Role of the covalent flavin linkage in monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a prototypical member of a recently recognized family of amine-oxidizing enzymes that all contain covalently bound flavin. Mutation of the covalent flavin attachment site in MSOX produces a catalytically inactive apoprotein (apoCys315Ala) that forms an unstable complex with FAD (K(d) = 100 muM), similar to that observed with wild-type apoMSOX where the complex is formed as an intermediate during covalent flavin attachment. In situ reconstitution of sarcosine oxidase activity is achieved by assaying apoCys315Ala in the presence of FAD or 8-nor-8-chloroFAD, an analogue with an approximately 55 mV higher reduction potential. After correction for an estimated 65% reconstitutable apoprotein, the specific activity of apoCys315Ala in the presence of excess FAD or 8-nor-8-chloroFAD is 14% or 80%, respectively, of that observed with wild-type MSOX. Unlike oxidized flavin, apoCys315Ala exhibits a high affinity for reduced flavin, as judged by results obtained with reduced 5-deazaFAD (5-deazaFADH(2)) where the estimated binding stoichiometry is unaffected by dialysis. The Cys315Ala.5-deazaFADH(2) complex is also air-stable but is readily oxidized by sarcosine imine, a reaction accompanied by release of weakly bound oxidized 5-deazaFAD. The dramatic difference in the binding affinity of apoCys315Ala for oxidized and reduced flavin indicates that the protein environment must induce a sizable increase in the reduction potential of noncovalently bound flavin (DeltaE(m) approximately 120 mV). The covalent flavin linkage prevents loss of weakly bound oxidized FAD and also modulates the flavin reduction potential in conjunction with the protein environment.     

A two-component flavin-dependent monooxygenase involved in actinorhodin biosynthesis in Streptomyces coelicolor

The two-component flavin-dependent monooxygenases belong to an emerging class of enzymes involved in oxidation reactions in a number of metabolic and biosynthetic pathways in microorganisms. One component is a NAD(P)H:flavin oxidoreductase, which provides a reduced flavin to the second component, the proper monooxygenase. There, the reduced flavin activates molecular oxygen for substrate oxidation. Here, we study the flavin reductase ActVB and ActVA-ORF5 gene product, both reported to be involved in the last step of biosynthesis of the natural antibiotic actinorhodin in Streptomyces coelicolor. For the first time we show that ActVA-ORF5 is a FMN-dependent monooxygenase that together with the help of the flavin reductase ActVB catalyzes the oxidation reaction. The mechanism of the transfer of reduced FMN between ActVB and ActVA-ORF5 has been investigated. Dissociation constant values for oxidized and reduced flavin (FMNox and FMNred) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMNred from ActVB to ActVA-ORF5 without involving a particular interaction between the two protein components. In full agreement with these data, we propose a reaction mechanism in which FMNox binds to ActVB, where it is reduced, and the resulting FMNred moves to ActVA-ORF5, where it reacts with O2 to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.     

Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.     

Molecular modeling reveals the possible importance of a carbonyl oxygen binding pocket for the catalytic mechanism of p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase catalyzes the hydroxylation of an aromatic substrate and uses flavin as a cofactor. The reaction probably occurs via a flavin 4a-hydroperoxide intermediate. In this study the crystal structure of 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin, an analogue of the flavin 4a-hydroperoxide intermediate, was fitted to the active site in the crystal structure of the p-hydroxybenzoate hydroxylase-3,4-dihydroxybenzoate complex. This model of an important catalytic intermediate fitted very well in the active site of p-hydroxybenzoate hydroxylase. The most striking result was that whereas with the normal flavin, the 0-4 of the flavin ring makes only poor hydrogen bonds with the protein, with the flavin 4a-hydroperoxide analogue, the same 0-4 makes strong hydrogen bonds with the NH groups of Gly-46 and Val-47. These two NH groups form a carbonyl oxygen binding pocket which has a geometry almost identical to the oxyanion hole found in several proteases. The possible consequences of this model for the reaction mechanism of p-hydroxybenzoate hydroxylase are discussed.     

Flavin-photosensitized reactions of retinal and stilbene

1. Retinol and stilbene are both isomerized when they are illuminated anaerobically in the presence of flavins. 2. Triplet quenchers (e.g. oxygen, potassium iodide and paramagnetic ions) inhibit the reaction more efficiently than they quench flavin fluorescence. At 77 degrees C in a diethyl ether-isopentane-ethanol (5:5:2) glass retinol quenches flavin phosphorescence, but not its fluorescence. 3. For the stilbene reaction cis/trans photostationary-state mixtures are obtained with different flavins and these are linearly related to the phosphorescence transition energies of the flavins used. 4. The reaction involves the triplet state of flavin and a scheme for the reaction is suggested. 5. The dependence of the rate of reaction on substrate concentration is explicable in terms of this scheme. 6. The photobleaching of rhodopsin sensitized by flavin is also demonstrated.     

NADPH-flavin reductase in human erythrocytes and the reduction of methemoglobin through flavin by the enzyme

A NADPH-dehydrogenase of human erythrocytes was exhaustively purified to a homogeneous protein judging from the electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate. Studies on the specificity for the electron acceptor of this enzyme suggest that flavins serve as the natural and direct electron acceptor. The enzyme showed a broad specificty for flavins and the Michaelis constants for flavins were estimated to be 5 × 10^?5 M for both FMN and riboflavin. Rapid reduction of methemoglobin by the enzyme in the presence of flavin was demonstrated, and the reduction was explained by the reduction of flavin by the enzyme, and subsequent non-enzymatic reduction of methemoglobin by the reduced flavin. The therapeutic significance of flavins was discussed with reference to the flavin reductase activity in hereditary methemoglobinemia.     

Laser-flash-photolysis studies of p-cresol methylhydroxylase. Electron-transfer properties of the flavin and haem components.

p-Cresol methylhydroxylase, a heterodimer consisting of one flavoprotein subunit and one cytochrome c subunit, may be resolved into its subunits, and the holoenzyme may then be fully reconstituted from the pure subunits. In the present study we have characterized the reduction kinetics of the intact enzyme and its subunits, by using exogenous 5-deazariboflavin semiquinone radical generated in the presence of EDTA by the laser-flash-photolysis technique. Under anaerobic conditions the 5-deazariboflavin semiquinone radical reacts rapidly with the native enzyme with a rate constant approaching that of a diffusion-controlled reaction (k = 2.8 X 10(9) M-1 X s-1). Time-resolved difference spectra at pH 7.6 indicate that both flavin and haem are reduced initially by the deazariboflavin semiquinone radical, followed by an additional slower intramolecular electron transfer (k = 220 s-1) from the endogenous neutral flavin semiquinone radical to the oxidized haem moiety of the native enzyme. During the steady-state photochemical titration of the native enzyme at pH 7.6 with deazariboflavin semiquinone radical generated by light-irradiation the haem appeared to be reduced before the protein-bound flavin and was followed by the formation of the protein-bound anionic flavin radical. This result suggests that the redox potential of the haem is higher than that of the flavin, and that deprotonation of the flavin neutral radical occurred during the photochemical titration. Reduction kinetics of the flavoprotein and cytochrome subunits were also investigated by laser-flash photolysis. The protein-bound flavin of the isolated flavin subunit was reduced rapidly by the deazariboflavin semiquinone radical (k = 2.2 X 10(9) M-1 X s-1), as was the haem of the pure cytochrome c subunit (k = 3.7 X 10(9) M-1 X s-1). Flash-induced difference spectra obtained for the flavoprotein and cytochrome subunits at pH 7.6 were consistent with the formation of neutral flavin semiquinone radical and reduced haem, respectively. Investigation of the kinetic properties of the neutral flavin semiquinone radical of the flavoprotein subunit at pH 7.6 and at longer times (up to 5s) were consistent with a slow first-order deprotonation reaction (k = 1 s-1) of the neutral radical to its anionic form.     

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.     

Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8

Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.     

Identity of the emitter in the bacterial luciferase luminescence reaction: binding and fluorescence quantum yield studies of 5-decyl-4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate as a model

The excited state of 4a-hydroxy-4a,5-dihydroFMN has been postulated to be the emitter in the bacterial bioluminescence reaction. However, while the bioluminescence quantum yield of the luciferase emitter is about 0.16, chemiluminescence and fluorescence quantum yields of earlier flavin models mimicking the luciferase emitter were no more than 10(-5). To further examine the proposed chemical identity of the luciferase emitter, 5-decyl-4a-hydroxy-4a,5-dihydroFMN was prepared as a new flavin model. Both the wild-type Vibrio harveyi luciferase and a catalytically active alphaC106A mutant formed complexes with the flavin model at a 1:1 molar ratio with K(d) values at 2.4 and 1.2 microM, respectively. This flavin model inhibited the activity of both luciferases, suggesting that it was bound to the enzyme active center. While the free flavin model was itself only very weakly fluorescent, its binding to either luciferase species resulted in markedly enhanced fluorescence, peaking at 440 nm. The fluorescence quantum yields of 5-decyl-4a-hydroxy-4a,5-dihydroFMN bound to wild-type and alphaC106A luciferases were 0.08 and 0.05, respectively, which are about 50% of the respective emitter bioluminescence quantum yields of these two luciferases. The present findings clearly demonstrated that the luciferase active site was suitable for marked enhancement of fluorescence of 4a-hydroxyflavin and, hence, provides a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN, in its exited state, as the luciferase emitter.     

Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin

The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.     

Adenosine diphosphate moiety does not participate in structural changes for the signaling state in the sensor of blue-light using FAD domain of AppA

A sensor of blue light using FAD (BLUF) protein is a flavin adenine dinucleotide (FAD) based new class blue-light sensory flavoprotein. The BLUF domain of AppA was reconstituted in vitro from apoprotein and flavin adenine dinucleotide, flavin adenine mononucleotide or riboflavin. The light-induced FTIR spectra of the domain reconstituted from various flavins and the 13C-labeled apoprotein showed that identical light-induced structural changes occur in both the flavin chromophore and protein for the signaling state in all of the reconstituted holoproteins. The results showed that an adenosine 5'-dinucleotide moiety is not required for signaling-state formation in a BLUF domain.     

Characterization of SgcE6, the flavin reductase component supporting FAD-dependent halogenation and hydroxylation in the biosynthesis of the enediyne antitumor antibiotic C-1027

The C-1027 enediyne antitumor antibiotic from Streptomyces globisporus possesses an ( S )-3-chloro-5-hydroxy-β-tyrosine moiety, the chloro- and hydroxy-substituents of which are installed by a flavin-dependent halogenase SgcC3 and monooxygenase SgcC, respectively. Interestingly, a single flavin reductase, SgcE6, can provide reduced flavin to both enzymes. Bioinformatics analysis reveals that, similar to other flavin reductases involved in natural product biosynthesis, SgcE6 belongs to the HpaC-like subfamily of the Class I flavin reductases. The present study describes the steady-state kinetic characterization of SgcE6 as a strictly NADH- and FAD-specific enzyme.     

Effect of flavin structure and redox state on catalysis by and flavin-pterin energy transfer in Escherichia coli DNA photolyase

5-DeazaFAD bound to a hydrophobic site in apophotolyase and formed a stable reconstituted enzyme, similar to that observed with FAD. Although stoichiometric incorporation was observed, the flavin ring modification in 1-deazaFAD interfered with normal binding, decreased protein stability, and prevented formation of a stable flavin radical, unlike that observed with FAD. The results suggest that an important hydrogen bond is formed between the protein and N (1) in FAD, but not N (5), and that there is sufficient space at the normal flavin binding site near N (5) to accommodate an additional hydrogen but not near N (1). Catalytic activity was observed with enzyme containing 5-deazaFADH2 (42% of native enzyme) or 1-deazaFADH2 (11% of native enzyme) as its only chromophore, but no activity was observed with the corresponding oxidized flavins, similar to that observed with FAD and consistent with a mechanism where dimer cleavage is initiated by electron donation from excited reduced flavin to substrate. The protein environment in photolyase selectively enhanced photochemical reactivity in the fully reduced state, as evidenced by comparison with results obtained in model studies with the corresponding free flavins. Phosphorescence was observed with free or photolyase-bound 5-deazaFADH2, providing the first example of a flavin that exhibits phosphorescence in the fully reduced state. Formation of an enzyme-substrate complex resulted in a nearly identical extent of quenching of 5-deazaFADH2 phosphorescence (85.1%) and fluorescence (87.5%). The data are consistent with a mechanism involving exclusive reaction of substrate with the excited singlet state of 5-deazaFADH2, analogous to that proposed for FADH2 in native enzyme. Direct evidence for singlet-singlet energy transfer from enzyme-bound 5-deazaFADH2 to 5,10-CH(+)-H4folate was provided by the fact that pterin fluorescence was observed upon excitation of 5-deazaFADH2, accompanied by a decrease in 5-deazaFADH2 fluorescence. On the other hand, the fluorescence of enzyme-bound pterin was quenched by 5-deazaFADox, consistent with energy transfer from pterin to 5-deazaFADox. In each case, the spectral properties of the chromophores were consistent with the observed direction of energy transfer and indicated that transfer in the opposite direction was energetically unlikely. Unlike 5-deazaFAD, energy transfer from pterin to FAD is energetically feasible with FADH2 or FADox. The results indicate that the direction of flavin-pterin energy transfer at the active site of photolyase can be manipulated by changes in the flavin ring or redox state which alter the energy level of the flavin singlet.     

An N1-hydrogen bonding model for flavin coenzyme.

A model flavin possessing a specific hydrogen bond to the N1-position has been synthesized. The redox potential has been measured in aqueous buffer and found to be shifted +21 mV as compared to a similar flavin lacking this hydrogen bond. The reaction of the N1-hydrogen-bonding model with sulfite and 1-benzyl-dihydronicotinamide were examined and compared with the non-hydrogen-bonded flavin. The N1-hydrogen bond did not accelerate the rate of sulfite ion or hydride addition to N5, however the N5-sulfite complex was stabilized by nearly 4-fold over a non-hydrogen-bonding model. The model flavins were also studied computationally.     

Structural basis for the inactivation of Thermus thermophilus proline dehydrogenase by N-propargylglycine

The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 A resolution structure of Thermus thermophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the -amino group of Lys99 via a three-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25 degrees , which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this alpha,beta-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a 1,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a three-carbon chain. The presumed nonenzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely.     

The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain

The sequences of nitric-oxide synthase (NOS) flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR), with the exception of a few regions. One such region is the C terminus; all NOS isoforms are 20-40 amino acids longer than CPR, forming a "tail" that is absent in CPR. To investigate its function, we removed the 21-amino acid C-terminal tail from murine macrophage inducible NOS (iNOS) holoenzyme and from a flavin domain construct. Both the truncated holoenzyme and reductase domain exhibited cytochrome c reductase activities that were 7-10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% faster than the intact form. Using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins and from the flavin to the heme domain is 2-5-fold faster in the absence of the C-terminal tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO catalysis, is 5-fold less stable in truncated iNOS. Although both CPR and intact NOS can exist in a stable, one electron-reduced semiquinone form, neither the truncated holoenzyme nor the truncated flavin domain demonstrate such a form. We propose that this C-terminal tail curls back to interact with the flavin domain in such a way as to modulate the interaction between the two flavin moieties.     

NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.     

A unique catalytic mechanism for UDP-galactopyranose mutase

The flavoenzyme uridine 5'-diphosphate (UDP)-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). The latter is an essential precursor to the cell wall arabinogalactan of Mycobacterium tuberculosis. The catalytic mechanism for this enzyme had not been elucidated. Here, we provide evidence for a mechanism in which the flavin cofactor assumes a new role. Specifically, the N5 of the reduced anionic flavin cofactor captures the anomeric position of the galactose residue with release of UDP. Interconversion of the isomers occurs via a flavin-derived iminium ion. To trap this putative intermediate, we treated UGM with radiolabeled UDP-Galp and sodium cyanoborohydride; a radiolabeled flavin-galactose adduct was obtained. Ultraviolet-visible spectroscopy and mass spectrometry indicate that this product is an N5-alkyl flavin. We anticipate that the clarification of the catalytic mechanism for UGM will facilitate the development of anti-mycobacterial agents.     

High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 mumols NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase.