Calorimetric studies of flavin-binding proteins: FMN and FAD binding to hen egg riboflavin-binding proteins

Systematic thermodynamic studies have been conducted for flavin (FMN, FAD) binding to purified riboflavin-binding proteins from hen egg white and egg yolk. These studies were conducted under a variety of temperature (14, 26, and 38 °C), pH (4.5, 5.5, 6.5, 7.4, and 9.0), and buffer conditions, and an extensive thermodynamic profile was constructed. Enthalpies of binding FMN to white riboflavin-binding protein and yolk riboflavin-binding protein were ?19.3 and ?14.4 kcal/mol, respectively, at pH 7.4 and 38 °C. FAD bound to white and yolk riboflavin-binding proteins under the same conditions with ΔH values of ?11.7 and ?6.0, respectively. Binding constants of about 10⁵ and 10⁴ were obtained for FMN and FAD, respectively, and were the same for both proteins under all conditions studied. Using established thermodynamic relationships, we were able to calculate entropy and free energy changes. Entropies indicated a large degree of ordering in the system upon flavin binding with FMN (about ?40 cal/mol/ °C) twice as large as FAD (about ?15 to ?25 cal/mol/ °C), which may indicate a structured solvent interaction with the charged phosphate group, or steric limitations placed on the ribityl side chain in the bound state. Our thermodynamic data support the idea that flavin binding is a mixture of forces, with no one predominant. Analysis of the data suggests that the nucleotide may bind both as the mono- or dianion, that flavin binding occurs with no significant change in the pK of any functional group in the system, except at low pH for FAD binding, and that the temperature variation of the enthalpy change is quite small. These findings are combined with other published data to outline a general scheme of flavin binding with a histidine residue implicated in hydrogen bonding to the adenine portion of FAD, which may be in the unstacked form.     

Identification and quantitative determination of the flavin component of soluble hydrogenase from Alcaligeneseutrophus

The flavin component of soluble hydrogenase (hydrogen: NAD⁺ oxidoreductase, EC 1.12.1.2) from $\text{Alcaligenes}\text{eutrophus}$ was identified as FMN by thin layer chromatography in two solvent systems and by binding studies with apoflavodoxin from $\text{Megasphaera}\text{elsdenii}$. The flavin of hydrogenase reacted rapidly with apoflavodoxin with almost complete quenching of the fluorescence at 525 nm. Quantitative determination of FMN was performed by fluorimetric titration with a standardized solution of apoflavodoxin. From the determined FMN content of different enzyme preparations and from the percentage of stimulation of hydrogenase activity by exogenous FMN it is concluded that hydrogenase contains 2 FMN per molecule.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

Entamoeba histolytica: flavins in axenic organisms.

The individual flavin species of axenic Entamoeba histolytica were assayed: separated riboflavin was assayed by the lumiflavin method; flavin-adenine dinucleotide (FAD), by an enzymatic method; flavin mononucleotide (FMN) was calculated from the difference, total flavin minus FAD and riboflavin. The amount of flavin in micrograms per grams fresh cells follows: total flavin, 7.6 ± 0.9 calculated as riboflavin; riboflavin, 1.6 ± 0.7; FMN, 6.6 ± 0.5; and FAD, 1.2 ± 0.1. Recalculated to nanomoles per milligrams total amebal protein these values were: total flavin, 0.21; riboflavin, 0.04; FMN, 0.15; and FAD, 0.02. The identity of each flavin was confirmed by a paper chromatographic method. Analyses on Panmede, the main source of flavins in the TP-S-1 medium, indicate that it contains all three forms of flavin. Its contribution to growth medium in micrograms per milliliters: riboflavin, 2.1 ± 0.3; FMN, 0.6 ± 0.1; and FAD, 0.4 ± 0.1. The in vivo biosynthesis of FMN and FAD from riboflavin by E. histolytica is demonstrated. A new and convenient method was found to separate riboflavin from flavin nucleotides in tissue extracts.     

Understanding the FMN cofactor chemistry within the Anabaena Flavodoxin environment

The chemical versatility of flavin cofactors within the flavoprotein environment allows them to play main roles in the bioenergetics of all type of organisms, particularly in energy transformation processes such as photosynthesis or oxidative phosphorylation. Despite the large diversity of properties shown by flavoproteins and of the biological processes in which they are involved, only two flavin cofactors, FMN and FAD (both derived from the 7,8-dimethyl-10-(1′-D-ribityl)-isoalloxazine), are usually found in these proteins. Using theoretical and experimental approaches we have carried out an evaluation of the effects introduced upon substituting the 7- and/or 8-methyls of the isoalloxazine ring in the chemical and oxido-reduction properties of the different atoms of the ring on free flavins and on the photosynthetic Anabaena Flavodoxin (a flavoprotein that replaces Ferredoxin as electron carrier from Photosystem I to Ferredoxin-NADP⁺ reductase). In Anabaena Flavodoxin both the protein environment and the redox state contribute to modulate the chemical reactivity of the isoalloxazine ring. Anabaena apoflavodoxin is shown to be designed to stabilise/destabilise each one of the FMN redox states (but not of the analogues produced upon substitution of the 7- and/or 8-methyls groups) in the adequate proportions to provide Flavodoxin with the particular properties required for the functions in which it is involved in vivo. The 7- and/or 8-methyl groups of the ixoalloxazine can be discarded as the gate for electrons exchange in Anabaena Fld, but a key role in this process is envisaged for the C6 atom of the flavin and the backbone atoms of Asn58.     

The binding and spectral alterations of oxidized flavin mononucleotide by bacterial luciferase

The binding of oxidized flavin mononucleotide (FMN) to bacterial luciferase was studied by equilibrium dialysis. A Scatchard plot of the data indicates a single FMN binding site per luciferase molecule, with a dissociation constant of 2.4 × 10^?4 M at 2° in 0.05 M Bis-Tris, 0.2 M NaCl, pH 7.0. The visible absorbance spectrum of luciferase-bound FMN is altered considerably relative to the spectrum of free FMN. The spectrum of the bound flavin shows an apparent splitting of the 443-nm peak yielding well-defined maxima at 458 nm and 434 nm.     

Insights into the role of F26 residue in the FMN: ATP adenylyltransferase activity of Staphylococcus aureus FAD synthetase

The bifunctional flavin adenine dinucleotide synthetase (FADS) synthesizes the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) co-factors essential for the function of flavoproteins. The Staphylococcus aureus FADS (SaFADS) produces FMN from riboflavin (RF) by ATP:riboflavin kinase (RFK) activity at its C-terminal domain. The N-terminal domain converts FMN to FAD under a reducing environment by FMN:ATP adenylyltransferase (FMNAT) activity which is reversible (FAD pyrophosphorylase activity). Herein, we investigated the role of F26 residue of the 24-GFFD-28 motif of SaFADS FMNAT domain, mostly conserved in the reducing agent-dependent FADSs. The steady-state kinetics studies showed changes in the K_m^ATP values for mutants, indicating that the F26 residue is crucial for the FMNAT activity. Further, the FMNAT activity of the F26S mutant was observed to be higher than that of the wild-type SaFADS and its other variants at lower reducing agent concentration. In addition, the FADpp activity was inhibited by an excess of FAD substrate, which was more potent in the mutants. The altered orientation of the F26 side-chain observed in the molecular dynamics analysis suggested its plausible involvement in stabilizing FMN and ATP substrates in their respective binding pockets. Also, the SaFADS ternary complex formed with reduced FMN exhibited significant structural changes in the β4n-β5n and L3n regions compared to the oxidised FMN bound and apo forms of SaFADS. Overall, our data suggests the functional role of F26 residue in the FMNAT domain of SaFADS.     

Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii.

1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine: (see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8 due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. 3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form.     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

Last in, first out: the role of cofactor binding in flavodoxin folding

Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I(1) <=> unfolded apoflavodoxin <=> I(2) <=> native apoflavodoxin. I(1)an off-pathway molten globule-like is intermediate that populates during denaturant-induced equilibrium unfolding; I(2) is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I(1) + FMN <=> unfolded apoflavodoxin + FMN <=> I(2) + FMN <=> native apoflavodoxin + FMN<=> holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I(1),I(2), and unfolded apoflavodoxin do not interact to a significantextent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur.     

The physiological role of riboflavin transporter and involvement of FMN-riboswitch in its gene expression in Corynebacterium glutamicum

Riboflavin is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which work as cofactors of numerous enzymes. Understanding the supply system of these cofactors in bacteria, particularly those used for industrial production of value added chemicals, is important given the pivotal role the cofactors play in substrate metabolism. In this work, we examined the effect of disruption of riboflavin utilization genes on cell growth, cytoplasmic flavin levels, and expression of riboflavin transporter in Corynebacterium glutamicum. Disruption of the ribA gene that encodes bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase in C. glutamicum suppressed growth in the absence of supplemental riboflavin. The growth was fully recovered upon supplementation with 1 μM riboflavin, albeit at reduced intracellular concentrations of FMN and FAD during the log phase. Concomitant disruption of the ribA and ribM gene that encodes a riboflavin transporter exacerbated supplemental riboflavin requirement from 1 μM to 50 μM. RibM expression in FMN-rich cells was about 100-fold lower than that in FMN-limited cells. Mutations in putative FMN-riboswitch present immediately upstream of the ribM gene abolished the FMN response. This 5′UTR sequence of ribM constitutes a functional FMN-riboswitch in C. glutamicum.     

The single NqrB and NqrC subunits in the Na+-translocating NADH: Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae each carry one covalently attached FMN

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe–2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na⁺-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na⁺-NQR contains approximately 1.7 mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na⁺-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na⁺-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Metabolic and bioprocess engineering of the yeast Candida famata for FAD production

Flavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play an important role in metabolism as cofactors for oxidoreductases and other enzymes. Flavin nucleotides have applications in the food industry and medicine; FAD supplements have been efficiently used for treatment of some inheritable diseases. FAD is produced biotechnologically; however, this compound is much more expensive than riboflavin. Flavinogenic yeast Candida famata synthesizes FAD from FMN and ATP in the reaction catalyzed by FAD synthetase, a product of the FAD1 gene. Expression of FAD1 from the strong constitutive promoter TEF1 resulted in 7- to 15-fold increase in FAD synthetase activity, FAD overproduction, and secretion to the culture medium. The effectiveness of FAD production under different growth conditions by one of these recombinant strains, C. famata T-FD-FM 27, was evaluated. First, the two-level Plackett–Burman design was performed to screen medium components that significantly influence FAD production. Second, central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum FAD yield. FAD production varied most significantly in response to concentrations of adenine, KH₂PO₄, glycine, and (NH₄)₂SO₄. Implementation of these optimization strategies resulted in 65-fold increase in FAD production when compared to the non-optimized control conditions. Recombinant strain that has been cultivated for 40 h under optimized conditions achieved a FAD accumulation of 451 mg/l. So, for the first time yeast strains overproducing FAD were obtained, and the growth media composition for maximum production of this nucleotide was designed.     

Purification and characterization of a flavin reductase from the biodesulfurizing bacterium Mycobacterium goodii X7B

Dibenzothiophene (DBT) in fossil fuels can be efficiently biodesulfurized by a thermophilic bacterium Mycobacterium goodii X7B. Flavin reductase DszD, which catalyzes the reduction of oxidated flavin by NAD(P)H, is indispensable for the biodesulfurization process. In this work, a flavin reductase DszD in M. goodii X7B was purified to homogeneity, and then its encoding gene dszD was amplified and expressed in Escherichia coli. DszD is a homodimer with each subunit binding one FMN as cofactor. The K_m values for FMN and NADH of the purified recombinant DszD were determined to be 6.6 ± 0.3 μM and 77.9 ± 5.4 μM, respectively. The optimal temperature for DszD activity was 55 °C. DszD can use FMN or FAD as substrate to generate FMNH₂ or FADH₂ as product. DszD was coexpressed with DBT monooxygenase DszC, the enzyme catalyzing the first step of the biodesulfurization process. It was indicated that the coexpressed DszD could effectively enhance the DszC catalyzed DBT desulfurization reaction.     

The Biosynthesis of Flavin Cofactors in Listeria monocytogenes

Listeria monocytogenes is riboflavin auxotrophic, but it has two genes envisaged to transform riboflavin into FMN and FAD after its uptaked by specialized transporters. One encodes a bifunctional type I FAD synthase (FADS, herein LmFADS-1), while the other produces a protein similar to type I at the FMN:ATP adenylyltransferase (FMNAT) site but with a shorter C-terminal that lacks any riboflavin kinase (RFK) motif. This second protein is rare among bacteria and has been named FADS type II (LmFADS-2). Here we present a biochemical and biophysical study of LmFADS-1 and LmFADS-2 by integrating kinetic and thermodynamic data together with sequence and structural prediction methods to evaluate their occurrence in Listeria, as well as their function and molecular properties. Despite LmFADS-1 similarities to other type I FADSs, (i) its RFK activity has not riboflavin substrate inhibition and occurs under reducing and oxidizing conditions, (ii) its FMNAT activity requires strong reducing environment, and (iii) binding of reaction products, but not substrates, favors binding of the second ligand. LmFADS-2 produces FAD under oxidizing and reducing environments, but its C-terminus module function remains unknown. Listeria species conserve both FADSs, being sequence identity high within L. monocytogenes strains. Our data exemplify alternative strategies for FMN and FAD biosynthesis and homeostasis, envisaging that in Listeria two FADSs might be required to fulfill the supply of flavin cofactors under niches that can go from saprophytism to virulence. As FADSs are attractive antimicrobial targets, understanding of FADSs traits in different species is essential to help in the discovery of specific antimicrobials.     

Purification and characterization of recombinant FAD synthetase from Neurospora crassa

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of ～8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of ～31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and k_cat of 1356 nmol/min/mg and 0.69sec^?1 respectively. Steady state kinetic analysis of NcFADS exhibited a K_m of NcFADS for FMN is 2.7 μM and for MgATP^?2 is 88.7 μM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent K_d of 20.8 μM for FMN and 16.6 μM for MgATP^?2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the T_m is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.     

Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 1. Kinetic evidence for cooperative effects associated with the binding of inorganic phosphate and the 5'-phosphate moiety of the cofactor

The pathway(s) by which the flavin cofactor binds to the apoflavoprotein is the subject of some debate. The crystal and NMR structures of several different flavodoxins have provided some insight, although there is disagreement about the location of the initial interaction between the flavin mononucleotide (FMN) and the apoflavodoxin and the degree of protein conformational change associated with cofactor binding [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332; Steensma, E., and van Mierlo, C. P. M. (1998) J. Mol. Biol. 282, 653-666]. Binding kinetics using stopped-flow spectrofluorimetry and phosphate competition studies were used to develop a model for flavin binding to the flavodoxin from Desulfovibrio vulgaris. In the presence of phosphate, the time course of fluorescence quenching associated with FMN binding to apoflavodoxin was biphasic, whereas riboflavin, which lacks the 5'-phosphate group of FMN, displayed monophasic binding kinetics. When the concentration of phosphate in solution was increased, the FMN binding rates of the two phases behaved differently; the rate of one phase decreased, while the rate of the other increased. A similar increase in the single phase associated with riboflavin binding was also observed. This has led to the following model. The binding of the flavin isoalloxazine ring to its subsite is dependent on the presence of a phosphate group in the phosphate-binding subsite. When phosphate is in the buffer solution, FMN can bind in either of two ways: by the initial insertion of the 5'-phosphate group followed by ring binding or, when inorganic phosphate from solution is bound, the insertion of the isoalloxazine ring first. Riboflavin, which lacks the phosphate moiety of FMN, binds only in the presence of inorganic phosphate, presumably due to the binding of this group in the phosphate-binding subsite. These results suggest that cooperative interactions exist between the phosphate subsite and the ring-binding region in the D. vulgaris flavodoxin that are necessary for isoalloxazine ring binding.