The streptococcal flavoprotein NADH oxidase. I. Evidence linking NADH oxidase and NADH peroxidase cysteinyl redox centers

The FAD-containing NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the four-electron reduction of O2----2H2O, has been purified by an improved procedure for analyses of its structural and redox properties. The enzyme is apparently a dimer of two identical subunits, each containing 1 mol of FAD. Dithionite reduction of the enzyme proceeds in two distinct phases corresponding to approximately 0.5 and 1.1 eq/FAD, respectively. Thiol assays of the NADH oxidase, reduced anaerobically with 1 eq of NADH/FAD prior to denaturation, are consistent with the presence of a single redox-active cysteinyl residue/subunit. Analysis of the cysteinyl peptides of the oxidase, identified in tryptic digests of the enzyme labeled metabolically with [35S]cysteine, reveals a sequence which is closely related to the redox-active cysteinyl peptide sequence recently determined for the streptococcal flavoprotein NADH peroxidase. A second cysteinyl peptide sequence, when aligned with residues 3-17 of the peroxidase NH2-terminal sequence, reveals identity in 7 of 15 positions and satisfies several of the criteria described for ADP-binding structures. Additional probes of the structural and redox properties of the NADH oxidase, including visible circular dichroism spectroscopy and sensitivity to inactivation by hydrogen peroxide, provide further evidence for a fundamental structural connection between flavin-dependent NADH oxidase and peroxidase functions.     

The streptococcal flavoprotein NADH oxidase. II. Interactions of pyridine nucleotides with reduced and oxidized enzyme forms

Anaerobic addition of 0.5 eq of NADH/FAD to the streptococcal NADH oxidase produces a redox form spectrally similar to that obtained with 0.5 eq of dithionite/FAD. The second phase of the titration, however, in addition to reducing the flavin with 1 eq of NADH/FAD, leads to the appearance of a long-wavelength absorbance band centered at 725 nm. Reductive titrations of the enzyme with 3-acetylpyridine-adenine dinucleotide, which has a redox potential 72 mV more positive than that of NADH, yield a similar reduced enzyme species. Dithionite reduction of the NADH oxidase followed by titration with NAD+ partially mimics the long-wavelength absorbance of the NADH-reduced enzyme but also leads to the oxidation of 1 FADH2/dimer. NADH is not formed, however, and a similar result is obtained when the dithionite-reduced oxidase is titrated with the nonreducible substrate analog 3-aminopyridine-adenine dinucleotide. These data indicate that the FADH2 oxidation observed is intramolecular and suggest that the active centers of the two apparently identical subunits/dimer are not equivalent. These results also demonstrate that bound pyridine nucleotides can modulate the redox manifold of the NADH oxidase and, when taken together with the effects of these ligands on pre-steady-state behavior, suggest an important regulatory aspect of the catalytic redox function of this unique flavoprotein.     

Active-site structural comparison of streptococcal NADH peroxidase and NADH oxidase. Reconstitution with artificial flavins.

The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.     

Reactivity of chicken liver xanthine dehydrogenase containing modified flavins

Native FAD was removed from chicken liver xanthine dehydrogenase (XDH) and replaced with a number of artificial flavins of different redox potential. Dithionite titration of the 2-thio-FAD- or 4-thio-FAD (high potential)-containing enzymes showed that the first center to be reduced was the flavin. With native enzyme, iron-sulfur centers are the first to be reduced. With the low potential flavin, 6-OH-FAD, the enzyme-bound flavin was the last center to be reduced in reductive titration with xanthine. These shifts in the reduction profile support the hypothesis that the distribution of reducing equivalents in multi-center oxidation-reduction enzymes of this type is determined by the relative potentials of the centers. The reaction of molecular oxygen with fully reduced 2-thio-FAD XDH or 4-thio-FAD XDH resulted in 5 electron eq being released in a fast phase and one in a slow phase. Reduction of these enzymes by xanthine was limited at a rate comparable to that for the release of urate from native XDH. Xanthine/O2 turnover with these enzymes (and native XDH) resulted in approximately 40-50% of the xanthine reducing equivalents appearing as superoxide. Steady state turnover experiments involving all modified flavin-containing enzymes, as well as native enzyme, showed that shifting the flavin potential either positive or negative relative to FAD caused a decrease in catalytic activity in the xanthine/NAD reductase reaction. In the case of the xanthine/O2 reductase activity, there is no simple obvious relationship between the activity and the redox potential of the reconstituted flavin.     

Mechanistic studies of p-hydroxybenzoate hydroxylase reconstituted with 2-Thio-FAD

2-Thio-FAD (oxygen substituent at position 2 is replaced by sulfur) was used to reconstitute the apoenzyme of p-hydroxybenzoate hydroxylase. The 2-thio-FAD enzyme differs from native enzyme in several respects. While the native enzyme catalyzes the fully coupled hydroxylation of p-hydroxybenzoate, the 2-thio-FAD enzyme shows no hydroxylation of this substrate, instead reducing molecular oxygen to hydrogen peroxide. The rate of reduction of 2-thio-FAD p-hydroxybenzoate hydroxylase by NADPH in the presence of substrate was 7-fold faster than with the native enzyme. However, the oxygen reactivity of the reduced 2-thio-FAD enzyme was less than 1% that of native enzyme. This slow oxygen reaction results in the very high KmO2 observed in steady state kinetic studies of the modified enzyme. Stopped flow studies of the oxygen reaction of the reduced 2-thio-FAD enzyme in the presence of substrate confirmed the formation of a transient intermediate. The spectrum of this intermediate is very similar to those of the flavin-C(4a) adducts obtained with 2-thio-FMN lactate oxidase. This evidence suggests that reduced 2-thio-FAD p-hydroxybenzoate hydroxylase forms a flavin-C(4a)-hydroperoxide on reaction with oxygen in a reaction analogous to that with native enzyme, but that the resulting peroxyflavin is incompetent as an oxygenating species, breaking down instead to oxidized 2-thio-FAD enzyme and hydrogen peroxide.     

Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.     

Purification and properties of NADH oxidase from Bacillus megaterium

NADH oxidase, which catalyzes the oxidation of NADH, with the consumption of a stoichiometric amount of oxygen, to NAD+ and hydrogen peroxide was purified from Bacillus megaterium by 5'-AMP Sepharose affinity chromatography to homogeneity. The enzyme is a dimeric protein containing 1 mol of FAD per mol of subunit, Mr = 52,000. The absorption maxima of the native enzyme (oxidized form) were found at 270, 383, and 450 with a shoulder at 475 nm in 50 mM KPi buffer, pH 7.0. The visible absorption bands at 383 and 450 nm disappeared on the addition of NADH under anaerobic conditions and reappeared upon the introduction of air. Thus, the non-covalently bound FAD functioned as a prosthetic group for the enzyme. We tentatively named this new enzyme NADH oxidase (NADH:oxygen oxidoreductase, hydrogen peroxide forming). This enzyme stereospecifically oxidizes the pro-S hydrogen at C-4 of the pyridine ring of NADH.     

Purification and some properties of cyclohexylamine oxidase from a Pseudomonas sp.

Cyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp. capable of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc electrophoresis, and the molecular weight was found to be approximately 80,000 by gel filtration. The enzyme catalyzed the following reaction: cyclohexylamine+O2+H2O leads to cyclohexanone+NH3+H2O2. The enzyme thus can be classified as an amine oxidase; it utilized oxygen as the ultimate electron acceptor. The pH optimum of the reaction was 6.8 and the apparent Km value for cyclohexylamine was 2.5 X 10(-4) M. The enzyme was highly specific for the deamination of alicyclic primary amines such as cyclohexylamine, but was found to be inactive toward ordinary amines used as substrates for amine oxidases. The enzyme solution was yellow in color and showed a typical flavoprotein spectrum; the addition of cyclohexylamine under anaerobic conditions caused reduction of the flavin in the native enzyme. The flavin of the prosthetic group was identified as FAD by thin layer chromatography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observation that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione.     

Purification and characterization of an NADH oxidase from Eubacterium ramulus

An NADH oxidase from the strictly anaerobic Eubacterium ramuluswas purified to homogeneity. The enzyme is composed of two types of subunits with molecular masses of 40 and 30 kDa. The molecular mass of the native enzyme is 450 kDa according to gel filtration and PAGE analysis. Six to eight mol of FAD were found per mol of native enzyme. The NADH-specific enzyme was inhibited by N-bromosuccinimide and sulfhydryl reagents such as N-ethylmaleimide, CuCl(2) or ZnCl(2). The physiological function of the purified enzyme is unclear, but the demonstration of NADH-dependent O(2)-consumption suggests that it plays a role in the scavenging of oxygen.     

Studies on the structure and mechanism of Streptococcus faecium L-alpha-glycerophosphate oxidase

An FAD-containing L-alpha-glycerophosphate oxidase has been purified to homogeneity from Streptococcus faecium. The purified protein exists as a dimer (subunit Mr = 65,000); each subunit contains 1 mol of FAD. The enzyme contains no iron, as determined by atomic absorption spectroscopy. The alpha-glycerophosphate oxidase reacts reversibly with sulfite to form a covalent N(5) adduct; it preferentially binds the anionic form of the native oxidized FAD, and it also stabilizes the p-quinonoid form of 8-mercapto-FAD. The enzyme shows an unusually high reactivity with ferricyanide in the absence of oxygen; however, there is no evidence for any superoxide ion (O2-.) generation under standard assay conditions. Dithionite titrations of the enzyme reveal an unusual pH dependence for the stabilization of the flavin semiquinone; only at pH 8.5 does significant anionic semiquinone accumulate. L-alpha-Glycerophosphate rapidly reduces the enzyme-bound FAD; in addition, a small amount of catalytically insignificant red semiquinone appears under these conditions. The 5-deaza-FAD-reconstituted enzyme is also reduced by substrate, strongly suggesting that a radical mechanism is not involved in the oxidation of alpha-glycerophosphate. Furthermore, nitroethane anion reduces the native enzyme; this observation suggests that an electron transfer mechanism involving a substrate carbanion is possible with this enzyme.     

Inhibitory Effect of Ellagitannin Metabolites on IgE-Mediated Allergic Responses in RBL-2H3 Cells

A flavoprotein functional as an NADH oxidase has NADH peroxidase activity in the presence of free-FAD. The NADH peroxidase activity was observed in cell free extracts from aerobically grown cells in the presence of free FAD. The enzyme that has this NADH peroxidase activity was purified to homogeneity, and had exactly the same properties as the NADH oxidase.     

Molecular cloning and analysis of the gene encoding the NADH oxidase from Streptococcus faecalis 10C1. Comparison with NADH peroxidase and the flavoprotein disulfide reductases.

The gene encoding the streptococcal flavoprotein NADH oxidase (NOXase), which catalyzes the four-electron reduction of O2-->2H2O, has been cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The deduced NOXase protein sequence corresponds to a molecular mass of 48.9 kDa and contains three previously sequenced cysteinyl peptides obtained with the purified enzyme. In Escherichia coli, the expressed nox gene produced a catalytically active product, which retained its immunoreactivity to affinity-purified NOXase antisera. Alignment of the NOXase protein sequence with that of streptococcal NADH peroxidase (NPXase) revealed that the proteins are 44% identical. Among the most highly conserved segments is a sequence containing Cys42; this residue is known to exist as a stabilized cysteine-sulfenic acid (Cys-SOH) in NPXase and serves as the non-flavin redox center. In addition, three previously identified NPXase segments, known to be involved in FAD and NAD(P)-binding in other pyridine nucleotide-linked flavoprotein oxidoreductases, are strongly conserved in NOXase. Overall, the extensive homology observed between NOXase and NPXase suggests that the monomer chain fold of the oxidase closely resembles that of the peroxidase. Both sequences share limited but significant homology to those of glutathione reductase and other members of the flavoprotein disulfide reductase family. These and other considerations suggest that these two unusual streptococcal flavoproteins constitute a distinct class of FAD-dependent oxidoreductases, the flavoprotein peroxide reductases, easily contrasted with enzymes such as glutathione reductase and thioredoxin reductase.     

Coenzyme A-disulfide reductase from Staphylococcus aureus: evidence for asymmetric behavior on interaction with pyridine nucleotides

An unusual flavoprotein disulfide reductase, which catalyzes the NADPH-dependent reduction of CoASSCoA, has recently been purified from the human pathogen Staphylococcus aureus [delCardayré, S. B., Stock, K. P., Newton, G. L., Fahey, R. C., and Davies, J. E. (1998) J. Biol. Chem. 273, 5744-5751]. Coenzyme A-disulfide reductase (CoADR) lacks the redox-active protein disulfide characteristic of the disulfide reductases; instead, NADPH reduction yields 1 protein-SH and 1 CoASH. Furthermore, the CoADR sequence reveals the presence of a single putative active-site Cys (Cys43) within an SFXXC motif also seen in the Enterococcus faecalis NADH oxidase and NADH peroxidase, which use a single redox-active cysteine-sulfenic acid in catalysis. In this report, we provide a detailed examination of the equilibrium properties of both wild-type and C43S CoADRs, focusing on the role of Cys43 in the catalytic redox cycle, the behavior of both enzyme forms on reduction with dithionite and NADPH, and the interaction of NADP+ with the corresponding reduced enzyme species. The results of these analyses, combined with electrospray mass spectrometric data for the two oxidized enzyme forms, fully support the catalytic redox role proposed for Cys43 and confirm that this is the attachment site for bound CoASH. In addition, we provide evidence indicating dramatic thermodynamic inequivalence between the two active sites per dimer, similar to that documented for the related enzymes mercuric reductase and NADH oxidase; only 1 FAD is reduced with NADPH in wild-type CoADR. The EH2.NADPH/EH4.NADP+ complex which results is reoxidized quantitatively in titrations with CoASSCoA, supporting a possible role for the asymmetric reduced dimer in catalysis.     

NADH oxidase activity of mitochondrial apoptosis-inducing factor

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFDelta1-120) exhibit NADH oxidase activity, whereas superoxide anion (O(2)(-)) is formed. AIFDelta1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFDelta1-120, which lacks FAD, has no NADH oxidase activity. However, native AIFDelta1-120, apoAIFDelta1-120, and the reconstituted (FAD-containing) holoAIFDelta1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFDelta1-120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFDelta1-120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.     

Differential reactivity of the two active site cysteine residues generated on reduction of pig heart lipoamide dehydrogenase.

Reduction of the active center disulfide bond in the flavoprotein pig heart lipoamide dehydrogenase generates two sulfur moieties which are chemically inequivalent in the 2-electron reduced form of the enzyme. Thus 1 cysteine residue is at least 13-fold more reactive than its partner toward iodoacetamide at pH 7.6. This selectivity was demonstrated by reaction of the 2-electron reduced enzyme with a low concentration of iodo[1-14C]acetamide under anaerobic conditions. The formation of a monolabeled derivative is accompanied by the reappearance of a spectrum of oxidized bound flavin, clearly different from that of the native enzyme. Alkylation of the remaining cysteine residues with iodo[12C]acetamide enabled the isolation of a tryptic version of the active center disulfide peptide. A single chymotryptic cleavage between the 2 alkylated cysteine residues generated a cationic and an anionic fragment containing 7% and 93% of the radioactivity of the purified tryptic peptide, respectively. The monolabeled derivative is catalytically inactive toward reduced or oxidized lipoamide, but is approximately 2-fold better as a transhydrogenase than the native protein using NADH and acetylpyridine adenine dinucleotide as substrates. Anaerobic titration with NADH leads to reduction of the flavin with concomitant formation of long wavelength absorption of low intensity. No intermediate reduced states were detected in this titration analogous to the red 2-electron form observed with the native enzyme. Similarly, intermediates during reduction of the enzyme by 1 eq of dithionite have not been detected.     

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.     

Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.     

Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.     

Surface Glycolipids in Shoot-forming Culture of Nicotiana umbratica

Phthalate oxygenase was induced in Rhodococcus erythropolis S-1, a Gram-positive bacterium, when this bacterium was cultured in a medium containing phthalate as a sole carbon source. The enzyme was purified 118-fold with 4.7% activity yield. The purified enzyme appeared homogenous on native PAGE. This enzyme is a large protein (213 kDa), a tetramer of identical 56kDa monomers, and a flavoprotein containing FAD with NADH-dependent dioxygenase activity. The enzyme is specific for phthalate and other closely related aromatic compounds. Optimum pH and temperature were 6.5 and 40°C. The K_m for phthalate and NADH were 0.040 mM and 0.069 mM. The enzyme catalyzes dihydroxylation of phthalate to form 3,4-dihydro-3,4-dihydroxyphthalate with consumption of NADH and oxygen.