depend on a single bifunctional enzyme, the FAD synthetase (FADS) (8Manstein D.J. Pai E.F. J. Biol. Chem. 1986; 261: 16169-16173Abstract Full Text PDF PubMed Google Scholar, 9Mack M. van Loon A.P. Hohmann H.P. J. Bacteriol. 1998; 180: 950-955Crossref PubMed Google Scholar). 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 PPi, 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 (10Efimov I. Kuusk V. Zhang X. McIntire W.S. Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (58) Google Scholar). In the adenylylation process, FMN is proposed to bind after ATP and the PPi to be released preceding FAD (10Efimov I. Kuusk V. Zhang X. McIntire W.S. Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (58) Google Scholar). The two enzymatic activities differ in their specificity for divalent cations, optimal pH, and temperature (8Manstein D.J. Pai E.F. J. Biol. Chem. 1986; 261: 16169-16173Abstract Full Text PDF PubMed Google Scholar, 11Hagihara T. Fujio T. Aisaka K. Appl. Microbiol. Biotechnol. 1995; 42: 724-729Crossref PubMed Scopus (33) Google Scholar, 12Nakagawa S. Igarashi A. Ohta T. Hagihara T. Fujio T. Aisaka K. Biosci. Biotechnol. Biochem. 1995; 59: 694-702Crossref PubMed Scopus (24) Google Scholar). The presence of Mg2+ improves the turnover of both processes but, although low concentrations (<1 mm) enhance the kinase activity, much larger concentrations (∼10 mm) are required for maximal FAD production (8Manstein D.J. Pai E.F. J. Biol. Chem. 1986; 261: 16169-16173Abstract Full Text PDF PubMed Google Scholar). 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 (10Efimov I. Kuusk V. Zhang X. McIntire W.S. Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (58) Google Scholar). The only structure reported for an FADS is that of Thermotoga maritima (TmFADS), both free and in complex with several substrates (13Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Proteins. 2003; 52: 633-635Crossref PubMed Scopus (22) Google Scholar, 14Wang W. Kim R. Yokota H. Kim S.H. Proteins. 2005; 58: 246-248Crossref PubMed Scopus (32) Google Scholar). One of these structures shows simultaneous binding of AMP in the N-terminal domain and ADP and FMN in the C-terminal domain. Thus, the protein folds in two almost independent domains, each one hosting one ATP-binding site, whereas only a flavin-binding site, located in the C-terminal domain, was detected (14Wang W. Kim R. Yokota H. Kim S.H. Proteins. 2005; 58: 246-248Crossref PubMed Scopus (32) Google Scholar). However, in the C-terminal domain the ribityl and phosphate of the flavin are not placed in the putative active site. This is probably due to the disorder in the protein regions hosting the flavin. The C-terminal domain shows structural homology with Homo sapiens and Schizosaccharomyces pombe RFKs (14Wang W. Kim R. Yokota H. Kim S.H. Proteins. 2005; 58: 246-248Crossref PubMed Scopus (32) Google Scholar, 15Frago S. Martínez-Júlvez M. Serrano A. Medina M. BMC Microbiol. 2008; 8: 160Crossref PubMed Scopus (44) Google Scholar, 16Karthikeyan S. Zhou Q. Mseeh F. Grishin N.V. Osterman A.L. Zhang H. Structure. 2003; 11: 265-273Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 17Bauer S. Kemter K. Bacher A. Huber R. Fischer M. Steinbacher S. J. Mol. Biol. 2003; 326: 1463-1473Crossref PubMed Scopus (50) Google Scholar) and can catalyze the phosphorylation of RF (15Frago S. Martínez-Júlvez M. Serrano A. Medina M. BMC Microbiol. 2008; 8: 160Crossref PubMed Scopus (44) Google Scholar). The N-terminal region presents remote similarities with nucleotydyltransferases (18Krupa A. Sandhya K. Srinivasan N. Jonnalagadda S. Trends Biochem. Sci. 2003; 28: 9-12Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), but it does not appear to be self-sufficient to transform FMN into FAD (15Frago S. Martínez-Júlvez M. Serrano A. Medina M. BMC Microbiol. 2008; 8: 160Crossref PubMed Scopus (44) Google Scholar). The TmFADS structure shows a large distance between the reported flavin-binding site and the adenylylation site (14Wang W. Kim R. Yokota H. Kim S.H. Proteins. 2005; 58: 246-248Crossref PubMed Scopus (32) Google Scholar). Although the asymmetric unit is a dimer, it does not appear functional. These observations, together with the fact that the FMN produced in the phosphorylation process has to be released before rebinding as substrate for the second reaction (10Efimov I. Kuusk V. Zhang X. McIntire W.S. Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (58) Google Scholar), make it logical to propose the presence of a second flavin-binding site, located in the N-terminal domain. The structural model of FADS from Corynebacterium ammoniagenes (CaFADS) shows the main structural arrangements present in TmFADS (13Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Proteins. 2003; 52: 633-635Crossref PubMed Scopus (22) Google Scholar, 14Wang W. Kim R. Yokota H. Kim S.H. Proteins. 2005; 58: 246-248Crossref PubMed Scopus (32) Google Scholar), and it also shows the disposition of some loops and a 310-helix at the C-terminal domain that are missed in the TmFADS structure. Additionally, the C-terminal domain of CaFADS showed an insertion around residue 230 with regard to TmFADS. This model also suggests the presence of a novel putative second flavin-binding site in the N-terminal domain (15Frago S. Martínez-Júlvez M. Serrano A. Medina M. BMC Microbiol. 2008; 8: 160Crossref PubMed Scopus (44) Google Scholar) (see Fig. 1B). This site would be located in the proximity of the adenine nucleotide-binding site and might bind FMN for adenylylation. The present study provides a detailed thermodynamic analysis of the binding of adenine nucleotide and flavin ligands to CaFADS and to its individually cloned C terminus domain. It confirms the presence of two flavin-binding sites in CaFADS and allows assigning affinities to the different flavin and adenine nucleotide-binding sites. Cloning, Overexpression, and Purification of CaFADS and Its C-terminal Domain-WT CaFADS was cloned, overexpressed, and purified as previously described (15Frago S. Martínez-Júlvez M. Serrano A. Medina M. BMC Microbiol. 2008; 8: 160Crossref PubMed Scopus (44) Google Scholar). The separately cloned C-terminal domain (Δ(1–182)FADS) was purified using a similar protocol, replacing the DEAE-cellulose chromatography with a Superose 12 gel filtration (GE Healthcare). Spectral Analysis-UV-visible spectra were recorded on a Cary-100 spectrophotometer. To determine the extinction coefficient (∈) of CaFADS in 50 mm Tris/HCl, pH 8.0, and 20 mm sodium phosphate, pH 7.0, the UV-visible absorbance spectrum of the protein was recorded in each buffer. The samples were subsequently diluted with 7.5 m guanidinium hydrochloride in 20 mm sodium phosphate, pH 6.5, to 6 m guanidinium hydrochloride. The absorbance spectra were again recorded. Protein concentration was calculated using the theoretical ∈280 nm for the denatured protein (19Gasteiger E. Hoogland C. Gattiker A. Duvaud S. Wilkins M.R. Appel R.D. Bairoch A. Protein Identification and Analysis Tools on the ExPASy Server. Humana Press, Totowa, NJ2005Crossref Google Scholar). The ∈280 nm in each buffer was obtained from the initial spectra. For Δ(1–182)FADS, the theoretical ∈280 nm of 14 mm–1 cm–1 was used (19Gasteiger E. Hoogland C. Gattiker A. Duvaud S. Wilkins M.R. Appel R.D. Bairoch A. Protein Identification and Analysis Tools on the ExPASy Server. Humana Press, Totowa, NJ2005Crossref Google Scholar). Difference spectroscopy measurements were carried out in 50 mm Tris/HCl, pH 8.0. The reference cuvette, containing buffer, and the sample cuvette, containing 5–6 μm FADS (3 μm for titration with RF), were stepwise titrated with aliquots of 1–10 μl of the corresponding flavin solutions (∼180 μm RF, 750–1500 μm FMN, and 450–600 μm FAD). For the measurements in the presence of AMPPNP, a nonhydrolyzable ATP analogue, both cuvettes contained 1 mm AMPPNP and 10 mm MgCl2. Dissociation constants and difference extinction coefficients were obtained by nonlinear regression fit of the experimental data to the theoretical equation for a 1:N stoichiometric complex (20Sancho J. Gomez-Moreno C. Arch. Biochem. Biophys. 1991; 288: 231-238Crossref PubMed Scopus (45) Google Scholar),ΔAbs=Δ∈l2N(N[FADS]+[L]+Kd)-(N[FADS]+[L]+Kd)2-4N[FADS][L]Eq. 1 where Δ∈ is the change in the flavin extinction coefficient upon ligand binding to FADS, l is the cuvette path length, [FADS] and [L] are the total concentrations of FADS and flavin, respectively, Kd is the dissociation constant, and N is the number of flavin-binding sites in FADS. The errors estimated for Kd and Δ∈ were ±15%. CD spectra were recorded in a Chirascan spectropolarimeter (Applied Photophysics Ltd.) at 5 °C in the far-UV (7 μm FADS in 5 mm sodium phosphate, pH 7.0, 0.1-cm cuvette) and in the near-UV (20 μm FADS in 20 mm sodium phosphate, pH 7.0, 0.4-cm cuvette). Near-UV CD spectra were also recorded at 20 °C with saturating concentrations of ATP, ADP, and/or FMN, at both 0 and 10 mm MgCl2. Fluorescence emission spectra were recorded in an Aminco-Bowman Series 2 spectrometer in 20 mm sodium phosphate, pH 7.0, at 25 °C, exciting the protein aromatic residues at 280 nm. Differential Scanning Calorimetry-The heat capacity of FADS (ΔCP) was measured as a function of temperature at pH 7.0 with a VP-DSC microcalorimeter (MicroCal LLC). Thermal denaturation scans were performed with 8 and 24 μm degassed FADS solutions in 20 mm sodium phosphate, pH 7.0, at a scanning rate of 1 °C/min from 10 to 100 °C. Reversibility of the unfolding process was checked by sample reheating after cooling inside the calorimetric cell. The unfolding of FADS was not reversible, and therefore only a van’t Hoff model-independent analysis was performed (21Freire E. Methods Enzymol. 1995; 259: 144-168Crossref PubMed Scopus (97) Google Scholar, 22Freire E. Methods Mol. Biol. 1995; 40: 191-218PubMed Google Scholar). High Sensitivity Isothermal Titration Calorimetry (ITC)-Measurements were carried out using a high precision VP-ITC system (MicroCal LLC). Typically, a ∼300 μm FAD or FMN solution or a 300–500 μm ADP, ATP, or AMPPNP solution was used to titrate ∼10–20 μm FADS. Because of the low solubility of Lumiflavin (LF, a RF analogue lacking the ribityl chain) and RF, FADS solutions of ∼5 and ∼10 μm were titrated with LF and RF ∼90 and ∼175 μm, respectively. Both the ligand and FADS were dissolved in the same buffer (20 mm phosphate, pH 7.0, with variable MgCl2 concentrations: 0, 0.8, or 10 mm) and degassed. This buffer was selected because of its low ionization enthalpy, which prevents any buffer influence on the observed enthalpy of binding caused by possible de/protonation events. Titrations of FADS·nucleotide or FADS·FAD complexes were carried out by stepwise injections of the ligand into a mixture of FADS and a saturating concentration of the adenine nucleotide (400–500 μm) and/or FAD (80–100 μm). Each titration was initiated by a 4-μl injection followed by 25–28 stepwise injections of 10 μl. The heat evolved after each ligand injection was obtained from the integral of the calorimetric signal. The heat caused by the binding reaction was obtained as the difference between the heat of reaction and the corresponding heat of dilution, the latter estimated as a constant heat throughout the experiment and included as an adjustable parameter in the analysis. The association constant (Ka), the enthalpy change (ΔH), and the stoichiometry (N) were obtained through nonlinear regression of the experimental data to a model for one or two independent binding sites implemented in Origin 7.0. The dissociation constant (Kd), the free energy change (ΔG), and the entropy change (ΔS) were obtained from basic thermodynamic relationships. Usually, when N = 2 there was not enough information in the titrations for using a model with two different binding sites and averaged binding parameters are given. The estimated error was ±15% in Kd and ± 0.3 kcal/mol in ΔH and –TΔS. To analyze a possible interaction between FADS and Mg2+, 20 μm FADS was titrated with a 300 μm MgCl2 solution. An additional titration of FADS in the presence of 40 μm EDTA with 900 μm MgCl2 was carried out to exclude putative Mg2+-binding sites in FADS already being loaded. Additionally, a solution of 40 μm EDTA was titrated with 900 μm MgCl2 to obtain the parameters for the EDTA-Mg2+ interaction. Finally, ITC experiments involving the dilution of 440 μm FADS into 20 mm sodium phosphate pH 7.0 buffer were also performed to test the enzyme oligomerization state (23Velázquez-Campoy A. Leavitt S.A. Freire E. Methods Mol. Biol. 2004; 261: 35-54PubMed Google Scholar, 24Burrows S.D. Doyle M.L. Murphy K.P. Franklin S.G. White J.R. Brooks I. McNulty D.E. Scott M.O. Knutson J.R. Porter D. Young P.R. Hensley P. Biochemistry. 1994; 33: 12741-12745Crossref PubMed Scopus (145) Google Scholar). Spectral Properties of CaFADS-CaFADS presented an UV-visible maximum at 279 nm. The ∈279 nm was 28.1 mm–1 cm–1 in 50 mm Tris/HCl, pH 8.0, and 25.6 mm–1 cm–1 in 20 mm sodium phosphate, pH 7.0. The far-UV CD spectrum of CaFADS showed a negative-positive couplet (208–191 nm) typical of a secondary α-helix structure. The negative band around 222 nm, also typical of α-helix, appeared like a shoulder (Fig. 2A), probably because of an important content of β-sheet (Fig. 1B). The near-UV CD spectrum (Fig. 2A) was sensible to substrate binding. Changes were more evident in the simultaneous presence of FMN, ADP, or ATP, and Mg2+ and suggest that binding takes place by influencing the environment of aromatic residues. Excitation of CaFADS at 280 nm produced a fluorescence emission band centered at 330 nm, indicating that, in general, the Trp residues of the protein are contained in an apolar environment. Binding of Flavins to FADS Followed by Differential Spectroscopy-Titration of FADS with RF, FMN, and FAD produced the appearance of visible difference spectra (Fig. 2B), indicating changes in the dielectric environment of the isoalloxazine upon interaction with FADS. Although titration with RF or FMN produced similar difference spectra, the spectrum elicited upon interaction with FAD was different (Fig. 2B). The magnitude of the difference spectra reached saturation (Fig. 2D), allowing the determination of Kd and Δ∈. Fitting of the data to Equation 1 suggested two independent binding sites for RF with an average Kd (Kd,av) of 5.2 μm, whereas a single binding site was detected for FMN and FAD with Kd values of 13.1 and 2.3 μm, respectively. The saturating concentration of AMPPNP-Mg2+ produced a considerable increase in Δ∈ when FADS was titrated with RF and, especially, with FMN (Fig. 2C). AMPPNP-Mg2+ considerably increased the FADS affinity for RF while decreasing that for FMN (Kd of 0.6 and 90.5 μm, respectively). However, AMPPNP-Mg2+ did not affect the FAD interaction (Fig. 2). FADS Thermal Denaturation Followed by Differential Scanning Calorimetry-FADS thermal denaturation exhibited two partially overlapping transitions (Tm,ap = 41.1 °C), suggesting that the unfolding processes of the N- and C-terminal domains are not fully cooperative. By direct integration of the signal, a calorimetric ΔH of 165 kcal/mol and an unfolding ΔCP of 4.2 kcal/K·mol were obtained. A statistical analysis of globular proteins indicates that the unfolding ΔCP and the unfolding ΔH at 60 °C scale with protein size according to: ΔCP = 13.9·NR cal/(K·mol·res) and ΔH(60 °C) = 0.698·NR kcal/(mol·res), where NR is the number of residues in the protein (25Robertson A.D. Murphy K.P. Chem. Rev. 1997; 97: 1251-1268Crossref PubMed Scopus (545) Google Scholar). FADS, with 338 residues, has an estimated unfolding ΔCP of 4.7 kcal/K·mol and an unfolding ΔH(60 °C) of 236 kcal/mol. They compare well with the experimental values of 4.2 kcal/K·mol and 245 kcal/mol (extrapolation at 60 °C). This suggests that the protein is folded in solution with no significant unstructured regions. A second cycle of thermal denaturation indicated that the unfolding process was not reversible, the less stable domain being responsible for the nonreversibility. The more stable domain showed a reversible thermal denaturation with a Tm of 43.8 °C. The van’t Hoff analysis provides a van’t Hoff unfolding ΔH of 84 kcal/mol. A value of 0.51 for the van’t Hoff-calorimetric ΔH ratio also indicates that the thermal transition is not fully cooperative and that at least two domains unfold independently. Finally, the lack of effect on Tm,ap upon increasing protein concentration (from 8 to 24 μm) indicated that FADS is in the monomeric state at both concentrations, in agreement with the FADS dilution experiment by ITC. ITC Analysis of the Interaction of FADS with Mg2+-Direct titration of the enzyme with MgCl2 did not produce any calorimetric change related to an interaction between FADS and Mg2+. This indicates that CaFADS does not show specific binding of free Mg2+. ITC Analysis of the Binding of Adenine Nucleotides to FADS-The interaction of WT FADS with ATP, ADP, and AMPPNP was analyzed by ITC at pH 7.0 and 25 °C at different [Mg2+] (Fig. 3 and Table 1). A single ATP-binding site with a K ATPd of 10 μm was detected for FADS in the absence of Mg2+. The presence of this cation produced the appearance of a second ATP-binding site and increased the Kd,avATP (Table 1). Similarly, FADS showed a single ADP-binding site in the absence of Mg2+, the affinity four times lower than that of ATP (Table 1) and two ADP-binding sites in the presence of Mg2+ without altering Kd,avADP. AMPPNP interacted with FADS considerably more weakly than ATP and without major [Mg2+] effects. Therefore, AMPPNP is not a suitable ATP analogue for the purpose of this study.TABLE 1Stoichiometry and interaction parameters for the FADS-nucleotide and FADS-flavin interaction determined by ITCFADS ligandNKd0 mm Mg2+0.8 mm Mg2+10 mm Mg2+0 mm Mg2+0.8 mm Mg2+10 mm Mg2+μmATP12210.041.655.3ADP12243.048.838.9AMPPNP1275.781.4FAD1112.01.80.8FMN1113.03.11.6RF2227.66.92.8LF2216.97.53.3 Open table in a new tab Direct titrations also allowed the determination of the enthalpy and entropy components of the interactions (Fig. 4). A favorable enthalpy change drove the interaction of ATP, ADP, and AMPPNP with FADS over the unfavorable entropic contribution, although the magnitude was considerably smaller for the latter. Increasing [Mg2+] resulted in an slightly less favorable enthalpic binding contribution and a less unfavorable entropic one (Fig. 4). However, especially for ADP and AMPPNP, ΔG remained remarkably insensitive to [Mg2+] through entropy/enthalpy compensation. ITC Analysis of the Binding of Flavins to FADS-The interaction of WT FADS with its RF, FMN, and FAD substrates/products, as well as with LF, was also analyzed (Fig. 5 and Table 1). A single FADS-binding site for FMN and FAD was determined, independently of [Mg2+], with Kd values in the range of 1–3 μm for both flavins. Binding of FMN and FAD was driven by a large enthalpy change but at high cost in entropy (Fig. 6). Increasing [Mg2+] hardly influenced the interaction with FMN, but the interaction with FAD resulted in being slightly less favored by the enthalpic contribution and less unfavored by the entropic one (Fig. 6).FIGURE 6Thermodynamic dissection of the interaction between FADS with FAD, FMN, RF, and LF. The other conditions were as described in the legend to Fig. 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two binding sites were detected when FADS was titrated with RF and LF, both binding only slightly more weakly than FMN or FAD (Table 1). Binding of RF and LF was also enthalpically driven with a very small opposing entropic contribution that was favorable in the presence of Mg2+ (unlike interactions with FAD and FMN) (Fig. 6). This is consistent with the considerably less polar nature of RF and LF. ITC Analysis of Flavin Binding to Preformed FADS·Nucleotide Complexes-A single FAD-binding site was unequivocally determined in the presence of saturating concentrations of ADP or AMPPNP (Table 2 and Fig. 7). AMPPNP had minor effects on the binding parameters, but the presence of ADP considerably reduced the FADS affinity for FAD at low [Mg2+] (Table 2). This reduction in the affinity is the result of considerably less favorable enthalpic and less unfavorable entropic binding contributions (Figs. 6 and 8). However, at high [Mg2+], binding affinity of FAD to FADS·ADP was similar to that to free FADS because of the less favorable enthalpic contribution being compensated with a favorable binding entropy (Figs. 6 and 8).TABLE 2Stoichiometry and interaction parameters for ligand binding to FADS in the presence of a second ligand determined by ITCTitrating ligandSaturating ligandNKd0 mm Mg2+0.8 mm Mg2+10 mm Mg2+0 mm Mg2+0.8 mm Mg2+10 mm Mg2+μmFADAMPPNP114.00.6FADADP11137.624.40.9FMNAMPPNP118.30.9FMNADP2228.51.60.25site 10.06site 21.1FMNFAD1200FMNADP, FAD21.4RFAMPPNP228.73.1RFADP1222.60.81.5RFFAD213.5LFADP22aMost likely value based on the analysis of the enthalpy and entropy contributions.14.74.95.7ADPFAD1bVery weak interaction is observed.ATPFAD116.8a Most likely value based on the analysis of the enthalpy and entropy contributions.b Very weak interaction is observed. Open table in a new tab FIGURE 8Thermodynamic dissection of the interaction of the FADS·ADP preformed complex with FAD, FMN, RF, and LF. The other conditions were as described in the legend to Fig. 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The preformed FADS·ADP complex was able to bind two FMN molecules. Therefore, the presence of ADP promoted the appearance of a second FMN-binding site (Tables 2 and 3). The presence of Mg2+ enhanced the FADS·ADP complex affinity for FMN (Table 2). At the highest [Mg2+] assayed, the independent Kd values for the two FMN-binding sites could be determined. Additionally, the simultaneous presence of Mg2+ and ADP had an important effect in the enthalpic and entropic binding contributions (Figs. 6 and 8). Binding of FMN to the preformed FADS·ADP complex was enthalpically driven with an opposing entropic contribution in the absence of Mg2+. However, its presence promoted the enthalpic contribution to become less favorable and transformed the unfavorable entropy into a favorable one, making the interaction stronger. Binding of FMN to the FADS·AMPPNP complex showed similar affinity values to those for the FADS·ADP complex, but a second FMN-binding site was not observed (Table 2).TABLE 3Stoichiometry and interaction parameters for the (Δ1-182)FADS-ligand interaction determined by ITCTitrating ligandSaturating ligandNKd0 mm Mg2+10 mm Mg2+0 mm Mg2+10 mm Mg2+μmATP117095.4ADP01aNo interaction is observed under these conditions.32.8FAD00aNo interaction is observed under these conditions.bVery weak interaction is observed.FMN00bVery weak interaction is observed.bVery weak interaction is observed.FMNADP10.45RF111.87.2RFADP10.9a No interaction is observed under these conditions.b Very weak interaction is observed. Open table in a new tab Binding of RF to FADS preloaded with ADP pointed out the presence of a single RF-binding site that turned into two sites in the presence of Mg2+ (Table 2). The cation also causes a slight increase in the affinity for RF. Two binding sites to FADS·AMPPNP were also detected. The favorable enthalpic and, especially, the unfavorable entropic contributions became considerably less intense upon increasing [Mg2+] (Fig. 8). ITC Analysis of Competitive Ligand Binding to Preformed FADS Complexes-A set of experiments in the presence of an excess of FAD as competitive ligand was carried out to establish the binding domain of ligands with a single binding site (Table 2). According to previous modeling studies (15Frago S. Martínez-Júlvez M. Serrano A. Medina M. BMC Microbiol. 2008; 8: 160Crossref PubMed Scopus (44) Google Scholar) and to our own data, FAD must bind at the N-terminal domain, blocking both the adenine nucleotide and the putative flavin nucleotide-binding sites of this domain. Under FAD saturation and lacking Mg2+, FMN binding was hardly detected (Table 2). Because in the presence of ADP and 10 mm MgCl2 FADS binds two FMN molecules (Table 2), FADS was simultaneously saturated with ADP and FAD at 10 mm MgCl2 and titrated with FMN. Two FMN-binding sites were also detected, but the FMN interaction was weaker than in the single ADP presence (Table 2). Two RF-binding sites were detected in the FAD·FADS complex with a Kd,avRF of 13.5 μm (Table 2). Therefore, FAD makes the interaction of FADS with one of the RF molecules slightly weaker. In the FADS·FAD complex, a single binding site for ATP or ADP was again observed (Tables 1 and 2). ATP affinity was not affected, but the presen" @default.
• W2022784171 created "2016-06-24" @default.
• W2022784171 creator A5021335979 @default.
• W2022784171 creator A5023025738 @default.
• W2022784171 creator A5091692002 @default.
• W2022784171 date "2009-03-01" @default.
• W2022784171 modified "2023-10-03" @default.
• W2022784171 title "The Puzzle of Ligand Binding to Corynebacterium ammoniagenes FAD Synthetase" @default.
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