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PubMed2003

Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten.

The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-gamma]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten, which may be liberated following cardiomyocyte destruction during the inflammation of the heart.

Acute Disease↗
PubMed1998

4-Hydroxybenzoyl-CoA reductase (dehydroxylating) from the denitrifying bacterium Thauera aromatica--prosthetic groups, electron donor, and genes of a member of the molybdenum-flavin-iron-sulfur proteins.

4-Hydroxybenzoyl-CoA reductase catalyzes an important reaction in the anaerobic metabolism of phenolic compounds, i.e. the reductive removal of an aromatic hydroxyl group. The prosthetic groups and the natural electron donor of the enzyme were investigated and the genes were cloned and sequenced. The enzyme is a molybdenum-flavin-iron-sulfur protein of subunit composition of alpha2beta2gamma2. It contains approximately 1.3 flavin nucleotide, probably FAD, 1.9 Mo, 15 Fe, and 12.5 acid-labile sulfur. Sequence interpretation suggests that the native enzyme contains two [4Fe-4S] and four [2Fe-2S] clusters. A 9.8-kDa ferredoxin with two [4Fe-4S] clusters functions as the natural electron donor. The genes coding for the three subunits, hcrABC, show high similarities to other molybdenum-flavin-iron-sulfur proteins of the xanthine oxidase family, notably to the three putative 4-hydroxybenzoyl-CoA reductase genes in Rhodopseudomonas palustris. In addition, there are close similarities to three open reading frames (orf) in E. coli. A major difference is the presence of an additional domain in the beta-subunit (HcrB, 35 kDa) probably carrying an additional iron-sulfur cluster. The 82-kDa alpha-subunit (HcrA) contains a Mo-cofactor-binding site. The 17-kDa gamma-subunit (HcrC) harbors two [2Fe-2S] clusters. Upstream of the hcrCAB region, an ORF was found coding for a regulatory protein of the MarR family. Downstream of the hcrCAB region lies an ORF presumably coding for a hydrophobic permease.

Amino Acid Sequence↗
PubMed2003

Marinobacterium sp. strain DMS-S1 uses dimethyl sulphide as a sulphur source after light-dependent transformation by excreted flavins.

Marinobacterium sp. strain DMS-S1 is a unique marine bacterium that can use dimethyl sulphide (DMS) as a sulphur source only in the presence of light. High-performance liquid chromatography (HPLC) analyses of the culture supernatant revealed that excreted factors, which could transform DMS to dimethyl sulphoxide (DMSO) under light, are FAD and riboflavin. In addition, FAD appeared to catalyse the photolysis of DMS to not only DMSO but also methanesulphonate (MSA), formate, formaldehyde and sulphate. As strain DMS-S1 can use sulphate and MSA as a sole sulphur source independently of light, the excretion of flavins appeared to support the growth on DMS under light. Furthermore, three out of 12 marine bacteria from IAM culture collection were found to be able to grow on DMS with the aid of photolysis by the flavins excreted. This is the first report that bacteria can use light to assimilate oceanic organic sulphur compounds outside the cells by excreting flavins as photosensitizers.

Alteromonadaceae↗
PubMed2001

Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction.

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-A resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 A from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora. This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.

Amino Acid Sequence↗
PubMed2000

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: conversion of FMN to 8-formyl-FMN.

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of alpha-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH(3)- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.

Amino Acid Sequence↗
PubMed2000

The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation.

Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases.

Binding Sites↗
PubMed1996

Interaction of bacterial luciferase with 8-substituted flavin mononucleotide derivatives.

Bacterial luciferase catalyzes the emission of visible light from the reaction of reduced flavin, molecular oxygen, and an n-alkyl aldehyde. The mechanism of the reaction was probed by measuring the electronic effects of various substituents at the 8-position of the flavin ring system. Substituent effects were obtained for CH3-, Cl-, CH3O-, CH3S-, F-, and H- on the rate of formation and decay of the hydroperoxyflavin intermediate and the time courses for the emission of visible light. The rate constant for the decay of light emission increases for the series Cl < F < H < CH3S < CH3 < CH3O. These results are not compatible with a standard Baeyer-Villiger type mechanism for the chemical transformation, but they are consistent with a decrease in the electron density at the reaction center of the flavin moiety during the rate-limiting step of the reaction.

Catalysis↗
PubMed1999

Homology between egg white sulfhydryl oxidase and quiescin Q6 defines a new class of flavin-linked sulfhydryl oxidases.

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. Reduced proteins are the preferred thiol substrates of this secreted enzyme. The egg white oxidase shows an average 64% identity (from randomly distributed peptides comprising more than 30% of the protein sequence) to a human protein, Quiescin Q6, involved in growth regulation. Q6 is strongly expressed when fibroblasts enter reversible quiescence (Coppock, D. L., Cina-Poppe, D., Gilleran, S. (1998) Genomics 54, 460-468). A peptide antibody against Q6 cross-reacts with both the egg white enzyme and a flavin-linked sulfhydryl oxidase isolated from bovine semen. Sequence analyses show that the egg white oxidase joins human Q6, bone-derived growth factor, GEC-3 from guinea pig, and homologs found in a range of multicellular organisms as a member of a new protein family. These proteins are formed from the fusion of thioredoxin and ERV motifs. In contrast, the flavin-linked sulfhydryl oxidase from Aspergillus niger is related to the pyridine nucleotide-dependent disulfide oxidoreductases, and shows no detectable sequence similarity to this newly recognized protein family.

Amino Acid Sequence↗
PubMed1999

Role of the flavin midpoint potential and NAD binding in determining NAD versus oxygen reactivity of xanthine oxidoreductase.

Xanthine oxidoreductase from bovine milk can be prepared in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH), depending on the number of protein cysteines versus cystines. Enzyme forms differ in respect to their oxidizing substrates; XDH prefers NAD to molecular oxygen, whereas XO only reacts significantly with oxygen. The preference for oxidizing substrate is partially explained by thermodynamics. Unlike XDH, the midpoint potential of the FAD, the center at which oxygen and NAD react, is too high in XO to efficiently reduce NAD (Hunt, J., Massey, V., Dunham, W.R., and Sands, R.H. (1993) J. Biol. Chem. 268, 18685-18691). To distinguish between changes in thermodynamics and in substrate binding, samples of both XO and XDH have been prepared in which the native FAD has been replaced with an FAD analog of different redox potential, 1-deaza-FAD or 8-CN-FAD. Reductive titrations indicate that both 1-deaza-XO and 1-deaza-XDH have a flavin midpoint potential similar to native XDH and that 8-CN-XO and 8-CN-XDH each have a flavin potential higher than XO. Both the low potential 1-deaza-XO and the high potential 8-CN-XDH contain essentially no xanthine/NAD activity. However, 1-deaza-XDH does exhibit xanthine/NAD activity, and 8-CN-XO has normal xanthine/oxygen activity. The binding of NAD to oxidized XO and XDH was investigated by ultrafiltration and isothermal titration calorimetry. The Kd for the binding of NAD to XDH was determined to be 280 +/- 145 microM by ultrafiltration and 160 +/- 40 microM by isothermal titration calorimetry. No evidence for the binding of NAD to XO by either method could be obtained. A low flavin midpoint potential is necessary but not sufficient for dehydrogenase activity.

Animals↗
PubMed2004

Catalytic mechanism of Chlamydia trachomatis flavin-dependent thymidylate synthase.

Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H(4)folate are products of the reaction. Production of H(4)folate rather than H(2)folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA(-) E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH(2)H(4)folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.

Amino Acid Sequence↗
PubMed1997

A Raman study on the C(4)=O stretching mode of flavins in flavoenzymes: hydrogen bonding at the C(4)=O moiety.

Raman spectroscopy was used to investigate the hydrogen bonding at the C(4)=O moiety of the isoalloxazine nucleus in a series of flavins and flavoproteins. Isotope effects of Raman bands confirmed that the band observed around 1,710 cm(-1) is mainly derived from C(4)=O stretching vibrational mode. A linear correlation was observed between the frequency of C(4)=O stretching and the chemical shift of 13C(4), suggesting that the data from both Raman and NMR spectroscopies reflect a common perturbation, i.e., hydrogen bonding. The maximum difference of C(4)=O frequency among flavins and flavoproteins examined is 36 cm(-1) [1,723 cm(-1) for riboflavin-binding protein (Kim, M. and Carey, P.C. (1993) J. Am. Chem. Soc. 115, 7015-7016) and 1,687 cm(-1) for the complex of medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA]; the maximum difference of 40-70 kJ/mol in the hydrogen bonding strength at the C(4)=O exists among flavoproteins. By use of an empirical linear correlation between the frequency of C=O stretching and the bond length of the C=O, it is estimated that the maximum difference in the bond length among flavoproteins treated here is ca. 0.017 A. The hydrogen bonding at the C(4)=O in medium-chain and short-chain acyl-CoA dehydrogenases becomes stronger upon complexation with substrate analogs. Since the hydrogen bonding at the C(4)=O is expected to enhance the electron-accepting capacity of the N(5) position, substrate-binding itself probably raises the reactivity of flavin, through enhancing the hydrogen bonding.

Crystallography, X-Ray↗
PubMed1976

Structure of flavin adducts with acetylenic substrates. Chemistry of monoamine oxidase and lactate oxidase inhibition.

The photoreaction of flavoquinones (lumiflavin, riboflavin, FMN etc. and their 3-alkylated derivatives) with propargylamine-type acetylenic substrates, R4 -Calpha identical to Cbeta -CgammaHR3 -NR2R1, yields a mixture of two adducts,which result from covalent Calpha fixation of the Cgamma-deprotonated substrate to either position C(4a) or N(5) in the flavin nucleus. The N(5) adduct is a dihydroflavin-5-trimethine-cyanine with very intense (xi greater than 20000 M-1 cm-1) absorption maxima in the region 380-450 nm depending on the R1,R2. This absorption allows recognition of minute amounts of this species of flavocyanine even in complex mixtures. Flavocyanines can be reconverted to starting flavin by base. It spectral properties are identical with those obtained for the pargyline-flavin inhibitor complex from bovine kidney or pig liver monoamine oxidase.

Acetylene↗
PubMed1980

Binding of Cibacron blue F3GA to the flavin and NADH sites in cytochrome b5 reductase.

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran--Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 microM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 microM NADH + 10 microM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitude at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran--Sepharose: the NADH binding site and flavin binding site. For the latter it is clear that isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, and FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochem. 90, 563--569].

Animals↗
PubMed1980

A flavin-mononucleotide-binding site in Hansenula anomala nicked flavocytochrome b2, requiring the association of two domains.

Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4.

Ascomycota↗
PubMed1983

A cysteine cluster critical for flavin binding in flavocytochrome b2 from Baker's yeast.

We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome b2, a reaction which leads to the loss of flavin-binding capacity ('inactivation') [D. Pompon and F. Lederer (1982) Eur. J. Biochem. 129, 143-137]. For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit. In this work we report the results of sequence studies which elucidate the nature of the modification. The modified protein was cleaved with cyanogen bromide and the peptides separated on Sephadex G-100 and SP-Sephadex C-25. 14C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting. It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion. Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule. Only two of the possible cross-links were found. These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme. These results, combined with those obtained in the affinity labeling reaction of holoenzyme with bromopyruvate [Alliel et al. (1982) Eur. J. Biochem. 122, 553-558], show that the three residues are located in or close to the active site. Their possible role is discussed.

Binding Sites↗
PubMed1986

Regulation of dehydrogenases/one-electron transferases by modification of flavin redox potentials. Effect of product binding on semiquinone stabilization in yeast flavocytochrome b2.

Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L-lactate cytochrome c oxidoreductase (or flavocytochrome b2) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements. In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18 degrees C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one-electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate.

Electron Spin Resonance Spectroscopy↗
PubMed1995

Characterization of the two unique human anti-flavin monoclonal immunoglobulins.

Form A of two previously described human monoclonal anti-riboflavin IgGs, the GAR [Farhangi, M. & Osserman, E. F. (1976) N. Engl. J. Med. 294, 177-183] and DOT [Merlini, G., Bruening, R., Kyle, R. & Osserman, E. F. (1990) Mol. Immunol. 27, 385-394], has been characterized in terms of binding properties and primary structure. Both forms were isolated as immunocomplexes with bound riboflavin and gave a reconstitutable apoprotein. The riboflavin-reconstituted IgGs showed a similar visible absorption spectrum, with a marked resolution of the 445-nm band and a ratio 445-nm/370-nm peaks of 1.13 for DOT and 1.19 for GAR. Both proteins bind riboflavin, FMN and FAD with a molar ratio ligand/protein of 2:1. DOT and GAR share a very similar affinity for the flavinic ligands; the Kd values for riboflavin and FMN are in the range 1 nM; that for FAD is an order of magnitude higher. DOT and GAR do not form an adduct between the nucleophilic group sulfite and the N(5) position of the flavin, and do not stabilize any flavinic semiquinone during reduction with the xantine/xantine oxidase benzylviologen system. The primary structure of fragment antigen binding (Fab) DOT and heavy-chain variable region (VH) GAR determined in the present study and that already known for the light-chain variable region (VL) GAR [Kiefer, C. R., McGuire, B. S., Osserman, E. F. & Garver, F. A. (1983) J. Immunol. 131, 1871-1875] evidenced that the two IgGs are assembled with VL and VH chains of different subgroups; a lambda III/HIII pair in GAR, and a lambda II/HI pair in DOT. Although less similar each other than to the counterparts of the same subclasses, DOT and GAR share an exclusive identity in the VH CDR3 region.

Amino Acid Sequence↗
PubMed1997

Two-component flavin-dependent pyrrole-2-carboxylate monooxygenase from Rhodococcus sp.

Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus. An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate. During purification of the enzyme, this activity was separated into two protein components which were both purified to apparent homogeneity. A small monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro the NADH and FAD-dependent reduction of cytochrome c and had an NADH-oxidase activity. The second component, a 54-kDa protein with a trimeric native structure had no enzymatic activity by itself, but exhibited a pyrrole-2-carboxylate-dependent oxygen consumption when it was complemented with the reductase component, FAD, and NADH. This indicated that the large protein referred to as oxygenase was responsible for the oxygen-dependent hydroxylation of the substrate. The rate of an uncoupled NADH oxidation without hydroxylation of the substrate was found to be strongly dependent on the molar ratio of both components. The uncoupling was nearly completely suppressed by a 5-7-fold molar excess of the oxygenase component. The small protein was N-terminally blocked. It was thus proteolytically digested and four of the resulting peptides were sequenced comprising 47 amino acids. The sequences of these fragments were similar to the sequences reported for the small component of different two-component flavin monooxygenases. Furthermore, the N-terminus of the oxygenase component showed high sequence similarity to the second, usually large subunit of these enzymes and to two single-component flavin monooxygenases. Thus, the enzyme from Rhodococcus sp. designated as pyrrole-2-carboxylate monooxygenase belongs to the recently discovered new class of two-component flavin aromatic monooxygenases. Some of the basic properties of both components were determined and their interaction during catalysis was investigated.

Amino Acid Sequence↗