Susana Frago

Structural analysis of FAD synthetase from Corynebacterium ammoniagenes

BackgroundThe prokaryotic FAD synthetase family – a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure.ResultsSequences of nearly 800 prokaryotic species were aligned. Those related with bifunctional FAD synthetase activities showed conservation of several consensus regions and highly conserved residues. A 3D model for the FAD synthetase from Corynebacterium ammoniagenes (CaFADS) was generated. This model confirms that the N-terminal and C-terminal domains are related to nucleotydyltransferases and riboflavin kinases, respectively. Models for the interaction of CaFADS with its substrates were also produced, allowing location of all the protein substrates in their putative binding pockets. These include two independent flavin binding sites for each CaFADS activity.ConclusionFor the first time, the putative presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation activity, is shown. Additionally, these models suggest the functional relevance of some residues putatively involved in the catalytic processes. Their relevant roles were analysed by site-directed mutagenesis. A role was confirmed for H28, H31, S164 and T165 in the stabilisation of the P groups and the adenine moiety of ATP and, the P of FMN for the adenylylation. Similarly, T208, N210 and E268 appear critical for accommodation of the P groups of ATP and the ribityl end of RF in the active site for the phosphorylation process. Finally, the C-terminal domain was shown to catalyse the phosphorylation process on its own, but no reaction at all was observed with the individually expressed N-terminal domain.

Oligomeric State in the Crystal Structure of Modular Fad Synthetase Provides Insights Into its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 A resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules-a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity-that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 A away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.

Flavodoxin-Mediated Electron Transfer from Photosystem I to Ferredoxin-NADP+ Reductase in Anabaena: Role of Flavodoxin Hydrophobic Residues in Protein−Protein Interactions†

Three surface hydrophobic residues located at the Anabaena flavodoxin (Fld) putative complex interface with its redox partners were replaced by site-directed mutagenesis. The effects of these replacements on Fld interaction with both its physiological electron donor, photosystem I (PSI), and its electron acceptor, ferredoxin-NADP+ reductase (FNR), were analyzed. Trp57, Ile59, and Ile92 contributed to the optimal orientation and tightening of the FNR:Fld and PSI:Fld complexes. However, these side chains did not appear to be involved in crucial specific interactions, but rather contributed to the obtainment of the optimal orientation and distance of the redox centers required for efficient electron transfer. This supports the idea that the interaction of Fld with its partners is less specific than that of ferredoxin and that more than one orientation is efficient for electron transfer in these transient complexes. Additionally, for some of the analyzed processes, WT Fld seems not to be the most optimized molecular species. Therefore, subtle changes at the isoalloxazine environment not only influence the Fld binding abilities, but also modulate the electron exchange processes by producing different orientations and distances between the redox centers. Finally, the weaker apoflavodoxin interaction with FNR suggests that the solvent-accessible region of FMN plays a role either in complex formation with FNR or in providing the adequate conformation of the FNR binding region in Fld.

Circulating levels of insulin-like growth factor-II/mannose-6-phosphate receptor in obesity and type 2 diabetes

OBJECTIVE The extracellular domain of the insulin-like growth factor II/mannose-6-phosphate receptor (IGF-II/M6P-R) is present in the circulation, but its relationship with plasma IGF-II is largely unknown. As IGF-II appears to be nutritionally regulated, we studied the impact of obesity, type 2 diabetes (T2D) and weight loss on circulating levels of IGF-II and its soluble receptor. METHODS Twenty-three morbidly obese non-diabetic subjects were studied before and after gastric banding (GB), reducing their BMI from 59.3+/-1.8 to 52.7+/-1.6 kg/m(2). Lean controls (n=10, BMI 24.2+/-0.5 kg/m(2)), moderately obese controls (n=21, BMI 31.8+/-1.0 kg/m(2)) and obese T2D patients (n=20, BMI 32.3+/-0.8 kg/m(2)) were studied before and after a hyperinsulinaemic euglycaemic clamp. RESULTS Morbidly obese subjects had elevated IGF-II/M6P-R and IGF-II levels, which both decreased following GB (IGF-II/M6P-R: from 0.97+/-0.038 to 0.87+/-0.030 nmol/l, P=0.001; IGF-II: from 134+/-7 to 125+/-6 nmol/l, P=0.01), as did fasting plasma glucose and insulin (P<0.05). However, the metabolic parameters correlated with neither IGF-II nor IGF-II/M6P-R. Obese diabetics had increased IGF-II/M6P-R as compared with lean and obese controls (0.82+/-0.031 vs. 0.70+/-0.033 vs. 0.74+/-0.026 nmol/l; P<0.03) and levels were unaffected by clamp. In the latter cohort, IGF-II/M6P-R but not IGF-II correlated with HbA1c, and fasting plasma C-peptide, insulin and glucose (0.34<r<0.45; P<0.05). In all subjects, BMI correlated with IGF-II/M6P-R (r=0.57; P<0.001) and IGF-II (r=0.39; P<0.005). IGF-II/M6P-R and IGF-II were not associated. CONCLUSION Serum IGF-II/M6P-R is up-regulated in morbid obesity, down-regulated by weight loss and elevated in moderately obese T2D. However, although plasma IGF-II was also reduced following GB, the two peptides were not statistically correlated. No acute effect of insulin was seen. These findings indicate that the IGF-II/M6P-R is nutritionally regulated, independently of IGF-II.

An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution

Parental genetic conflict may have exploited changes in the coding of a protein loop in a growth factor receptor. Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation. Many mammals have imprinted alleles, where the paternal or maternal version is solely expressed during reproduction. In humans, one such imprinted gene set is the growth promoter insulin-like growth factor 2 (IGF2) and its binding inhibitor, IGF2R mannose 6-phosphate/IGF2 receptor. To avoid parental conflict in fetal growth, imprinting regulates expression of these genes so that expression of IGF2R in the fetus quenches IGF2 and prevents fetus overgrowth through high-affinity binding of IGF2 to IGF2R. Williams et al. (p. 1209) demonstrate that high-affinity binding of IGF2 to IGF2R is present in placental and marsupial mammals; absent in birds and fish; and present, with a 10-fold lower affinity, in monotreme (egg-laying) mammals. The appearance of exonic splicing enhancers in exon 34 of the IGF2R of monotremes appears to have been a key mutational event leading to the establishment of higher affinity, which may have been driven by selection to minimize parental conflict.

The puzzle of ligand binding to Corynebacterium ammoniagenes FAD synthetase.

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.

Role of Key Residues at the Flavin Mononucleotide (FMN):Adenylyltransferase Catalytic Site of the Bifunctional Riboflavin Kinase/Flavin Adenine Dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes

In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP(+) reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.

Key Residues at the Riboflavin Kinase Catalytic Site of the Bifunctional Riboflavin Kinase/FMN Adenylyltransferase From Corynebacterium ammoniagenes

Many known prokaryotic organisms depend on a single bifunctional enzyme, encoded by the RibC of RibF gene and named FAD synthetase (FADS), to convert Riboflavin (RF), first into FMN and then into FAD. The reaction occurs through the sequential action of two activities present on a single polypeptide chain where the N-terminus is responsible for the ATP:FMN adenylyltransferase (FMNAT) activity and the C-terminus for the ATP: riboflavin kinase (RFK) activity. Sequence and structural analysis suggest that T208, N210 and E268 at the C-terminus RFK module of Corynebacterium ammoniagenes FADS (CaFADS) might be key during RF phosphorylation. The effect of site-directed mutagenesis on the RFK activity, as well as on substrates and products binding, indicates that T208 and N210 provide the RFK active-site geometry for binding and catalysis, while E268 might be involved in the catalytic step as catalytic base. These data additionally suggest concerted conformational changes at the RFK module of CaFADS during its activity. Mutations at the RFK site also modulate the binding parameters at the FMNAT active site of CaFADS, altering the catalytic efficiency in the transformation of FMN into FAD. This observation supports the hypothesis that the hexameric assembly previously revealed by the crystal structure of CaFADS might play a functional role during catalysis.

Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokaryotic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 133.47 A and a = b = c = 133.40 A, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 A resolution, respectively.