Production of flavin mononucleotide by metabolically engineered yeast Candida famata

Recombinant strains of the flavinogenic yeast Candida famata able to overproduce flavin mononucleotide (FMN) that contain FMN1 gene encoding riboflavin (RF) kinase driven by the strong constitutive promoter TEF1 (translation elongation factor 1α) were constructed. Transformation of these strains with the additional plasmid containing the FMN1 gene under the TEF1 promoter resulted in the 200-fold increase in the riboflavin kinase activity and 100-fold increase in FMN production as compared to the wild-type strain (last feature was found only in iron-deficient medium).Overexpression of the FMN1 gene in the mutant that has deregulated riboflavin biosynthesis pathway and high level of riboflavin production in iron-sufficient medium led to the 30-fold increase in the riboflavin kinase activity and 400-fold increase in FMN production of the resulted transformants. The obtained C. famata recombinant strains can be used for the further construction of improved FMN overproducers.     

Chorismate synthase of Neurospora crassa: a flavoprotein

Chorismate synthase is purified from Neurospora crassa. The final step is accomplished by preparative electrophoresis. Its purity is estimated at ≥90% on the basis of analytical polyacrylamide gel electrophoresis. The enzyme appears to be active in at least two multimeric states, with a subunit molecular weight of ~55,000. The purified enzyme preparation is absolutely dependent on the presence of a reducing system, which can readily be provided under aerobic conditions by NADPH plus FMN or under stringent anaerobic conditions by dithionite. The following evidence implicates a physiological role for FMN in N. crassa chorismate synthase activity: (a) a preferential stimulation of activity by NADPH and FMN over other pyridine and flavin nucleotides, respectively, in both impure and purified enzyme preparations; (b) an alteration of the Chromatographic pattern of the enzyme on diethylaminoethylcellulose by the addition of FMN to the elution buffer; (c) an apparent binding of FMN to the enzyme as exhibited by gel filtration in the presence of the substrate, 3-enolpyruvylshikimate 5-phosphate; (d) a requirement for preliminary incubation with FMN, in concert with the substrate, to eliminate a reaction lag (i.e., to activate the enzyme); (e) a substrate-dependent diaphorase activity exhibited by purified enzyme preparations in the presence of FMN and NADPH. The observed activation and alteration of Chromatographic behavior of chorismate synthase by FMN suggest that the flavin nucleotide influences the conformation of the enzyme. The ability to replace NADPH and FMN with dithionite suggests that FMN mediates the flow of electrons from a source of reducing power (NADPH) to some enzymic site important to the function of the enzyme. Hence, the diaphorase activity which is observed as intrinsic to chorismate synthase of N. crassa may be significant from the standpoint of catalysis or may have importance as a regulatory mechanism.     

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.     

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 the biosynthesis of cytochrome P-450 in rat liver--a probe with phenobarbital.

The reaction of NAD(P)H:flavin oxidoreductase (flavin reductase) from Photobacterium fischeri is proposed to follow a ping-pong bisubstrate-biproduct mechanism. This is based on a steady-state kinetic analysis of initial velocities and patterns of inhibition by NAD⁺ and AMP. The double reciprocal plots of initial velocities versus concentrations of FMN or NADH show, in both cases, a series of parallel lines. The Michaelis constants for NADH (FMN saturating) and FMN (NADH saturating) are 2.2 and 1.2 × 10^?4m, respectively. The product NAD⁺ has been found to be an inhibitor competitive with FMN but non-competitive with NADH. Using AMP as an inhibitor, noncompetitive inhibition patterns were observed with respect to both NADH and FMN as the varied substrate. In addition, the reductase was not inactivated by treatment with N-ethylmaleimide either alone or in the presence of FMN, but the enzyme was inactivated by N-ethylmaleimide in the presence of NADH. These findings suggest that flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis.     

Location of lysyl residues at the allosteric site of fructose 1,6-bisphosphatase

Various flavin analogs were used as alternate substrates or competitive inhibitors to characterize the FMN binding sites of the NADH- and NADPH-specific FMN oxidoreductases from Beneckea harveyi. Several polyhydroxyl compounds were found to be poor competitive inhibitors for the FMN sites of these enzymes. The FMN binding sites of the two enzymes were found to be quite similar. The NADH:FMN oxidoreductase binds FMN exclusively through the isoalloxazine ring. The methyl groups at positions 7 and 8 contribute significantly to this binding. Utilizing lumichrome as a competitive inhibitor of the FMN binding site and AMP as a competitive inhibitor of the NADH binding site, we were able to determine that the NADH:FMN oxidoreductase forms an active ternary complex with NADH binding first in an ordered mechanism. The NADPH oxidoreductase also binds FMN primarily through the isoalloxazine ring. Unlike their participation in reaction with the NADH-specfic enzyme, the methyl groups at positions 7 and 8 are not involved in binding. There was no significant binding of the ribityl phosphate moiety with either enzyme. Both enzymes have lower K_m values for lumiflavin than FMN.     

Identification of a 14mer RNA that recognizes and binds flavin mononucleotide with high affinity

Aptamers are nucleic acids developed by in vitro evolution techniques that bind to specific ligands with high affinity and selectivity. Despite such high affinity and selectivity, however, in vitro evolution does not necessarily reveal the minimum structure of the nucleic acid required for selective ligand binding. Here, we show that a 35mer RNA aptamer for the cofactor flavin mononucleotide (FMN) identified by in vitro evolution can be computationally evolved to a mere 14mer structure containing the original binding pocket and eight scaffolding nucleotides while maintaining its ability to bind in vitro selectively to FMN. Using experimental and computational methodologies, we found that the 14mer binds with higher affinity to FMN (K_D ~ 4 µM) than to flavin adenine dinucleotide (K_D ~ 12 µM) or to riboflavin (K_D ~ 13 µM),despite the negative charge of FMN. Different hydrogen-bond strengths resulting from differing ring-system electron densities associated with the aliphatic-chain charges appear to contribute to the selectivity observed for the binding of the 14mer to FMN and riboflavin. Our results suggest that high affinity and selectivity in ligand binding is not restricted to large RNAs, but can also be a property of extraordinarily short RNAs.     

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 Å 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 Å 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.     

Regulation of Riboflavin Biosynthesis in Bacillus subtilis Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by ribC

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.     

Absorption of the flavin dimers

The electronic absorption spectra of the flavomononucleotide (FMN) in aqueous solution and in glycerine-water solution with change of the dye concentration have been measured. The FMN dimer absorption spectrum, monomer absorption spectrum, dimerization constant K and molar fraction of the monomer were calculated. It was found that FMN dimerization constants in aqueous solution were K_a = 118.0 l/mol and in glycerine K_g = 20.5 l/mol. In the region of the monomer absorption band two dimer absorption bands appear, in accordance with the Kasha molecular exciton theory.     

The flavin mononucleotide content of oral bacteria related to the dry weight of dental plaque obtained from monkeys

The analysis of the protein and flavin mononucleotide (FMN) content of dental plaque obtained from monkeys proved to be, an effective method of estimating the dry weight of dental plaque samples. The FMN content of various bacterial cultures was also comparable with the FMN content of dental plaque. FMN could be measured in a suspending medium containing amines or protein; however, both of these saspending media interfered with the estimate of dry weight by protein determination.     

Crystal Structure of Reduced MsAcg,a Putative Nitroreductase from Mycobacterium smegmatis and a Close Homologue of Mycobacterium tuberculosis Acg

This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacteria''s adaptation to a dormancy state.     

高等植物乙醇酸氧化酶与黄素单核苷酸的松弛结合

The binding condition between flavin mononucleotide (FMN) and protein moiety of glycollate oxidase (GO) was investigated in 11 species of higher plants from 7 families. A loose binding of FMN with GO was universally observed in all the plants. According to this character, a new efficient but mild method was established for preparation of FMN-free GO, which could produce 87.5% recovery of activity . FMN-free GO could be reactivated by adding FMN. For half reactivation 8×10 mol/L FMN was needed, and more than 5×10 mol/L for 100%. The result indicates that there exists a reversible dissociation balance between FMN and protein moiety of GO. Therefore, the concentration of FMN may act as a factor to regulate GO activity in higher plants.     

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.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Discovery and Characterization of FMN-Binding β-Glucuronidases in the Human Gut Microbiome

The human gut microbiota encodes β-glucuronidases (GUSs) that play key roles in health and disease via the metabolism of glucuronate-containing carbohydrates and drugs. Hundreds of putative bacterial GUS enzymes have been identified by metagenomic analysis of the human gut microbiome, but less than 10% have characterized structures and functions. Here we describe a set of unique gut microbial GUS enzymes that bind flavin mononucleotide (FMN). First, we show using mass spectrometry, isothermal titration calorimetry, and x-ray crystallography that a purified GUS from the gut commensal microbe Faecalibacterium prausnitzii binds to FMN on a surface groove located 30 Å away from the active site. Second, utilizing structural and functional data from this FMN-binding GUS, we analyzed the 279 unique GUS sequences from the Human Microbiome Project database and identified 14 putative FMN-binding GUSs. We characterized four of these hits and solved the structure of two, the GUSs from Ruminococcus gnavus and Roseburia hominis, which confirmed that these are FMN binders. Third, binding and kinetic analysis of the FMN-binding site mutants of these five GUSs show that they utilize a conserved site to bind FMN that is not essential for GUS activity, but can affect K_M. Lastly, a comprehensive structural review of the PDB reveals that the FMN-binding site employed by these enzymes is unlike any structurally characterized FMN binders to date. These findings reveal the first instance of an FMN-binding glycoside hydrolase and suggest a potential link between FMN and carbohydrate metabolism in the human gut microbiota.     

Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin

Contribution of three regions (phosphate-binding, 50’s and 90’s loops) of Anabaena apoflavodoxin to FMN binding and reduction potential was studied. Thr12 and Glu16 did not influence FMN redox properties, but Thr12 played a role in FMN binding. Replacement of Trp57 with Glu, Lys or Arg moderately shifted E_ox/sq and E_sq/hq and altered the energetic of the FMN redox states binding profile. Our data indicate that the side chain of position 57 does not modulate E_ox/sq by aromatic stacking or solvent exclusion, but rather by influencing the relative strength of the H-bond between the N(5) of the flavin and the Asn58-Ile59 bond. A correlation was observed between the isoalloxazine increase in solvent accessibility and less negative E_sq/hq. Moreover, E_sq/hq became less negative as positively charged residues were added near to the isoalloxazine. Ile59 and Ile92 were simultaneously mutated to Ala or Glu. These mutations impaired FMN binding, while shifting E_sq/hq to less negative values and E_ox/sq to more negative. These effects are discussed on the bases of the X-ray structures of some of the Fld mutants, suggesting that in Anabaena Fld the structural control of both electron transfer steps is much more subtle than in other Flds.     

Bacillus subtilis ypaA gene regulation mechanism by FMN riboswitch

We studied the regulation of the Bacillus subtilis ypaA gene-encoding riboflavin-transporter by FMN riboswitch. Using translational fusions of the leader region of wild-type ypaA gene with the lacZ-reporter gene in the leader region we showed that in vivo ypaA gene expression decreased more than 10-fold in the presence of endogenous FMN. Introduction of two nucleotide substitutions providing stabilization of the sequester hairpins results in almost complete repression of reporter gene expression. Using toeprint assay in vitro it has been shown that FMN presence inhibits the formation of the 30S initiation complexint the ypaA gene leader mRNA. Our results support the model of ypaA gene regulation whereby FMN binding with the ypaA gene leader sequence results in translation suppression through the sequestering of the SD-sequence.     

The thermodynamics of flavin binding to the apoflavodoxin from Azotobacter vinelandii

A thermodynamic study of the binding of flavins (FMN, FAD, 8-carboxylic acid-riboflavin) to the purified apoflavodoxin from Azotobacter vinelandii has been conducted. The binding of FMN was studied at a number of temperatures (10,15, 20, 25, and 30 °C), pH's (6.0, 7.4, and 9.0), and buffer conditions. The binding of FAD was studied at pH 7.4 and 25 °C under a number of buffer conditions. The binding of 8-carboxylic acid-riboflavin to the apoflavodoxin and the binding of FMN to the dimeric form of the apoflavodoxin were investigated at pH 7.4 and 25 °C. Enthalpies of binding for FMN, FAD, and 8-carboxylic- acid-riboflavin were ?28.3, ?16.6, and ?14.0 kcal mol^?1, respectively. The enthalpy of binding of FMN to the dimeric form of the apoflavodoxin was ?22.2 kcal mol of binding sites^?1. Binding constants of about 10⁸,10⁶, and 10⁶ were obtained for the binding of FMN, FAD, and 8-carboxylic acid-riboflavin, respectively. Using established thermodynamic relationships free energy and entropy changes were calculated. The entropy data indicate that a large degree of ordering of the system occurs upon flavin binding. The pH data suggest that FMN may bind in both the mono-and dianion forms, and that binding doesn't change the pK_a of any functional group in the system. It appears that the phosphate group is probably responsible for approximately half the binding enthalpy observed for the binding of FMN. The temperature-dependence data over the temperature range studied is biphasic, centered at 20 °C, indicating that flavin binding occurs to the protein in two thermodynamic states corresponding to the two heat capacities observed. These findings are used to discuss a model for flavin binding.     

Primary Reactions of the LOV2 Domain of Phototropin Studied with Ultrafast Mid-Infrared Spectroscopy and Quantum Chemistry

Phototropins, major blue-light receptors in plants, are sensitive to blue light through a pair of flavin mononucleotide (FMN)-binding light oxygen and voltage (LOV) domains, LOV1 and LOV2. LOV2 undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine and the FMN C(4a) atom. Here, the primary reactions of Avena sativa phototropin 1 LOV2 (AsLOV2) were studied using ultrafast mid-infrared spectroscopy and quantum chemistry. The singlet excited state (S1) evolves into the triplet state (T1) with a lifetime of 1.5 ns at a yield of ∼50%. The infrared signature of S1 is characterized by absorption bands at 1657 cm⁻¹, 1495-1415 cm⁻¹, and 1375 cm⁻¹. The T1 state shows infrared bands at 1657 cm⁻¹, 1645 cm⁻¹, 1491-1438 cm⁻¹, and 1390 cm⁻¹. For both electronic states, these bands are assigned principally to C=O, C=N, C-C, and C-N stretch modes. The overall downshifting of C=O and C=N bond stretch modes is consistent with an overall bond-order decrease of the conjugated isoalloxazine system upon a π-π^∗ transition. The configuration interaction singles (CIS) method was used to calculate the vibrational spectra of the S1 and T1 excited ππ^∗ states, as well as respective electronic energies, structural parameters, electronic dipole moments, and intrinsic force constants. The harmonic frequencies of S1 and T1, as calculated by the CIS method, are in satisfactory agreement with the evident band positions and intensities. On the other hand, CIS calculations of a T1 cation that was protonated at the N(5) site did not reproduce the experimental FMN T1 spectrum. We conclude that the FMN T1 state remains nonprotonated on a nanosecond timescale, which rules out an ionic mechanism for covalent adduct formation involving cysteine-N(5) proton transfer on this timescale. Finally, we observed a heterogeneous population of singly and doubly H-bonded FMN C(4)=O conformers in the dark state, with stretch frequencies at 1714 cm⁻¹ and 1694 cm⁻¹, respectively.