Vol. 174, No. 10

JOURNAL OF BACTERIOLOGY, May 1992, p. 3275-3281 0021-9193/92/103275-07$02.00/0 Copyright ©D 1992, American Society for Microbiology

Purification and Properties of the NADH Reductase Component of Alkene Monooxygenase from Mycobacterium Strain E3 FRANS J. WEBER,1* WILLEM J. H. VAN BERKEL,2 SYBE HARTMANS,1 AND JAN A. M. DE BONT' Division of Industrial Microbiology, Department of Food Science, 1 and Department of Biochemistry,2 Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 26 November 1991/Accepted 2 March 1992

Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 191%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone.

Mycobacterium strains E3 (10) and Li (14) contain an inducible alkene monooxygenase (AMO) which oxidizes short-chain alkenes to epoxides. Cells containing AMO may be of interest in the production of optically pure epoxides. 1,2-Epoxypropane, for example, is produced almost exclusively in the R form (99%) by Mycobacterium strain Li (11, 32). The capacity of these mycobacteria to degrade ethene or vinyl chloride may also have other applications, for instance, in ethene removal during fruit storage (31b) or vinyl chloride removal from industrial waste gases (14). When AMO activity in crude extracts is plotted against the protein concentration in the assay, a sigmoidal relationship is observed (15), indicating that AMO, like related enzymes, is a multicomponent enzyme. The soluble methane monooxygenase (MMO) from the type I methanotroph Methylococcus capsulatus (Bath) (5) has been resolved into three components: an oxygenase, an NADH reductase, and a regulatory protein. Very similar enzymes have been reported for the type II methanotrophs Methylosinus trichosporium OB3b (7, 27) and Methylosinus sporium 5 (22). The facultative methanotroph Methylobacterium strain CRL26 possesses a two-component enzyme which has been purified and does not require the regulatory component (20). The purified oxygenase and NADH:acceptor reductase of Methylobacterium strain CRL26 show close similarity to the corresponding components of M. capsulatus (Bath) MMO (20, 16). These MMOs have a much broader substrate specificity than AMO, oxidizing methane, alkanes, and alkenes, as well as aromatic hydrocarbons. Furthermore, they form racemic epoxides from alkenes (32). Therefore, AMO differs from the monooxygenases so far reported. Fractionation of crude extracts from both Mycobacterium strains (by DEAE-Sepharose CL6B) revealed two fractions which were both required for AMO activity. One fraction was inhibited by acetylene, indicating that it contains an oxygenase component (15, 24). The other fraction contained a ferricyanide-reducing activity which probably is the

NADH reductase component of AMO, as it was present only in extracts of ethene-grown cells (15). Mutants lacking AMO activity were obtained by using vinyl chloride as a mutagenic agent (13). Complementation assays using the partially purified reductase and oxygenase components showed that one of the mutants lacked only the reductase component (AMO-). This mutant can be used in a complementation assay to determine NADH reductase activity specifically. This assay has the advantage that partial purification of the oxygenase component for a specific NADH reductase assay is not necessary, thereby facilitating purification of the reductase. In this report, we describe the purification and characterization of the NADH reductase of AMO.

MATERIALS AND METHODS Chemicals and reagents. NADH, NADPH, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and dithiothreitol (DTT) were from Boehringer, Mannheim, Germany. Glycerol (87%) and Tween 80 were from Merck, Darmstadt, Germany. DEAE-Sepharose CL6B, Sephadex G10 and G200, Mono Q, and Superose 12 were obtained from Pharmacia, Uppsala, Sweden, and hydroxylapatite was from Bio-Rad, Richmond, Calif. Filtron Corporation (Northborough, Mass.) supplied membrane filters with a cutoff of 10 kDa (Omega NMWL 10K). All other chemicals were of analytical grade. Organisms and growth. Mycobacterium strain E3 has been isolated on ethene (10) and was grown on mineral medium with ethene as the carbon source (15) in a 10-liter fermentor at 350 rpm, pH 7.0, and 30°C. Ethene was supplied as a 2% (vol/vol) mixture in air at a rate of 1 liter/min. Three times a week, 7 liters was withdrawn from the fermentor and replaced with new mineral medium. Cells were harvested by centrifugation (10 min, 16,000 x g, 4°C) and washed once with 50 mM potassium phosphate buffer (pH 7.0) containing 0.1% Tween 80. Portions (25 ml) of cell paste were stored at -30°C until use. The AMO- mutant was grown on epoxyethane (50 ml) in

* Corresponding author. 3275

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WEBER ET AL.

5-liter Erlenmeyer flasks containing 0.5 liter of mineral medium and sealed with rubber stoppers. Precipitation with ammonium sulfate. To determine the precipitation behavior of the AMO components, cell extract was brought to 15% ammonium sulfate saturation. After 45 min at 4°C, precipitated proteins were collected by centrifugation (16,000 x g, 10 min). The ammonium sulfate concentration of the supernatant was subsequently increased stepwise, and precipitated proteins were collected after each addition. The precipitated proteins were dissolved in a total of 20 ml and desalted by membrane dialysis (18 h, 4°C) against 5 liters of 50 mM potassium phosphate buffer (pH 7.0). Reductase and oxygenase activities were determined by using a complementation assay with an excess of mutant and acetylene-inactivated cell extract, respectively. Purification of NADH reductase. All procedures were performed at 4°C unless stated otherwise. Chromatographic columns were equilibrated with the starting buffer. AMO buffer refers to 50 mM potassium phosphate buffer (pH 7.0) containing 10% (vol/vol) glycerol (87%), 0.1% (wt/vol) Tween 80, and 1 mM DTT. Portions (25 ml) of cell paste were broken by a single passage through a prechilled (-30°C) 25-ml X-Press (AB Biox, Goteborg, Sweden). Six portions of broken cells were pooled, thawed, and diluted to a total volume of 300 ml with AMO buffer. The final concentrations of glycerol and DTT were brought to 8.7% (vol/vol) and 1 mM, respectively. (Glycerol is omitted during the cell breakage because it reduces the pressure at which the cells are broken). DNase I (50 ,ug) was added to reduce the viscosity. Cell debris and unbroken cells were removed by centrifugation (49,000 x g, 20 min). When necessary, this was repeated until a clear supernatant was obtained. Step 1: ammonium sulfate precipitation. The supernatant was brought to 50% ammonium sulfate saturation by slow addition of solid ammonium sulfate. After 45 min of stirring, the precipitated proteins were removed by centrifugation (16,000 x g, 20 min). The supernatant was brought to 90% saturation with solid ammonium sulfate. After an additional 45 min, the precipitated proteins were collected by centrifugation and redissolved in 100 ml of AMO buffer. This was dialyzed against 10 liters of AMO buffer (3 h, 4°C) to remove most of the ammonium sulfate. Step 2: DEAE-Sepharose. The redissolved and desalted pellet was loaded on a DEAE-Sepharose CL6B column (30 by 4.8 cm) equilibrated with AMO buffer. After application, a 0 to 0.8 M NaCl gradient in 3 liters of the same buffer was started at a flow rate of 2.5 ml/min. Fractions of 20 ml were collected, and those containing NADH reductase activity (0.35 M NaCI) were pooled. Step 3: hydroxylapatite. The pooled NADH reductase fractions from the DEAE-Sepharose column were applied to a hydroxylapatite column (40 by 2.6 cm) and eluted with a gradient of 50 to 300 mM potassium phosphate buffer (pH 7.0) at 0.8 ml/min with a total volume of 1 liter with the same additions as AMO buffer. Fractions containing NADH reductase activity (100 mM potassium phosphate buffer) were pooled and concentrated by ultrafiltration under nitrogen at a pressure of 2 atmospheres. Step 4: Sephadex G200 gel filtration. The concentrate of about 5 ml was applied to a Sephadex G200 column (70 by 1.7 cm) and eluted with AMO buffer containing 0.2 M NaCl at a flow rate of 0.1 ml/min. The fractions with NADH reductase activity were pooled and concentrated by ultrafiltration until a protein concentration of about 2 mg/ml was obtained.

J. BACTERIOL.

Enzyme assay. AMO activity was determined as described previously (15). Epoxypropane formation rates were determined by analyzing 0.2-ml headspace samples with a gas chromatograph. Owing to the sigmoidal effect of the protein concentration on AMO activity, it is important to use the same protein concentration in the activity assay when comparing enzyme activities. NADH reductase activity could be determined in two ways, by monitoring the reduction of the artificial electron acceptor ferricyanide (6) in 50 mM potassium phosphate buffer (pH 7.3) or by using a complementation assay containing saturating amounts of extract of the epoxyethanegrown AMO- mutant, which lacks the NADH reductase component. Oxygenase activity was determined by using acetyleneinactivated cell extract from wild-type cells as described

previously (15). Protein determination. Protein was determined with the Folin-Ciocalteu reagent by using the micro-assay of Peterson (21). This method uses sodium dodecyl sulfate (SDS) to eliminate the interference of Tween 80 with the assay. Bovine serum albumin was used as the standard. Flavin identification. Flavin was assayed by high-performance liquid chromatography on a C-18 column (20 by 0.3 cm) with water-methanol (80:20) containing 5 mM ammonium acetate (pH 6.0) as the mobile phase at a flow rate of 0.5 ml/min. The flavin was dissociated from the protein by boiling in a 1% (wt/vol) SDS solution for 10 min. Precipitated proteins were removed by centrifugation (5 min, 10,000 x g). The flavin was identified by comparison of the retention time with those of FAD and FMN standards. Iron and acid-labile-sulfide determination. Iron was determined by using 4,7-diphenyl-1,10-phenanthroline disulfonic acid. To a 500-,u diluted sample containing 0.3 mg of protein, 50 RI of ,B-mercaptoethanol, 250 ,ul of 0.4 M sodium acetate-acetic acid buffer (pH 4.0), 50 pI of 10% (wt/vol) SDS, and 50 pul of 20 mM bathophenanthroline solution were added. The extinction was measured after 10 min at 535 nm. (NH4)2Fe(SO4)2 was used as the standard. Acid-labile sulfide was determined by the method of Brumby et al. (4). Prior to addition of N,N'-dimethyl-pphenylenediamine and FeCl3, the sample was incubated for 2 h with the alkaline-zinc reagent as suggested by Suhara et al. (29). Na2S, freeze-dried to remove water, was used as the standard. SDS-PAGE and molecular weight determination. SDSpolyacrylamide gel electrophoresis (PAGE) was used to determine the subunit molecular weight and purity of the NADH reductase. A 12.5% (wt/vol) separation slab gel (180 by 140 by 1 mm) was used with the discontinuous buffer system of Laemmli (17). Proteins were stained with Coomassie brilliant blue R-250. Carbonic anhydrase, egg albumin, bovine serum albumin, and phosphorylase b were used as standards. The molecular weight of the native protein was determined by means of gel filtration on a Superose 12 fast protein liquid chromatography column (30 by 1.0 cm) at a flow rate of 0.30 ml/min. The buffer used was 20 mM potassium phosphate (pH 7.0) containing 8.7% (vol/vol) glycerol, 0.1% (wt/vol) Tween 80, 1 mM DTT, and 0.25 M NaCl. Protein standards (kit MSII) from Serva (Heidelberg, Germany) were used to calibrate the column. Amino acid composition and NH2-terminal sequence deter. mination. Tween 80, which interferes with the determination, was removed from the samples by anion-exchange chromatography. NADH reductase (2 mg) was loaded on a

NADH REDUCTASE OF AMO FROM MYCOBACTERIUM STRAIN E3

VOL. 174, 1992

Mono Q (HR 5/5) fast protein liquid chromatography column equilibrated with 20 mM Tris-HCI buffer (pH 7.5) with no additions. Tween 80 was removed by washing the column with 30 volumes of buffer. The protein was eluted with a gradient of 0 to 1 M NaCl in the same buffer. The NADH reductase was subsequently dialyzed against 5 liters of Milli Q water overnight at 4°C. This sample was hydrolyzed in 6 N HC1 for 16 h at 105°C. HCl was removed by evaporation using a rotary evaporator at 40°C, and the resulting precipitate was used for analysis on a Biotronic LC 6000E amino acid analyzer equipped with a separation program for physiological solutions. No modifications for cysteine or methionine were performed, and tryptophan was destroyed by the method used, so that these amino acids could not be determined. For NH2-terminal amino acid sequence determination, Tween 80 was removed by the same procedure, starting with 0.5 mg of protein. The sequence was determined on an Applied Biosystems 470A protein sequencer equipped on line with a model 120A phenylthiohydantoin analyzer. Absorption spectroscopy. Absorption spectra were recorded on a Cary 14 spectrophotometer equipped with a thermostat-equipped cell holder. Anaerobic titrations were done in a 1-ml anaerobic quartz cuvette. The samples in the cuvette were made anaerobic by 10 cycles of evacuation with a rotary vacuum pump and flushing with argon. Oxygen-free additions were made by using a gas-tight Hamilton syringe. Titrations were carried out in AMO buffer at 20°C. Fluorescence emission and excitation spectra were recorded on an Aminco SPF-500 fluorimeter. EPR spectroscopy. Electron paramagnetic resonance (EPR) spectra were recorded on a Bruker ER200 spectrophotometer at a temperature of 20 K. The following settings were used: microwave frequency, 9,329 MHz; modulation frequency, 100 kHz; modulation amplitude, 0.8 mT; microwave power, 8 mW. RESULTS

Recovery of activity after gel filtration. After gel filtration on a Sephadex G10 column in the presence of 8.7% (vol/vol) glycerol and 1 mM DTT, almost no NADH reductase activity was recovered (6%, using the mutant complementation assay). This effect was also seen with other chromatographic techniques; e.g., with DEAE-Sepharose CL6B anion-exchange chromatography only 20% of the activity was recovered. Addition of various cofactors, metals, or the salt peak obtained after gel filtration did not restore reductase activity, indicating irreversible inactivation. Different buffers and stabilizing agents were tested to prevent loss of NADH reductase activity during gel filtration. Addition of 0.05% (wt/vol) Tween 80 to the elution buffer resulted in recovery of 86% of the reductase activity after Sephadex G10 gel filtration. A further increase in the TIween 80 concentration to 0.1% (wtlvol) resulted in almost quantitative recovery of activity (96%). Purification of the NADH reductase. NADH reductase was purified 920-fold by using standard chromatographic techniques, with 19% recovery of activity (Table 1). Most of the activity was lost during ammonium sulfate precipitation. In this procedure, AMO activity, as well as oxygenase activity, precipitated at 15 to 50% ammonium sulfate saturation but NADH reductase activity was present in all fractions (Fig. 1). The fraction precipitating between 50 and 90% saturation was used, as this fraction contained no oxygenase activity.

3277

TABLE 1. Purification of the NADH reductase component of AMO from Mycobacterium strain E3 Purification step

Trotae (mg)

Sp act (nmol

min-1 mg-l)a

Purification factor (fold)

Recovery (

Crude cell extract

5,130

2.5

1

100

(NH4)2S04 precipitation

1,254

5.0

2

48

76

30

35

942 2,300

377 920

23 19

59

DEAESepharose

3.1 1.1

Hydroxylapatite Sephadex G200

a Activities were determined by measuring epoxypropane formation from propene in the presence of an excess of AMO- mutant extract.

This resulted in loss of the reductase activity that precipitated below 50% ammonium sulfate saturation. Analysis of the purified enzyme by SDS-PAGE revealed the presence of a single band, corresponding to a polypeptide chain molecular mass of about 56 kDa (Fig. 2). This value was also obtained by gel filtration on a Superose 12 fast protein liquid chromatography column, indicating that NADH reductase is a monomeric protein. Stabilizing effect of Tween 80. The stabilizing effect of Tween 80 during chromatographic purification steps was further analyzed by using the purified reductase. Gel filtration on a Sephadex G10 column in the presence of Tween 80 yielded the fully active enzyme. In the absence of Tween 80, however, the enzyme was inactivated and a second peak of low molecular weight was obtained. This peak was yellow and contained FAD, the prosthetic group of the enzyme (also see below). The inactivation of the purified reductase during gel filtration was irreversible; addition of neither FAD nor the salt peak restored activity. Amino acid composition and NH2-terminal sequence. The amino acid composition of the NADH reductase is shown in 10.0 9.0

E

8.0

.E

7.0

06

6.0

^t

5.0

4._'>.

4.0

*r,

3.0

2.0 1.0 0.0 15 30 50 70 80 90 % Ammnoniun Sulfate saturation FIG. 1. Ammonium sulfate precipitation of AMO. The activities of the precipitated AMO and the reductase and oxygenase components at increasing ammonium sulfate saturations are shown. 0

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A

1 0.40

0.30 0

0.20

2

r

0.10 0.00 30'o

400

500

600

700

600

700

Wavelength (mrn)

FIG. 2. SDS-PAGE of the purified NADH reductase component of AMO from Mycobacterium strain E3. The slab gel was stained with Coomassie brilliant blue R250. The left lane contained protein molecular size markers (from top to bottom: phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; egg albumin, 45 kDa; carbonic anhydrase, 30 kDa). The right lane contained the purified NADH reductase (30 pg).

LL

4)

(r

Table 2. Cysteine, methionine, and tryptophan were not determined. The NH2-terminal amino acid sequence has been determined as Gly-Asp-Thr-Val-Thr-Val-Gln-Pro-Phe-Gly-AspThr-Phe-Pro-Val-Glu-Ser. This sequence does not show homology with the NH2-terminal sequences reported for the reductase component of the soluble MMOs from Methylosinus trichosporium OB3b (8) and Methylococcus capsulatus (Bath) (26) or naphthalene dioxygenase from Pseudomonas strain NCIB 9816 (12). Prosthetic groups. The absorption spectrum of the purified NADH reductase revealed the presence of prosthetic groups (Fig. 3A). The extinction coefficients of the absorption maxima at 384, 420, and 460 nm are, respectively, 12.1, 10.3, and 11.9 mM-1 cm-'. The absorption maxima at 384 and 460 nm indicate the presence of a flavin. This was confirmed by

300

500

400

Wavelength (rnm

FIG. 3. Spectral properties of the purified NADH reductase. (A) Curve 1 is the absorption spectrum of the purified reductase component (2 mg/ml), and curve 2 is that of the flavin liberated from the reductase by boiling in 1% SDS. (B) Fluorescence spectrum of the NADH reductase (2 mg/ml). Curve 1 is the excitation spectrum with emission at 525 nm, and curve 2 is the emission spectrum with excitation at 460 nm.

boiling the enzyme in the presence of 1% SDS and recording the absorption spectrum of the resulting yellow supernatant (Fig. 3A). High-performance liquid chromatography analysis of the extracted flavin and FMN and FAD reference solu-

TABLE 2. Amino acid composition of the purified NADH reductase component of AMO % of reductase component

Amino acid

Aspartate + asparagine Threonine Serine Glutamate + glutamine Proline

Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

ofI:

AMO

BDO

NDO

PHO

TDO

ALH

100 51 114

100 48 87 135 61 94 123 65 58 116 45 52 13 32 52

100 76 84 128 84 140 116 144 96 140 44 40 40 44 76

100 75 114 86 64 89 89 75 46 86 18 57 46 25 64

100 55 66 129 53 100 124 63 61 111 21 29 26 8 37

100 76 92 146 57 119 165 130 92 141 35 27 68 11 73

124 36 325 169 59 24 77 32 16 22 14 56

a The amino acid composition of the purified NADH reductase and those of related reductases are shown as percentages of aspartate-plus-asparagine content. Abbreviations: BDO, benzoate dioxygenase (33); NDO, naphthalene dioxygenase (12); PHO, phthalate dioxygenase (2); TDO, toluene dioxygenase (28); ALH,

alkane hydroxylase (31).

NADH REDUCTASE OF AMO FROM MYCOBACTERIUM STRAIN E3

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3279

0.50

300

400

500

600

700

3100

3300

3500

3700

3900

Wavelength (rn

FIG. 4. Absorption spectra of the NADH reductase after anaerobic reduction with NADH. From top to bottom: NADH reductase (31 pM) in its oxidized state and after anaerobic reduction with, respectively, 20, 40, and 60 p,M NADH and a slight excess of sodium dithionite.

tions revealed that the flavin prosthetic group was FAD. The concentration of FAD was determined at 1.05 mol/mol of enzyme, indicating that each molecule of NADH reductase contains one FAD molecule. The presence of FAD was also confirmed by recording the fluorescence emission and excitation spectra of the native enzyme (Fig. 3B). The excitation maxima at 370 and 450 nm and the emission maximum at 525 nm are characteristic for FAD. The absorption maximum at 410 nm (Fig. 3A) indicates the presence of a second prosthetic group. Colorimetric determinations revealed the presence of both iron and acid-labile sulfide in ratios of 2.17 and 2.03 mol/mol of enzyme, respectively, confirming the presence of an Fe-S cluster. Anaerobic reduction and EPR. Anaerobic titration of the reductase with NADH resulted in a decrease of the absorption maxima at 384, 410, and 460 nm and in the initial formation of a new maximum at about 650 nm typical of a neutral flavin semiquinone (Fig. 4). This spectrum highly resembles the NADH-reduced spectra reported for MMOs (18, 23). Further addition of a slight excess of sodium dithionite further reduced the flavin semiquinone, resulting in bleaching of the absorption at 650 nm. The EPR spectrum (Fig. 5) of the anaerobically fully reduced reductase revealed g values at 2.011, 1.921, and 1.876, which is typical for a [2Fe-2S] cluster (5, 23). DISCUSSION The AMO of Mycobacterium strain E3 oxidizes alkenes to epoxides but does not hydroxylate alkanes (31b) and therefore differs from the monooxygenases studied previously. The oxygenase component obviously is the most interesting protein to study in detail. However, as a simple and specific assay for the oxygenase component was not available, we chose to purify the reductase component first. The reductase can be easily and specifically assayed with a complementation assay by using a mutant lacking the reductase component. The reductase is induced only in cells grown on ethene (15) and is therefore expected to be specific for the AMO. Preliminary purification of the NADH reductase component resulted in substantial loss of enzyme activity. Addition of 0.1% (wt/vol) Tween 80 to the elution buffers resulted in almost quantitative recovery of reductase activity after the

Magnetic Field (mTesla)

FIG. 5. EPR spectrum of the NADH reductase. A 200-pl volume of NADH reductase (20 puM) was anaerobically reduced with a slight excess of sodium dithionite in AMO buffer.

various chromatographic purification steps. Tween 80 and other nonionic detergents are often used to keep membranebound proteins in solution. Although some of the related monooxygenases are indeed membrane bound (1, 30), AMO activity was not precipitated during ultracentrifugation experiments in the absence of Tween 80 or glycerol (15). This indicates that AMO is probably a soluble enzyme. With the purified reductase, it was shown that Tween 80 prevented irreversible dissociation of FAD. Loss of FAD during purification has also been reported for the reductase components of naphthalene dioxygenase from a Pseudomonas species, toluene dioxygenase of Pseudomonas putida (12, 28), and the 3-hydroxyphenylacetate 6-hydroxylase of a Flavobacterium sp. (31a). With these enzymes, however, the activity was restored by addition of FAD. By using Tween 80 to prevent inactivation, the NADH reductase was purified 920-fold with a recovery of 19%. As only the 50 to 90% ammonium sulfate fraction, containing about 50% of the reductase activity, is used in the purification protocol, significant loss of activity is inevitable. The precipitation of reductase activity in two fractions can be explained by assuming that the reductase can be present in two forms, either free or bound to the oxygenase component. Both forms could then precipitate differently, with the reductase-oxygenase complex (AMO) precipitating at 20 to 50% saturation and the unbound reductase precipitating at about 70 to 80% saturation. Although ammonium sulfate precipitation has a low recovery (48%) and purification factor (twofold), this precipitation step is essential for effective purification of the enzyme by anion-exchange chroma-

tography. The high purification factor of the homogeneous protein of 920-fold indicates that the NADH reductase content of the intracellular proteins is only 0.1%. Similarly low levels (0.1 to 0.3%) have been reported for the reductase components of the soluble MMOs from M. trichosporium OB3b (7) and M. sporium 5 (22) and the benzoate 1,2-dioxygenase from P. arvilla (33). The other components of these oxygenase systems are present at levels about 10-fold higher than that of the reductase. Fox et al. (7) assumed that, owing to the much higher electron transfer throughput of the reductase than the hydroxylation rate of the hydroxylase, lower concentrations of the reductase would prevent spontaneous

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dismutations owing to the formation of reactive reduced oxygen. Another explanation for the apparent low reductase content could be the existence of a second reductase for AMO. In a crude extract, AMO can use both NADH and NADPH as electron donors. With NADPH, a slightly higher activity is obtained than with NADH. The purified reductase can, however, use only NADH as an electron donor, indicating the presence of a second reductase component. The existence of a second reductase could also account for the broad ammonium sulfate precipitation range in which the reductase precipitates, assuming that the postulated second reductase could use both NADH and NADPH. Shafiee and Hutchinson (25) reported two different reductases for the 6-deoxyerythronolide B hydroxylase from Saccharopolyspora erythraea. One reductase was completely NADH dependent, whereas the other could use both NADH and NADPH. The purified reductase is a yellow-red monomeric protein of 56 kDa. The NADH reductase activity (in the presence of an excess of the mutant) is 2.3 ,umol of epoxypropane formed per min per mg of protein. This activity is lower than the activities of the purified MMO NADH reductases from M. tnchosporium OB3b, M. sporium 5, Methylobacterium strain CRL-26, and Methylococcus capsulatus (Bath) of 26.1, 17.5, 6.2, and 6.0 ,umol of epoxide formed min-' mg-, respectively (6, 7, 20, 22). Two prosthetic groups were identified: FAD and a [2Fe2S] cluster. Related reductase components with the same prosthetic groups have a molecular weight lower than that of the reductase from AMO (56 kDa). For the reductase components of the MMOs (6, 7, 20, 22), phthalate oxygenase from P. cepacia (2), 4-methoxybenzoate O-demethylase from P. putida (3), naphthalene dioxygenase from Pseudomonas strain NCIB 9816 (13), and benzoate 1,2-dioxygenase from P. arvilla (33), molecular masses of 34 to 44.6 kDa have been reported. Another difference from related reductases is the surprisingly high glycine content of the AMO reductase (about 25%; Table 2). Two redox groups, a flavin and a [2Fe-2S] cluster, are found in most multicomponent oxygenases and form a short electron transport chain to the oxygenase. The flavin, the first redox group, accepts two electrons from NAD(P)H; these electrons are then transferred to the [2Fe-2S] cluster, which accepts only one electron at a time and thus functions as a 2e-1/le-1 transformase (19). The electrons are finally transferred to the oxygenase. These two redox groups can be located on the same protein (the reductase component), as with AMO, the MMOs (5, 7, 20, 22), phthalate oxygenase (2), 4-methoxybenzoate O-demethylase (3), and benzoate 1,2-dioxygenase (33). The two redox centers can also be located on two different proteins (flavin on the reductase and the [2Fe-2S] cluster on a ferredoxin or rubredoxin type of protein), as for instance with alkane hydroxylase of P. oleovorans (31) and toluene dioxygenase and benzene dioxygenase from P. putida (9, 28). Recently a system requiring three redox groups has been described for the naphthalene dioxygenase from a Pseudomonas species (12). The reductase contained both a FAD and a [2Fe-2S] cluster, and a third redox group was located on the ferredoxin, which transfers its electrons to the oxygenase component. For the soluble MMOs from M. tnichosponum OB3b (7), M. sponium 5 (22), and M. capsulatus (Bath) (5), which contain a reductase with two redox groups, an additional protein has been reported. This protein contains no redox group and is thought to function as a regulatory protein.

Whether a third protein (regulatory or with a third redox group) is required for AMO activity is still unclear. There has been some indication of the involvement of a third component. When AMO was fractionated on a DEAESepharose column, two fractions, X and Y, were located which, when combined, showed AMO activity. When the reductase (Y) was assayed with an excess of fraction X (containing the oxygenase), a much lower specific activity was obtained than when it was assayed with the AMOmutant. This could indicate a requirement for a third component which is present in the mutant but only in a limiting amount in the combination of fractions X and Y. The possible requirement for a third component is therefore still unclear and will be the subject of further investigations. ACKNOWLEDGMENTS We thank Rick Koken for performing some of the experiments; W. F. Hagen, Department of Biochemistry (Agricultural University of Wageningen) for EPR measurements; W. Roelofson, Department of Microbiology (Agricultural University of Wageningen), for determination of amino acid composition; and R. Amons, State University of Leiden (SON gas phase sequencer facility) for amino acid sequence determination.

REFERENCES 1. Akent'eva, N. F., and R. I. Gvozdev. 1988. Purification and physicochemical properties of methane monooxygenase from membrane structures of Methylococcus capsulatus. Biokhimiya

53:79-84. (English translation.) 2. Batie, C. J., E. LaHaie, and D. P. Ballou. 1987. Purification and characterization of phthalate oxygenase and phthalate oxygen-

ase reductase from Pseudomonas cepacia. J. Biol. Chem.

262:1510-1518. 3. Bernhardt, F. H., H. Pachowsky, and H. Staudinger. 1975. A

4-methoxybenzoate O-demethylase from Pseudomonas putida, a new type of monooxygenase system. Eur. J. Biochem. 57:241256. 4. Brumby, P. E., R. W. Miller, and V. Massey. 1965. The content

and possible catalytic significance of labile sulfide in some

metalloflavoproteins. J. Biol. Chem. 240:2222-2228.

5. Colby, J., and H. Dalton. 1978. Resolution of the methane monooxygenase of Methylococcus capsulatus (Bath) into three components; purification and properties of component C, a flavoprotein. Biochem. J. 171:461-468. 6. Colby, J., and H. Dalton. 1979. Characterization of the second prosthetic group of the flavoenzyme NADH-acceptor reductase (component C) of the methane monooxygenase from Methylococcus capsulatus (Bath). Biochem. J. 177:903-908. 7. Fox, B. G., W. A. Froland, J. E. Dege, and J. D. Lipscomb. 1989.

8.

9.

10.

11.

Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph. J. Biol. Chem. 264:10023-10033. Fox, B. G., Y. Liu, J. E. Dege, and J. D. Lipscomb. 1991. Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. J. Biol. Chem. 266:540-550. Geary, P. J., F. Saboowalla, D. Patil, and R. Cammack. 1984. An investigation of the iron-sulfur proteins of benzene dioxygenase from Pseudomonasputida by electron-spin-resonance spectroscopy. Biochem. J. 217:667-673. Habets-Crutzen, A. Q. H., L. E. S. Brink, C. G. van Ginkel, J. A. M. de Bont, and J. Tramper. 1984. Production of epoxides from gaseous alkenes by resting-cell suspensions and immobilized cells of alkene utilizing bacteria. Appl. Microbiol. Biotechnol. 20:245-250. Habets-Crutzen, A. Q. H., S. J. N. Carlier, J. A. M. de Bont, D. Wistuba, V. Schurig, S. Hartmans, and J. Tramper. 1985. Stereospecific formation of 1,2-epoxypropane, 1,2-epoxybutane and 1-chloro-2,3-epoxypropane by alkene-utilizing bacteria. Enzyme Microb. Technol. 7:17-21.

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NADH REDUCTASE OF AMO FROM MYCOBACTERIUM STRAIN E3

12. Haigler, B. E., and D. T. Gibson. 1990. Purification and properties of NADH-ferredoxinNAp reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 172:457-464. 13. Hartmans, S., et al. Unpublished data. 14. Hartmans, S., J. A. M. de Bont, J. Tramper, and K. C. A. M. Luyben. 1985. Bacterial degradation of vinyl chloride. Biotechnol. Lett. 7:383-388. 15. Hartmans, S., F. J. Weber, D. P. M. Somhorst, and J. A. M. de Bont. 1991. Alkene monooxygenase from Mycobacterium: a multicomponent enzyme. J. Gen. Microbiol. 137:2555-2560. 16. Hou, C. T. 1986. Recent progress in research on methanotrophs and methane monooxygenases. Biotech. Gen. Eng. Rev. 4:145168. 17. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 18. Lund, J., M. P. Woodland, and H. Dalton. 1985. Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath). Eur. J. Biochem. 147:297305. 19. Orme-Johnson, W. H., and H. Beinert. 1969. Reductive titrations of iron-sulfur proteins containing two to four iron atoms. J. Biol. Chem. 244:6143-6148. 20. Patel, R. N. 1987. Methane monooxygenase: purification and properties of flavoprotein component. Arch. Biochem. Biophys. 252:229-236. 21. Peterson, G. L. 1977. A simplification of the protein assay method of Lowry et aL which is more generally applicable. Anal. Biochem. 83:346-356. 22. Pilkington, S. J., and H. Dalton. 1991. Purification and characterization of the soluble methane monooxygenase from Methylosinus sporium 5 demonstrates the highly conserved nature of this enzyme in methanotrophs. FEMS Microbiol. Lett. 78:103108. 23. Prince, R. C., and R. N. Patel. 1986. Redox properties of the flavoprotein of methane monooxygenase. FEBS Lett. 203:127130. 24. Prior, S. D., and H. Dalton. 1985. Acetylene as a suicide substrate and active site probe for methane monooxygenase from Methylococcus capsulatus (Bath). FEMS Microbiol. Lett. 29:105-109. 25. Shafiee, A., and C. R. Hutchinson. 1988. Purification and recon-

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stitution of the electron transport components for 6-deoxyerythronolide B hydroxylase, a cytochrome P-450 enzyme of macrolide antibiotic (erythromycin) biosynthesis. J. Bacteriol. 170: 1548-1553. 26. Stainthorpe, A. C., V. Lees, G. P. Salmond, H. Dalton, and J. C. Murrell. 1990. The methane monooxygenase gene cluster of Methylococcus capsulatus (Bath). Gene 91:27-34. 27. Stirling, D. I., and H. Dalton. 1979. Properties of the methane monooxygenase from extracts of Methylosinus trichosporium OB3b and evidence for its similarity to the enzymne from Methylococcus capsulatus (Bath). Eur. J. Biochem. 96:205-212. 28. Subramanian, V., T. N. Liu, W. K. Yeh, M. Narro, and D. T. Gibson. 1981. Purification and properties of NADH-ferredoxinTOL reductase, a component of toluene dioxygenase from Pseudomonas putida. J. Biol. Chem. 256:2723-2730. 29. Suhara, K., S. Takamori, M. Katagiri, K. Wada, H. Kobayashi, and H. Matsubara. 1975. Estimation of labile sulfide in ironsulfur proteins. Anal. Biochem. 68:632-636. 30. Tonge, G. N., D. E. F. Harrison, and I. J. Higgins. 1977. Purification and properties of the methane monooxygenase enzyme system from Methylosinus trichosporium OB3b. Biochem. J. 161:333-344. 31. Ueda, T., E. T. Lode, and M. J. Coon. 1972. Enzymatic w-oxidation, VI. Isolation of homogeneous reduced diphosphopyridine nucleotide-rubredoxin reductase. J. Biol. Chem. 247:2109-2116. 31a.van Berkel, W. J. H., and W. J. J. van den Tweel. 1991. Purification and characterisation of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacterium species. Eur. J. Biochem. 201:585-592. 31b.van Ginkel, C. G., H. G. J. Welten, J. A. M. de Bont, and H. A. M. Boerrigter. 1986. Removal of ethene to very low concentrations by immobilized Mycobacterium E3. J. Chem. Tech. Biotechnol. 36:593-598. 32. WeiJers, C. A. G. M., C. G. van Ginkel, and J. A. M. de Bont. 1988. Enantiomeric composition of lower epoxyalkanes produced by methane-, alkane-, and alkene-utilizing bacteria. Enzyme Microb. Technol. 10:214-218. 33. Yamaguchi, M., and H. Fujisawa. 1978. Characterization of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1. J. Biol. Chem. 253:8848-8853.

Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3.

Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 whe...
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