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PubMed1990

Multiplicity of liver microsomal flavin-containing monooxygenase in the guinea pig: its purification and characterization.

Two distinct forms (FMO-I and FMO-II) of flavin-containing monooxygenase were purified from the liver microsomes of guinea pig. The minimum molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 54,000 for FMO-I and 56,000 for FMO-II, respectively. Tryptic digestion of these enzymes gave different electrophoretic patterns, suggesting that FMO-I and -II have distinct amino acid sequences. The amino terminal sequence of FMO-II could not be estimated probably due to its blocking while that of FMO-I was determined to be highly homologous to the rabbit liver flavin-containing monooxygenase (J. Ozols, 1989, Biochem. Biophys. Res. Commun. 163, 49-55). Absorption maxima of FMO-I and -II were recorded at 368 and 440 nm and 381 and 456 nm, respectively. Molar ratios of FAD to both of these apoenzymes were shown to be one to one. Substrate specificity of FMO-I and -II was determined using 15 compounds as the substrate. The results showed two enzymes that exhibited overlapped but different specificity toward these substrates although FMO-I had lower activity than did FMO-II with all compounds except thiobenzamide. Of particular interest, only FMO-II showed considerably high activities for primary amines, n-octylamine, and n-decylamine. Immunoglobulin G raised against FMO-II could recognize FMO-I as well as FMO-II, but the reactivity of FMO-I toward the antibody was obviously lower than that of FMO-II. Electrophoresis followed by immunostaining revealed that microsomes of lung, kidney, urinary bladder, testis, and spleen contain the same protein as FMO-II and/or FMO-I. Only lung was shown to have an additional isozyme of FAD-monooxygenase with a molecular weight apparently higher than those of FMO-I and -II. These results strongly suggest that at least two forms of flavin-containing monooxygenases distinct from the lung-type isozyme are expressed in liver of guinea pigs.

Amino Acid Sequence↗

PubMed1980

Characterization of a NADH:flavin oxidoreductase induced by cholic acid in a 7 alpha-dehydroxylating intestinal Eubacterium species.

A NADH:flavin oxidoreductase was partially purified (seven-fold) from an intestinal Eubacterium species V.P.I. 12708 using Bio-Gel A 0.5-M and DEAE-cellulose column chromatography. Enzyme activity was measured spectrophotometrically at 340 nm under anaerobic conditions. A molecular weight of 260 000 was estimated by gel filtration chromatography. The partially purified enzyme preparation exhibited single displacement kinetics with respect to the substrates NADH and FAD. The pH optimum under these conditions was 6.8. NADH:flavin oxidoreductase showed an absolute specificity for NADH as electron donor. However, methylene blue, 2,6-dichlorophenolindophenol, K3Fe(CN)6, menadione, riboflavin, FMN and molecular oxygen served as alternate electron acceptors with varying degrees of efficiency. Acriflavin, rotenone, o-phenanthroline, p-chloromercuribenzoate, dicoumarol and 2,4-dinitrophenol inhibited enzyme activity. Surprisingly, 0.1 mM cholic acid, but not 0.1 mM deoxycholic acid, rapidly induced NADH:flavin oxidoreductase activity in growing cultures.

Cell Fractionation↗

PubMed2006

Activity coupling of Vibrio harveyi luciferase and flavin reductase (FRP): oxygen as a probe.

Several lines of evidence have been reported previously to document the ability of the Vibrio harveyi NADPH-specific flavin reductase FRP to directly transfer reduced riboflavin-5'-phosphate to luciferase for bioluminescence. This study aimed at characterizing further the kinetic properties of FRP in such a direct channeling system and investigating whether the complete direct transfer of reduced flavin was the exclusive pathway in the FRP:luciferase coupled bioluminescence reaction. To these ends, a new kinetic approach of oxygen variation was employed. Results indicated that increases in oxygen concentration led to gradual decreases of the peak bioluminescence intensity, K(m,FMN), and K(m,NADPH) of FRP in the coupled reaction. In comparison with theoretical schemes, these findings indicated that the FRP:luciferase coupled reaction can utilize reduced flavin by both free diffusion and direct transfer. The upper limits of the true K(m,FMN) and K(m,NADPH) of FRP in the direct transfer system were determined.

Enzyme Activation↗

PubMed2002

A bacterial flavin reductase system reduces chromate to a soluble chromium(III)-NAD(+) complex.

Biological reduction of carcinogenic chromate has been extensively studied in eukaryotic cells partly because the reduction produces stable chromium(III)-DNA adducts, which are mutagenic. Microbial reduction of chromate has been studied for bioremediation purposes, but little is known about the reduction mechanism. In eukaryotic cells chromate is mainly reduced non-enzymatically by ascorbate, which is usually absent in bacterial cells. We have characterized the reduction of chromate by a flavin reductase (Fre) from Escherichia coli with flavins. The Fre-flavin system rapidly reduced chromate, whereas chemical reduction by NADH and glutathione was very slow. Thus, enzymatic chromate reduction is likely the dominant mechanism in bacterial cells. Furthermore, the end-product was a soluble and stable Cr(III)-NAD(+) complex, instead of Cr(III) precipitate. Since intracellularly generated Cr(III) forms adducts with DNA, protein, glutathione, and ascorbate in eukaryotic cells, we suggest that the produced Cr(III) is primarily complexed to NAD(+), DNA, and other cellular components inside bacteria.

Chromates↗

PubMed1984

Rabbit lung flavin-containing monooxygenase is immunochemically and catalytically distinct from the liver enzyme.

Flavin-containing monooxygenase has been purified to homogeneity from lung microsomes of pregnant rabbits. Antibody to rabbit lung flavin-containing monooxygenase was raised in guinea pig. Ouchterlony double diffusion analysis with this antibody produced precipitin lines of identity with the purified rabbit lung enzyme and lung microsomes from pregnant and non-pregnant rabbits. No cross-reaction was seen with liver microsomes from rabbit or with purified pig liver enzyme. The tricyclic antidepressant drugs, imipramine and chlorpromazine, are not substrates for the rabbit lung enzyme, whereas they are rapidly oxidized by the pig liver enzyme. These results indicate that rabbit lung and liver flavin-containing monooxygenases differ in substrate specificity, are immunochemically distinct proteins, and may be different gene products.

Aniline Compounds↗

PubMed1990

The flavin-containing monooxygenase expressed in pig liver: primary sequence, distribution, and evidence for a single gene.

The primary sequence of the flavin-containing monooxygenase expressed in pig liver has been derived from the nucleotide sequence of cloned cDNA. The derived sequence is composed of 532 amino acids and represents a protein having a molecular weight of 58,952. The complete sequence was obtained from a single clone containing 2070 bases. A second clone, obtained from an independent library, yielded an identical sequence for the 1374 bases present. The amino acid composition compiled from the derived sequence is very similar to that obtained previously from the purified protein. In addition, a 10 amino acid sequence in a peptide formed from the purified protein by digestion with V8 protease exactly matches the derived sequence for residues 309-318. The flavin-containing monooxygenase expressed in pig liver is also expressed in pig lung and kidney as determined by analysis of both microsomal proteins and mRNA. The ratio of mRNA to protein for the enzyme in kidney is about 5 times greater than the same ratio for liver and about twice the ratio for lung. The reasons for these differences are not understood. Southern analysis of genomic DNA indicates that there is a single gene encoding the flavin-containing monooxygenase expressed in pig liver. Therefore, the broad activity of this enzyme in liver appears to be the result of the catalytic diversity of a single protein.

Amino Acid Sequence↗

PubMed2005

Augmenter of liver regeneration: a flavin-dependent sulfhydryl oxidase with cytochrome c reductase activity.

Augmenter of liver regeneration (ALR; hepatopoietin) is a recently discovered enigmatic flavin-linked sulfhydryl oxidase. An N-terminal His-tagged construct of the short form of the human protein has been overexpressed in Escherichia coli. Several lines of evidence suggest that, contrary to a recent report, human ALR is a disulfide-bridged dimer (linked via C15-C124) with two free cysteine residues (C74 and 85) per monomer. The C15-124 disulfides are not critical for dimer formation and have insignificant impact on the dithiothreitol (DTT) oxidase activity of ALR. Although the crystal structure of rat ALR shows a proximal disulfide (C62-C65) poised to interact with the FAD prosthetic group [Wu, C. K., Dailey, T. A., Dailey, H. A., Wang, B. C., and Rose, J. P. (2003) Protein Sci. 12, 1109-1118], only flavin reduction is evident during redox titrations of the enzyme. ALR forms large amounts of neutral semiquinone during aerobic turnover with DTT. This semiquinone arises, in part, by comproportionation between flavin centers within the dimer. Surprisingly, cytochrome c is about a 100-fold better electron acceptor for ALR than oxygen when DTT is the reducing substrate. These data suggest that this poorly understood flavoenzyme may not function as a sulfhydryl oxidase within the mitochondrial intermembrane space but may communicate with the respiratory chain via the mediation of cytochrome c.

Aerobiosis↗

PubMed1996

Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 2. Cystine disulfides involved in catalysis of peroxide reduction.

The two-component alkyl hydroperoxide reductase enzyme system from Salmonella typhimurium catalyzes the pyridine nucleotide-dependent reduction of alkyl hydroperoxide and hydrogen peroxide substrates. This system is composed of a flavoenzyme, AhpF, which is related to the disulfide-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor. We have demonstrated that NADH-linked reduction of AhpF under anaerobic conditions converts two cystine disulfide centers to their dithiol forms. The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl hydroperoxide. Disulfide bridges within oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys489, exist as free thiol groups in oxidized AhpF and play no role in catalysis. Removal of the N-terminal 202-amino acid segment containing the Cys129-Cys132 disulfide center obliterates the ability of AhpF to transfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC. NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin reduction; the reduction potentials of the disulfide centers are thus appropriately poised for electron transfer from NADH and flavin to disulfide-containing substrates (AhpC or DTNB), and ultimately to peroxides from AhpC. Blue, neutral flavin semiquinone is also generated in high yields during reductive titrations (91% yield during dithionite titrations), although the relatively slow formation of this species indicates its catalytic incompetence. A long wavelength absorbance band beyond 900 nm attributable to an FADH2-->NAD+ charge transfer interaction is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved in the catalytic cycle. A catalytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed.

Amino Acid Sequence↗

PubMed1997

Extent of Intramolecular Aromatic-Ring Stacking in Ternary Cu(2+) Complexes Formed by 2,2'-Bipyridyl or 1,10-Phenanthroline and Flavin Mononucleotide (FMN(2)(-))(1)(,)(2).

The stability constants of the 1:1 complexes formed between Cu(Arm)(2+), where Arm = 2,2'-bipyridyl or 1,10-phenanthroline, and flavin mononucleotide (= FMN(2)(-) = riboflavin 5'-phosphate) were determined by potentiometric pH titrations in aqueous solution at 25 degrees C and I = 0.1 M (NaNO(3)). The experimental conditions were carefully selected such that only the monomeric complex species formed. On the basis of previously established log K versus pK(a) straight-line plots (Chen, D.; et al. J. Chem. Soc., Dalton Trans. 1993, 1537-1546) for the corresponding ternary complexes of simple phosphate monoesters and phosphonate derivatives, R-PO(3)(2)(-), where R is a noncoordinating residue, it is shown that the stability of the ternary Cu(Bpy)(FMN) and Cu(Phen)(FMN) complexes is considerably higher than is expected on the basis of the basicity of the phosphate group of FMN(2)(-). By comparison with the stability of the ternary Cu(Arm)(G1P) complexes, where G1P = glycerol 1-phosphate, which had previously been studied (Liang, G.; et al. J. Am. Chem. Soc.1992, 114, 7780-7785) and in which the coordination sphere of Cu(2+) is identical with the one in Cu(Arm)(FMN), it can unequivocally be shown that the mentioned enhanced stability of the Cu(Arm)(FMN) complexes is solely due to the formation of intramolecular stacks; their formation degree reaches for Cu(Bpy)(FMN) and Cu(Phen)(FMN) about 80 and 90%, respectively. These, as well as recent results regarding the self-stacking of FMN(2)(-) (Bastian, M.; Sigel, H. Biophys. Chem. in press) show that the flavin moiety is ideally suited for stacking and charge-transfer interactions, which are so important for the flavin coenzymes in nature.

Journal Article↗

PubMedDate not supplied

Enantioselective S-oxygenation by flavin-containing and cytochrome P-450 monooxygenases.

The reaction of the modified Sharpless reagent, as well as microsomes and highly purified flavin-containing monooxygenase from hog liver, and cytochrome P-450IIB-1 from rat liver efficiently S-oxygenates 2-aryl-1,3-oxathiolanes with significant diastereoselectivity and enantioselectivity. The absolute configuration of the synthetic S-oxides and the enzyme-derived S-oxides was correlated by NMR analysis and by the sign of the Cotton effects obtained from circular dichroism studies. of the sulfides studied, the trans-S-oxide was the major diastereomer produced from monooxygenase-catalyzed biotransformations. In all cases examined, enantioselective S-oxygenation was observed although enantiomeric excess varied from 7 to 100%. In contrast to previous reports, the enantioselectivity of S-oxygenation catalyzed by cytochrome P-450IIB-1 was not always opposite to that of hog liver flavin-containing monooxygenase activity. The presence of the minor S-oxide diastereomers in each case was due to incomplete chiral processing by each monooxygenase and not to a competing achiral nonenzymatic process. The results suggest that the active site of hog liver flavin-containing monooxygenase places greater constraints than that of cytochrome P-450IIB-1 on substrate orientation, but in both cases trans-S-oxide formation is strongly preferred possibly due to steric interactions of the substrate and the active site.

Animals↗

PubMedDate not supplied

Flavin-containing monooxygenase-dependent stereoselective S-oxygenation and cytotoxicity of cysteine S-conjugates and mercapturates.

The metabolism of cysteine S-conjugates of both cis- and trans-1,3-dichloropropene in the presence of rat kidney microsomes and purified flavin-containing monooxygenase from hog liver was investigated in vitro. Preliminary studies with isolated rat kidney cells demonstrated that cysteine S-conjugates were quite toxic to the cells in a process which was consistent with a role of the flavin-containing monooxygenase in the bioactivation of the nephrotoxins. Putative S-oxide metabolites of cysteine S-conjugates were chemically synthesized, and diastereomers were separated and identified by spectroscopic means. The metabolic products of cysteine S-conjugates were identified by comparing the chemical properties of the metabolites with authentic synthetic cysteine S-conjugate S-oxides. Surprisingly, S-conjugate S-oxygenase activity was not observed with rat kidney microsomes but was present when cysteine S-conjugates were incubated with the highly purified flavin-containing monooxygenase from hog liver. The kinetic parameters indicated that considerable S-oxygenase stereoselectivity and structural selectivity was observed: cis cysteine S-conjugates were preferred substrates and N-acetylation of cysteine S-conjugates decreased substrate activity. S-Oxygenation was considerably diastereoselective and diastereoselectivity was much greater for cysteine S-conjugates with higher Vmax values. Cysteine S-conjugate S-oxides were not indefinitely stable, and under certain conditions, the S-oxides underwent a [2,3]-sigmatropic rearrangement to acrolein. Formation of acrolein or other electrophilic products from S-(chloropropenyl)cysteine conjugate S-oxides may contribute to the renal effects observed for S-(chloropropenyl)cysteine conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗

PubMedDate not supplied

N-oxygenation of primary amines and hydroxylamines and retroreduction of hydroxylamines by adult human liver microsomes and adult human flavin-containing monooxygenase 3.

Adult human liver microsomes catalyze the NADPH-dependent N-oxygenation of 10-N-(n-octylamino)-2-(trifluoromethyl)phenothiazine to the corresponding oximes through the intermediacy of the hydroxylamine. In the presence of adult human liver microsomes, the primary amine is stereoselectively converted to the cis-oxime, but addition of the alternative competitive substrate hydroxylamine hydrochloride apparently decreases the amount of aliphatic hydroxylamine retroreduction because an increase in oxime formation was observed. In the presence of hydroxylamine hydrochloride, however, the oxime product recovered was formed with very low stereoselectivity. Studies on the biochemical mechanism of oxime formation suggested that cis-oxime formation in the presence of adult human liver microsomes was largely dependent on the human flavin-containing monooxygenase (form 3). This conclusion is based on the effects of incubation conditions on product formation when compared to results observed in the presence of cDNA-expressed human FMO3. The retroreduction of the intermediate hydroxylamine was dependent on NADPH but was not catalyzed by human flavin-containing monooxygenase (form 3) or any one of seven prominent cytochromes P-450 that have been well-characterized in the human liver microsomes examined. The results suggest that aliphatic primary amines are efficiently sequentially N-oxygenated in the presence of human liver microsomes to hydroxylamines and then to oximes mainly by the human flavin-containing monooxygenase. Retroreduction of the intermediate hydroxylamine is apparently facilitated by a novel but as yet poorly characterized enzyme system that does not employ any of the currently known well-characterized cytochrome P-450 enzymes present in adult human liver microsomes.

Adolescent↗

PubMed1996

Evidence that biliverdin-IX beta reductase and flavin reductase are identical.

A search of the database shows that human biliverdin-IX beta reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IX beta reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IX beta reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IX alpha. Although there is little obvious sequence identity between biliverdin-IX alpha reductase (BVR-A) and biliverdin-IX beta reductase (BVR-B), they do show weak immunological cross-reactivity. Both enzymes bind to 2',5'-ADP-Sepharose.

Amino Acid Sequence↗

PubMed1998

Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica.

The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN.

Animals↗

PubMed1996

Saccharomyces cerevisiae chorismate synthase has a flavin reductase activity.

Chorismate synthase (CS) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to form chorismate, which is the last common intermediate in the synthesis of the three aromatic amino acids phenylalanine, tyrosine and tryptophan. Despite the overall redox-neutral reaction, catalysis has an absolute requirement for reduced flavin. In the fungus Neurospora crassa, a flavin reductase (FR) activity able to generate reduced flavin mononucleotide in the presence of NADPH is an intrinsic feature of a bifunctional CS. In all bacterial and plant species investigated to date, purified CSs lack an FR activity and are correspondingly 8-10 kDa smaller than the N. crassa CS (on the basis of SDS-PAGE). The cloning of N. crassa CS and subsequent characterization of the purified heterologously expressed enzyme indicates that, surprisingly, the FR probably resides within a region conserved amongst both mono- and bifunctional CSs and is not related to non-homologous sequences which contribute to the larger molecular mass of the N. crassa CS. This information directed this work towards the smaller Saccharomyces cerevisiae CS, the sequence of which was known, although the protein has not been extensively characterized biochemically. Here the characterization of the S. cerevisiae CS is reported in more detail and it is shown that the protein is also bifunctional. With this knowledge, S. cerevisiae could be used as a genetic system for studying the physiological consequences of bifunctionality. The phylogenetic relationship amongst known CSs is discussed.

Amino Acid Sequence↗

PubMed2002

Photoactivation of the flavin cofactor in Xenopus laevis (6 - 4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance.

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6-4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6-4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair.

Animals↗

PubMed2001

Involvement of the flavin si-face tyrosine on the structure and function of ferredoxin-NADP+ reductases.

In ferredoxin-NADP(+) reductase (FNR), FAD is bound outside of an anti-parallel beta-barrel with the isoalloxazine lying in a two-tyrosine pocket. To elucidate the function of the flavin si-face tyrosine (Tyr-89 in pea FNR) on the enzyme structure and catalysis, we performed ab initio molecular orbital calculations and site-directed mutagenesis. Our results indicate that the position of Tyr-89 in pea FNR is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin. Moreover, most of FNR-like proteins displayed geometries for the si-face tyrosine phenol and the flavin, which correspond to the more negative free energy theoretical value. FNR mutants were obtained replacing Tyr-89 by Phe, Trp, Ser, or Gly. Structural and functional features of purified FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein. Moreover, hydrogen bonding through the Tyr-89 hydroxyl group may be responsible of the correct positioning of FAD and the substrate NADP(+)

Animals↗

PubMed2003

Structural studies on flavin reductase PheA2 reveal binding of NAD in an unusual folded conformation and support novel mechanism of action.

The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols. We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 A), which is the first structure of a member of this flavin reductase family. We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 A). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group (K(d) = 10 nm) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking. This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide. The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate. In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis.

Amino Acid Sequence↗