High-level expression of fully active yeast flavocytochrome b2 in Escherichia coli.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from the yeast Saccharomyces cerevisiae and three singly substituted mutant forms (F254, R349 and K376) have been expressed in the bacterium Escherichia coli. The enzyme expressed in E. coli contains the protohaem IX and flavin mononucleotide (FMN) prosthetic groups found in the enzyme isolated from yeast, has an electronic absorption spectrum identical with that of the yeast protein and an identical Mr value of 57,500 estimated by SDS/polyacrylamide-gel electrophoresis. N-Terminal amino-acid-sequence data indicate that the flavocytochrome b2 isolated from E. coli begins at position 6 (methionine) when compared with mature flavocytochrome b2 from yeast. The absence of the first five amino acid residues appears to have no effect on the enzyme-catalysed oxidation of L-lactate, since Km values for the yeast- and E. coli-expressed wild-type enzymes were identical within experimental error. The F254 mutant enzyme expressed in E. coli also showed kinetic parameters essentially the same as those found for the enzyme from yeast. The R349 and K376 mutant enzymes had no activity when expressed in either yeast or E. coli. The yield of flavocytochrome b2 from E. coli is estimated to be between 500- and 1000-fold more than from a similar wet weight of yeast (this high level of expression results in E. coli cells which are pink in colour). The increased yield has allowed us to verify the presence of FMN in the R349 mutant enzyme. The advantages of E. coli as an expression system for flavocytochrome b2 are discussed.     

Determination of the role of the Carboxyl-terminal leucine-122 in FMN-binding protein by mutational and structural analysis

Mutants of flavin mononucleotide-binding protein (FMN-bp) were made by site-directed mutagenesis to investigate the role of carboxyl-terminal Leu122 of the pairing subunit in controlling redox potentials, binding the prosthetic group, and forming the tertiary and quaternary structure. We compared the oxidation-reduction potentials, FMN-binding properties, and higher structures of wild-type FMN-bp and four mutant proteins (L122Y, L122E, L122K and L122-deleted). We found that the redox potentials were affected by mutations. Also, the affinities of L122E, L122K and L122 deletion mutant apoproteins for FMN were lower than for the wild-type apoprotein, whereas the affinity of L122Y for FMN was increased. Analytical ultracentrifugation showed that the dissociation constants for dimerization of L122E and L122K were larger than for wild-type FMN-bp, whereas the dissociation constants for L122Y and the deletion mutant were lower than for the wild type. Finally, we determined the higher structures of L122Y, L122E and L122K mutants by X-ray crystallography. Our results show that the mutation of Leu122 in FMN-bp changes midpoint potentials, dissociation constants for FMN, and dimer formation, indicating that this residue is important in the pairing subunit.     

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L-lactate dehydrogenase).

The family of FMN-dependent, alpha-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate alpha-proton (so-called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins & flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122). This suggested the occurrence of a pKa increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and O4, a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pKa drop of about half a pH unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pKa increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.     

Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase.

Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR). Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme. By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for approximately 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase. The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.     

High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7

Flavin reductase HpaC(St) catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaC(St), which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaC(St) was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS-PAGE. The purified refolded HpaC(St) did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.     

Mutation of the heme-binding crevice of flavocytochrome b2 from Saccharomyces cerevisiae: altered heme potential and absence of redox cooperativity between heme and FMN centers.

Kinetic and thermodynamic properties of yeast flavocytochrome b2 (EC 1.1.2.3) are modified by the product pyruvate, which binds to the flavosemiquinone (FSQ) form of the prosthetic flavin and decreases the thermodynamic driving force for electron transfer from FSQ to heme. Pyruvate inhibits flavocytochrome b2, but the catalytic competence of pyruvate-ligated FSQ in intramolecular electron transfer to heme is unclear; one kinetic study suggested pyruvate prevented this reaction [Tegoni, M, Janot J.-M., & Labeyrie, F. (1990) Eur. J. Biochem. 190, 329-342], while laser flash photolysis indicated pyruvate was essential [Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555]. To address this problem, wild-type (WT) and mutant (L36I) flavocytochromes b2 have been expressed in Escherichia coli. Both forms incorporated heme and FMN prosthetic groups and were catalytically active. The mutation L36I was a conservative substitution within the heme-binding crevice and was designed to alter the midpoint potential (Em) of the heme to alter the pyruvate-FSQ/heme equilibrium. Potentiometric titrations yielded Em values (pH 7.0, 25 degrees C) of +8 and -28 mV for WT and L36I forms, respectively. The FMN midpoint potentials in the absence of pyruvate (-58 mV, n = 2) were identical within experimental error in WT and L36I species and were also identical (+5 mV, n = 1) in the presence of pyruvate. These results indicated the absence of redox cooperativity between FMN and heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.     

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.     

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.     

Kinetic studies on the role of Lys-171 and Lys-358 in the beta subunit of sarcosine oxidase from Corynebacterium sp. U-96

Heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96(SO-U96) contains non-covalent and covalent flavins. Lys-358 and Lys-171 in the beta subunit is present at non-covalent flavin adenine dinucleotide (FAD)- and covalent flavin monodinucleotide (FMN)-binding sites, respectively. The Lys-358 mutant, K358R showed 0.07% activity and higher apparent K(m) for sarcosine than the wild-type enzyme, but K358A and K358D mutants showed no activity, suggesting the importance of amino group of Lys358 in the sarcosine-binding to the enzyme. The Lys171 mutants, K171R, K171A and K171D showed 58, 39 and 32% activity of the wild-type enzyme, respectively. An apparent K(m) for oxygen and K(d) of enzyme-sulphite complex increased by the mutation. The rate of reduction of the FAD of K171 mutants with sarcosine did not change by the mutation. The stopped-flow photodiode array analyses of the anaerobic reduction with sarcosin of the wild-type and K171 mutant enzymes showed characteristic spectra of neutral and anionic semiquinones, especially for K171A enzyme. On the basis of these results, the reductive-half reaction of the wild-type and K171 mutant enzymes is explained by a mechanism involving the semiquinones. Low activity of K171 mutants is suggested to be derived from the low rate of oxidation of the reduced FMN in the enzyme.     

Crystallographic investigation of the role of aspartate 95 in the modulation of the redox potentials of Desulfovibrio vulgaris flavodoxin

The side chain of aspartate 95 in flavodoxin from Desulfovibrio vulgaris provides the closest negative charge to N(1) of the bound FMN in the protein. Site-directed mutagenesis was used to substitute alanine, asparagine, or glutamate for this amino acid to assess the effect of this charge on the semiquinone/hydroquinone redox potential (E(1)) of the FMN cofactor. The D95A mutation shifts the E(1) redox potential positively by 16 mV, while a negative shift of 23 mV occurs in the oxidized/semiquinone midpoint redox potential (E(2)). The crystal structures of the oxidized and semiquinone forms of this mutant are similar to the corresponding states of the wild-type protein. In contrast to the wild-type protein, a further change in structure occurs in the D95A mutant in the hydroquinone form. The side chain of Y98 flips into an energetically more favorable edge-to-face interaction with the bound FMN. Analysis of the structural changes in the D95A mutant, taking into account electrostatic interactions at the FMN binding site, suggests that the pi-pi electrostatic repulsions have only a minor contribution to the very low E(1) redox potential of the FMN cofactor when bound to apoflavodoxin. Substitution of D95 with glutamate causes only a slight perturbation of the two one-electron redox potentials of the FMN cofactor. The structure of the D95E mutant reveals a large movement of the 60-loop (residues 60-64) away from the flavin in the oxidized structure. Reduction of this mutant to the hydroquinone causes the conformation of the 60-loop to revert back to that occurring in the structures of the wild-type protein. The crystal structures of the D95E mutant imply that electrostatic repulsion between a carboxylate on the side chain at position 95 and the phenol ring of Y98 prevents rotation of the Y98 side chain to a more energetically favorable conformation as occurs in the D95A mutant. Replacement of D95 with asparagine has no effect on E(2) but causes E(1) to change by 45 mV. The D95N mutant failed to crystallize. The K(d) values of the protein FMN complex in all three oxidation-reduction states differ from those of the wild-type complexes. Molecular modeling showed that the conformational energy of the protein changes with the redox state, in qualitative agreement with the observed changes in K(d), and allowed the electrostatic interactions between the FMN and the surrounding groups on the protein to be quantified.     

The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as measured by NMR chemical shift temperature dependencies.

In the Clostridium beijerinckii flavodoxin, the reduction of the flavin mononucleotide (FMN) cofactor is accompanied by a local conformation change in which the Gly57-Asp58 peptide bond "flips" from primarily the unusual cis O-down conformation in the oxidized state to the trans O-up conformation such that a new hydrogen bond can be formed between the carbonyl group of Gly57 and the proton on N(5) of the neutral FMN semiquinone radical [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. This interaction is thought to contribute to the relative stabilization of the flavin semiquinone and may be at least partially responsible for the substantial separation of the midpoint potentials of the two one-electron reduction steps. Through a series of amino acid substitutions, the above cited study demonstrated the critical role of the often conserved glycine residue in this process. However, it has not been directly established experimentally as to whether these substitutions brought about the changes in the midpoint potentials by altering the strength of this hydrogen-bonding interaction as proposed. In this study, the relative strengths of the FMN N(5)H.O57 hydrogen bond in wild type and the G57A, G57N, and G57T mutants were evaluated by measuring the temperature dependency of the chemical shift for the proton on N(5) of the fully reduced cofactor by 1H-15N HSQC nuclear magnetic resonance spectroscopy. Based on the established correlation between the temperature coefficient of amide protons and the strength of hydrogen bonding in small peptides, the apparent strength of the N(5)H.O57 interaction was found to depend on the properties of the side chain at position 57. The glycine residue found in the wild-type flavodoxin appears to provide the strongest interaction while the beta-branched side chain in the G57T mutant provides the weakest. A good correlation was noted between the temperature coefficients of N(5)H and the one-electron reduction potential for the ox/sq couple as well as the binding free energy of the FMN semiquinone in this group of mutants. These results provide more direct quantitative evidence that support the previous hypothesis that this conformation change and the associated formation of the hydrogen bonding interaction with N(5)H of the reduced FMN represent an important means of stabilizing the neutral semiquinone and in modulating the oxidation-reduction potentials of the flavin cofactor in this and perhaps other flavodoxins.     

A new flavin radical signal in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae. An EPR/electron nuclear double resonance investigation of the role of the covalently bound flavins in subunits B and C

The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.     

Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1))

Trimethylamine dehydrogenase (TMADH) is an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cysteinyl FMN, which is bent by approximately 25 degrees along the N5-N10 axis of the flavin isoalloxazine ring. This unusual conformation is thought to modulate the properties of the flavin to facilitate catalysis, and has been postulated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30A mutant TMADH enzymes, both determined at 2.2 A resolution. Combined crystallographic and NMR studies reveal the presence of inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the deflavo forms of the recombinant wild-type and C30A enzymes that are generated in vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, contrary to expectation, the 6-S-cysteinyl FMN link is not responsible for the 25 degrees butterfly bending along the N5-N10 axis of the flavin in TMADH. Computational quantum chemistry studies strongly support the proposed role of the butterfly bend in modulating the redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wild-type and C30A proteins. Computational studies reveal a hitherto, unrecognized, contribution made by the S(gamma) atom of Cys-30 to substrate binding, and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.     

Modulation of the redox properties of the flavin cofactor through hydrogen-bonding interactions with the N(5) atom: role of alphaSer254 in the electron-transfer flavoprotein from the methylotrophic bacterium W3A1

The functional effects of hydrogen-bonding interactions at the N(5) atom of the flavin cofactors in the oxidized state have not been well established in flavoproteins. The unique properties of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) were used to advantage in this study to evaluate this interaction. In wETF, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized state of the flavin. The strength of this hydrogen bond was systematically altered by the substitution of alphaSer254 with threonine, cysteine, or alanine by site-directed mutagenesis. The anionic semiquinone form of the flavin, which is highly stabilized both thermodynamically and kinetically in the wild-type protein, was observed to accumulate in all three mutants. However, the midpoint potential for the first couple (Eox/sq) was significantly decreased for all of the mutants, and the kinetic barrier toward the reduction of the anionic semiquinone that is observed in the wild-type wETF was effectively abolished in the alphaS254T and alphaS254C mutants. Based on the observed changes in the Kd values and associated binding energies for the flavin, the amino acid replacements destabilize both the oxidized and semiquinone states of the flavin, but to a much greater extent for the anionic semiquinone state. The Eox/sq values for the alphaSer254 mutants follow a general trend with the strength of N(5) H-bond in the oxidized state as indicated by Raman spectral analyses. These results support the conclusion that the H-bonding interaction at the N(5) plays a key role in establishing the high Eox/sq and the unusually high stability of the anionic semiquinone state in wETF.     

Inactivation of C30A trimethylamine dehydrogenase by N-cyclopropyl-alpha-methylbenzylamine, 1-phenylcyclopropylamine, and phenylhydrazine.

Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methylotrophus (sp. W(3)A(1)) and its C30A mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine, N-cyclopropyl-alpha-methylbenzylamine, and 1-phenylcyclopropylamine. All three compounds irreversibly inactivated both the wild-type and C30A mutant enzymes, although phenylhydrazine was 10 times more potent than N-cyclopropyl-alpha-methylbenzylamine, which was much more potent than 1-phenylcyclopropylamine. The change in the UV--visible absorption spectra upon modification indicated that the flavin was modified. In the case of the C30A mutant, the absence of a covalent attachment of the flavin to the polypeptide has permitted LC-electrospray mass spectrometry of the reaction product to be undertaken, demonstrating new mass peaks corresponding to various chemically modified forms of the flavin cofactor. In the case of N-cyclopropyl-alpha-methylbenzylamine, masses corresponding to hydroxy-FMN and hydroxyriboflavin were detected. 1-Phenylcyclopropylamine inactivation of the C30A mutant produced three modified flavins, as evidenced by the electrospray mass spectrum: hydroxy-FMN, FMN plus C(6)H(5)COCH(2)CH(2), and hydroxy-FMN plus C(6)H(5)COCH(2)CH(2). Phenylhydrazine inactivation of the C30A mutant gave at least seven different modified flavins: hydroxyriboflavin, hydroxy-FMN, two apparently isomeric compounds corresponding to hydroxy-FMN plus one phenyl group, two apparently isomeric compounds corresponding to FMN plus one phenyl group, and FMN plus two phenyl groups. Covalent flavin adduct formation appears to be the only modification because dialysis of the inactive enzyme followed by reconstitution with FMN restores the enzyme activity to that of a noninactivated control.     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

Switching kinetic mechanism and putative proton donor by directed mutagenesis of glutathione reductase

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase, Tyr-177 (the residue corresponding to Tyr-197 in the NADPH-binding pocket of the homologous human enzyme) was changed to phenylalanine (Y177F), serine (Y177S), and glycine (Y177G). The catalytic activity of the Y177F mutant was very similar to that of the wild-type enzyme, but that of the Y177S and Y177G mutants was substantially diminished. However, all three mutants retained the ability to protect the reduced flavin from adventitious oxidation, indicating that Tyr-177 does not act as a simple "lid" on the NADPH-binding pocket and that the protection of the reduced enzyme must be due largely to burial of the isoalloxazine ring in the protein. The wild-type enzyme and Y177F mutant displayed ping-pong kinetics, but the Y177S and Y177G mutants appeared to have switched to an ordered sequential mechanism. This could be explained by supposing that the enzyme normally functions by a hybrid kinetic mechanism and that the Y177S and Y177G mutations diverted flux from the ping-pong loop favored by the wild-type enzyme to an ordered sequential loop. The necessary change in the partitioning of the common E-NADPH intermediate could be caused by a slowing of the formation of the EH2 intermediate on the ping-pong loop, or by the observed concomitant fall in the Km for glutathione favoring flux through the ordered sequential loop. In another experiment, His-439, thought to act as a proton donor/acceptor in the glutathione-binding pocket, was mutated to a glutamine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase

Tyrosine ammonia lyase (TAL) catalyzes the conversion of L-tyrosine to p-coumaric acid using a 3,5-dihydro-5-methylidene-4H-imidazole-4-one (MIO) prosthetic group. In bacteria, TAL is used for production of the photoactive yellow protein chromophore and for caffeic acid biosynthesis in certain actinomycetes. Here we biochemically examine wild-type and mutant forms of TAL from Rhodobacter sphaeroides (RsTAL). Kinetic analysis of RsTAL shows that the enzyme displays a 90-fold preference for L-tyrosine versus L-phenylalanine as a substrate. The pH-dependence of TAL activity with L-tyrosine and L-phenylalanine demonstrates a common protonation state for catalysis, but indicates a difference in charge-state for binding of either amino acid. Site-directed mutagenesis demonstrates that Ser150, Tyr60, and Tyr300 are essential for catalysis. Mutation of Ser150 to an alanine abrogates formation of the MIO prosthetic group, as shown by mass spectrometry, and prevents catalysis. The Y60F and Y300F mutants were inactive with both amino acid substrates, but bound p-coumaric and cinnamic acids with less than 12-fold changes in affinity compared the wild-type enzyme. Analysis of MIO-dithiothreitol adduct formation shows that the reactivity of the prosthetic group is not significantly altered by mutation of either Tyr60 or Tyr300. The mechanistic roles of Ser150, Tyr60, and Tyr300 are discussed in relation to the three-dimensional structure of RsTAL and related MIO-containing enzymes.     

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.