©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of a Novel Methyltransferase Responsible for Biosynthesis of Halomethanes and Methanethiol in Brassica oleracea(*)

Jihad M. Attieh , Andrew D. Hanson (§) , Hargurdeep S. Saini (¶)

From the (1) Institut de recherche en biologie végétale, Université de Montréal, 4101 rue Sherbrooke est, Montréal, Québec H1X 2B2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel S-adenosyl- L-methionine:halide/bisulfide methyltransferase (EC 2.1.1.-) was purified approximately 1000-fold to apparent homogeneity from leaves of Brassica oleracea. The enzyme catalyzed the S-adenosyl- L-methionine-dependent methylation of the halides iodide, bromide, and chloride to monohalomethanes and of bisulfide to methanethiol. The dual function of the enzyme was demonstrated through co-purification of the halide- and bisulfide-methylating activities in the same ratio and by studies of competition between the alternative substrates iodide and bisulfide. The purification procedure included gel filtration, anion exchange chromatography, and affinity chromatography on adenosine-agarose. Elution of the protein from a chromatofocusing column indicated a pI value of 4.8. The pH optimum of halide methylation (5.5-7.0) was different from that of bisulfide methylation (7.0-8.0). The molecular mass values for the native and denatured protein were 29.5 and 28 kDa, respectively, suggesting that the active enzyme is a monomer. The enzyme had the highest specificity constant for iodide and the next highest for bisulfide. Substrate interaction kinetics and product inhibition patterns were consistent with an Ordered Bi Bi mechanism.


INTRODUCTION

Interest in trace gas emissions has increased over the last two decades following the realization of their impact on atmospheric chemistry (1) . Halogen- and sulfur-containing organic gases have attracted particular attention because of their respective effects on the integrity of stratospheric ozone (2) and the formation of acid rain (3) .

Biological activity in the oceans is commonly viewed as a major source of organohalogen (4, 5) and organosulfur emissions (6, 7) . Terrestrial microorganisms, fungi, and a few higher plants also emit these gases (8, 9) . The biochemical bases for these emissions, however, remain poorly understood. Possible mechanisms involve spontaneous (10) or enzymatic (11, 12) reactions. A haloperoxidase-mediated incorporation of halides into a carbon skeleton was suggested as the main route for halomethane biosynthesis (11) but has never been actually shown to produce monohalomethanes. Subsequently, an enzymic mechanism involving a single-step conversion of halides into a monohalocarbon was proposed (13) ; this was supported by evidence for incorporation of the methionine methyl group into a monohalomethane (14) . Recently, it was reported (15) that a methyl chloride transferase from the marine red alga Endocladia muricata could carry out the transfer of a methyl group from S-adenosyl- L-methionine (AdoMet)() to a halide (X) as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Preliminary evidence was also presented for a similar activity in a wood-rotting fungus, Phellinus pomaceus, and a halophytic higher plant, Mesembryanthemum crystallinum.

Methanethiol was originally thought to come from methionine via a methioninase ( L-methionine methanethiol-lyase) reaction (12) . However, the observation that several bacterial isolates from soil and agricultural crops as well as cultures of marine algae evolved CHSH in the presence of bisulfide (HS) suggested that this gas may also be produced via a different mechanism (16) . Thiol methyltransferases, capable of catalyzing the following reaction, were later found in a variety of organisms including bacteria (16) , algae (17) , and mammals (18, 19) .

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

A survey of higher plants done in our laboratory (20) showed that many diverse species produce CHI when supplied with I, and CHSH when supplied with HS, in a manner similar to Reactions 1 and 2. The levels of the two activities were highly correlated among species. Plants from the family Brassicaceae exhibited some of the highest rates of CHI and CHSH production. To elucidate the biochemical basis for these environmentally important gas emissions, we developed an easy and reliable assay for their quantitation and proceeded to purify and characterize the halide- and bisulfide-methylating activities.


EXPERIMENTAL PROCEDURES

Plant MaterialPlants of Brassica oleracea cv. April Red were grown in a greenhouse under a photoperiod of at least 12 h and were watered and fertilized as necessary. The two most expanded basal leaves of 3-month-old plants were used for enzyme extraction. Chemicals S-Adenosyl- L-methionine was from Boehringer Mannheim. The inorganic phosphorus test kit, S-adenosyl- L-homocysteine, 5`-ADP-agarose, KI, KBr, and KCl were purchased from Sigma. Ammonium sulfide and authentic CHSH, CHBr, and CHI were obtained from Aldrich. Authentic CHCl was purchased from Liquid Carbonic (Scarborough, Ontario, Canada). Sephadex G-100, Sephadex G-25, gel filtration calibration kit, and Polybuffer 74 were from Pharmacia Biotech Inc. Calf intestinal alkaline phosphatase was purchased from New England Biolabs (Beverly, MA). The protein dye reagent, TEMED, ammonium persulfate, and bisacrylamide were all from Bio-Rad (Mississauga, Ontario, Canada). All other chemicals were of reagent grade. BuffersThe buffers used were as follows: 100 m M Tris acetate, pH 7.5, containing 10% glycerol (v/v) and 14 m M 2-mercaptoethanol (buffer A); 25 m M Tris acetate, pH 7.4, containing 10% glycerol and 14 m M 2-mercaptoethanol (buffer B); 25 m M Tris acetate, pH 7.4, containing 14 m M 2-mercaptoethanol (buffer C); buffer C with 100 m M NaCl (buffer D); 25 m M BisTris/iminodiacetic acid, pH 7.0 (buffer E); and Polybuffer 74/ iminodiacetic acid (1:10, v/v), pH 4.0 (buffer F). All buffers were filtered before use through a 0.22-µm membrane. Measurement of Halide/Bisulfide Methyltransferase Activity

Enzyme Assay

Since Iwas the preferred substrate among halides, it was used as the halide substrate throughout the purification procedure. The enzyme activity was assayed in a 1-ml mixture containing 0.5 m M AdoMet, 50 m M KI (halide methylation), or 20 m M (NH)S (bisulfide methylation) all prepared in buffer A. Enzyme preparations containing up to 200 µg of protein were used to start the reaction. The mixture was incubated in a 5-ml glass vial sealed with a screw cap fitted with a Teflon-lined septum (Supelco, Oakville, Ontario, Canada) and maintained on an orbital shaker (150 rpm) at room temperature. The reaction rate was linear for at least 45 min at all enzyme and substrate concentrations. A standard incubation time of 30 min was adopted.

Gas Chromatography

The products formed were analyzed by gas chromatography using a flame ionization detector. One-ml headspace samples were injected in a 210 0.3-cm stainless steel column packed with 80/100-mesh Porapak Q (Supelco) in a Hewlett-Packard 5890 series II gas chromatograph. Column temperatures were 160 °C for CHI and CHBr, 145 °C for CHSH, and 130 °C for CHCl. The carrier gas (helium, ultrapure) flow rate was 40 ml/min. The column was purged by heating to 200 °C between injections. Products were quantified by peak area and identified by comparison of their retention times with those of authentic methyl halides or CHSH, which were used to calibrate the instrument. Recoveries of 75 nl of CHI and 28 nl of CHSH injected to vials containing the assay mixture were 69 and 87%, respectively, after 30 min. The data presented here were not corrected for recoveries. The identity of CHI and CHSH in the headspace of enzyme assays was confirmed by mass spectrometry using a KRATOS MS 50 mass spectrometer operated in the electron impact mode. Protein EstimationTotal soluble proteins were determined by the method of Bradford (21) using the Bio-Rad protein reagent, following the microassay procedure, and using bovine serum albumin as the standard. Preparation of Adenosine-Agarose Affinity Gel5`-ADP-agarose (4 ml) was washed under vacuum according to the manufacturer's instructions and incubated with 800 units of calf intestinal alkaline phosphatase and 5 ml of calf intestinal alkaline phosphatase buffer (1 ), pH 7.5, in a total volume of 9 ml. The gel was dephosphorylated overnight at 37 °C in a continuously rotating reaction vial, transferred to a column, washed with 10 ml of buffer C containing 2 M NaCl, and washed again with 20 ml of buffer C alone. The washing was done in fractions of 10 ml, which were subsequently assayed for phosphate content. The dephosphorylation reaction was repeated until no more phosphate was detected. The gel was washed with an excess of deionized water before each reaction. Free phosphate was determined colorimetrically with the method of Fiske and Subbarow (22) . Purification of Halide/Bisulfide Methyltransferase

Extraction

All manipulations were carried out at 4 °C unless stated otherwise. Halide- and HS-methylating activities were monitored simultaneously throughout the purification procedure. Generally, 1 kg of freshly harvested leaves were cut into small squares, immediately frozen with liquid nitrogen, and then homogenized with buffer A (1:3, w/v) and polyvinylpolypyrrolidone (10%, w/w) for 5 min in a blender at full speed. The homogenate was filtered through eight layers of cheesecloth and centrifuged for 20 min at 15,000 g. The supernatant (crude extract) was recovered, and enzyme activity and protein concentration were assayed in an aliquot desalted by passage through a PD-10 column (Pharmacia). The crude extract was fractionated with solid (NH)SO, and the proteins that precipitated between 60 and 85% saturation, containing halide and bisulfide methyltransferase activities, were recovered by centrifugation at 12,000 g for 20 min.

Gel Filtration on Sephadex G-100

The pellet from the 60-85% (NH)SOfraction was resuspended in 15 ml of buffer B and applied to a Sephadex G-100 column (2.6 90 cm) that had been previously equilibrated in the same buffer. The column was eluted with buffer B at 25 ml/h, and 2-ml fractions were collected. The active fractions from this step were pooled and concentrated by diafiltration in a stirred Amicon cell fitted with a YM-30 membrane (Amicon, Danvers, MA).

Affinity Chromatography on Adenosine-Agarose

The concentrated sample was applied to an adenosine-agarose column (1.5 3 cm) that had been previously equilibrated with buffer B. The column was washed at a constant flow rate of 10 ml/h with 15 ml of buffer B, followed by 8 ml of the same buffer containing 300 m M (NH)SO. The salt was then removed by washing the column with buffer B. The halide- and bisulfide-methylating activities were recovered by elution with a 40-ml linear gradient of 0-4 m M AdoMet in buffer B. One-ml fractions were collected and assayed for activity and protein content. The halide and bisulfide methyltransferase activities eluted between 2 and 3 m M AdoMet. The remaining bound proteins were washed off the column with 2 M NaCl in buffer B; no methyltransferase activity was detected in this fraction.

Anion Exchange on Protein Pak Q

Active eluate from the affinity step was submitted to high performance liquid chromatography (HPLC) (Millipore, Milford, MA) on a 1 10-cm Protein Pak Q anion exchange column (Millipore) equilibrated with buffer C. The sample was loaded at a flow rate of 0.5 ml/min, and the column was washed with the same buffer until no further UV-absorbing material was eluted. Enzyme activity was then eluted with NaCl in buffer C in a two-step linear gradient of 0-200 m M in 80 min and 200-500 m M in 20 min, collecting 0.5-ml fractions.

Gel Filtration on Superdex 75

The fraction containing the first peak of halide and bisulfide methyltransferase activity from the previous step was loaded on an HPLC Superdex 75 gel filtration column (1 30 cm) (Pharmacia) previously equilibrated with buffer D. Proteins were eluted in the same buffer at a flow rate of 0.5 ml/min, collecting 0.5-ml fractions. At this level of purification the halide and bisulfide methyltransferase preparation was apparently free from contaminating proteins. ChromatofocusingAn enzyme preparation from the Sephadex G-100 gel filtration step was subjected to HPLC chromatofocusing on a Mono P HR (5/20) column (Pharmacia) previously equilibrated with buffer E. Unbound proteins were removed by washing the column with the same buffer. Enzyme activity was then eluted with a pH gradient of 7.0-4.0 generated with 45 ml of buffer F at a flow rate of 1 ml/min. One-ml fractions were collected in 0.2 ml of 500 m M Tris acetate buffer, pH 7.7, containing 50% glycerol. Molecular Weight DeterminationMolecular weight of the native halide/bisulfide methyltransferase was determined by gel filtration on a Sephadex G-100 column (2.5 44 cm) calibrated with the following markers: ribonuclease A ( M13,700), chymotrypsinogen A ( M25,000), ovalbumin ( M43,000), and albumin ( M67,000). The column void volume was determined with blue dextran 2000. Buffer B was used for column equilibration and elution. One-ml samples were applied to the column, which was operated at a flow rate of 20 ml/h. In order to achieve better resolution, column calibration was done in two steps, the first involving ribonuclease A and ovalbumin and the second involving albumin and chymotrypsinogen A. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Proteins from different purification steps were separated by SDS-PAGE under denaturing conditions (23) using 12% acrylamide gels. Gels were stained with silver nitrate or Coomassie Blue. The molecular mass of the denatured enzyme was determined from a plot of log molecular mass against the migration distance of standard proteins. Determination of Kinetic PropertiesThe kinetic properties of the halide/bisulfide methyltransferase were determined using a preparation purified through a Sephadex G-100 gel filtration step.

Alternative substrate competition experiments were performed by varying the concentration of one substrate at each of a series of concentrations of the other. The concentration of AdoMet was kept constant at 0.5 m M. Data were presented as double-reciprocal plots of initial velocity (v) versus varying substrate (S) concentrations.

Substrate interaction studies were done by fixing the concentration of one substrate while changing that of the other. Linear regressions were fitted to the data in double-reciprocal plots. Replots of the data were used to determine the kinetic parameters.


RESULTS

B. oleracea leaf extracts catalyzed the AdoMet-dependent methyl transfer to Xor HS. The enzyme responsible for both of the reactions was purified to homogeneity, and its dual function was demonstrated.

Purification of Halide/Bisulfide Methyltransferase

The enzyme was extracted from B. oleracea by subjecting leaf tissue to a freeze-thaw cycle prior to homogenization and was purified using (NH)SOprecipitation followed by gel filtration chromatography on Sephadex G-100, affinity chromatography on adenosine-agarose, and HPLC on Protein Pak Q anion exchange and Superdex 75 gel filtration columns. The specific activity of the enzyme was enriched more than 1000-fold over the crude preparation with a recovery of 0.35%. summarizes the purification procedure, and Fig. 1shows a typical pattern of proteins from various purification steps separated on SDS-PAGE and visualized by Coomassie Blue.


Figure 1: SDS-PAGE of fractions with halide/bisulfide methyltransferase activity from the successive steps of purification. Lane A, crude extract (10 µg); lane B, 60-85% (NH)SO(5 µg); lane C, Sephadex G-100 (5 µg); lane D, Amicon YM-30 (5 µg); lane E, adenosine-agarose (5 µg); lane F, Protein Pak Q (5 µg); lane G, Superdex 75 (1 µg). The molecular mass markers are indicated to the left in kDa. The gel was stained with high sensitivity Coomassie Brilliant Blue G-250.



Affinity chromatography on adenosine-agarose was efficient in removing most of the contaminating proteins (Fig. 2). Further purification was achieved by anion exchange HPLC, which resolved four peaks of halide/bisulfide methyltransferase activity (Fig. 3). The first and largest peak of activity eluting from this column contained only two proteins as seen on the SDS gel (Fig. 1). Further purification of this peak by HPLC on a Superdex 75 gel filtration column resolved two protein peaks, only one of which contained halide/bisulfide-methylating activity. This peak appeared free of contaminants when visualized on SDS-PAGE (Fig. 1). The Superdex-purified halide/bisulfide methyltransferase migrated as a single band on SDS-PAGE with an apparent molecular mass of 28,000 daltons. Since this is close to the value of 29,500 determined for the native protein on a G-100 gel filtration column, the active enzyme probably exists as a monomer.


Figure 2: Elution of halide/bisulfide methyltransferase from an adenosine-agarose affinity column. Specifically bound proteins were eluted in a 0-4 m M AdoMet ( SAM) gradient in buffer B. Fractions of the eluate were assayed for halide () and bisulfide () methyltransferase activities. Both activities overlapped up to fraction 55. Proteins were continuously monitored by absorbance at 280 nm ( solid line). Most of the increase in the absorbance with the gradient elution was due to the presence of AdoMet. nkat, nanokatals.



Demonstration of Dual Activity

The halide and bisulfide methyltransferase activities co-purified to homogeneity in the same ratio (1.26 ± 0.14) during a five-step procedure (, Figs. 2 and 3). Moreover, double-reciprocal plots of the data from competition kinetics between the halide and bisulfide substrates gave regression lines that intersected at the 1/ v axis (Fig. 4, A and B). These results established that the halide and bisulfide methylations are catalyzed by the same protein, halide/bisulfide methyltransferase.

Effect of pH on Halide/Bisulfide Methyltransferase

The halide/bisulfide methyltransferase activity was assayed over a wide range of pH, using buffers with overlapping ranges. The pH optimum for Imethylation was fairly broad, between 5.5 and 7.0 (Fig. 5 A), whereas that for HSmethylation was sharper and more alkaline, between 7.0 and 8.0 (Fig. 5 B). In addition, halide/bisulfide methyltransferase activity eluted at pH 4.8 upon chromatofocusing on a Mono P column, suggesting that the enzyme has a pI value of 4.8 ± 0.2.

Enzyme Stability

After Sephadex G-100 gel filtration, the enzyme was stable for over 2 months at -80 °C in buffer B. After affinity chromatography, the halide/bisulfide methyltransferase became extremely labile, losing all activity after overnight storage at -80 °C. Addition of 20% glycerol to the preparation and storage at -20 °C conserved 12% of the activity after 48 h. In contrast, after the Protein Pak Q anion exchange step, the enzyme retained more than 70% of its activity after 24 h and 55% after 48 h at 4 °C in buffer C containing 175 m M NaCl. Kinetic Analysis

Substrate Interaction Kinetics

Double-reciprocal plots with Ias the variable substrate at several fixed concentrations of AdoMet gave converging lines (Fig. 6 A). The same pattern was observed when HSwas the variable substrate at different fixed concentrations of AdoMet (Fig. 6 B).


Figure 6: Double-reciprocal plots of initial velocity ( v) versus substrate concentration for the methylation of iodide and bisulfide. The effect of iodide ( A) or bisulfide ( B) concentrations on the initial velocity of the methylation reaction at different fixed concentrations of AdoMet ( SAM) is shown. nkat, nanokatals.



Product Inhibition Kinetics

The order of substrate binding and product release was determined from product inhibition studies. S-Adenosyl- L-homocysteine was a competitive inhibitor with respect to AdoMet (Fig. 7 A) and noncompetitive with respect to iodide (Fig. 7 B) or bisulfide (Fig. 7 C). The kinetics of product inhibition for the gaseous CHI or CHSH could not be determined under the present assay conditions.


Figure 7: Product inhibition of halide/bisulfide methyltransferase reaction, presented as double-reciprocal plots. A, inhibition of the methylation reaction by S-adenosyl- L-homocysteine ( SAH) with respect to S-adenosyl- L-methionine; the concentration of iodide was fixed at 50 m M. B, inhibition of the methylation reaction by S-adenosyl- L-homocysteine with respect to iodide; the concentration of AdoMet was fixed at 400 µ M. C, inhibition of the methylation reaction by S-adenosyl- L-homocysteine with respect to bisulfide; the concentration of AdoMet was fixed at 400 µ M. nkat, nanokatals.



As calculated from replots of the data, the Vfor the formation of CHI was 1140 nanokatals/mg of protein; that for CHSH was 804 nanokatals/mg of protein. The respective Kvalues for Iand HSwere 1.3 and 4.7 m M. There were two different Kvalues for AdoMet depending on the second substrate used, 0.03 m M with Iand 0.226 m M with HS. Table II summarizes the kinetic parameters of the halide/bisulfide methyltransferase for its different substrates.


DISCUSSION

The halide and bisulfide methyltransferase activities from B. oleracea were purified to apparent homogeneity. The two activities co-purified at a constant ratio (1.26 ± 0.14) throughout the procedure (, Figs. 2 and 3). The kinetics of mutual inhibition between the substrates Iand HSwere characteristic of competitive inhibition (Fig. 4, A and B). Taken together, these results establish that both halide- and bisulfide-methylating activities reside on the same active site of a single protein, the halide/bisulfide methyltransferase. The homogeneous protein was obtained through a five-step procedure that resulted in an overall purification of approximately 1000-fold with a recovery of 0.35% (). Affinity chromatography on an adenosine-agarose column was the key step, in which the enzyme specifically bound to the matrix while most of the contaminating proteins were washed through during sample loading (Fig. 2). The enzyme was selectively eluted with the co-substrate AdoMet in a linear gradient resulting in a 1300-fold increase in the specific activity over the crude preparation. Adenosine, linked via its adenine-Cto agarose (ag-adenosine, Pharmacia), has been successfully used to purify other plant methyltransferases (24, 25) . Our adenosine-agarose matrix was highly stable and was repeatedly used without any loss of its binding or eluting efficiencies. Other affinity matrices prepared with either AdoMet or AdoHcy linked via their free amino or carboxyl groups to Affi-Prep 10 (Bio-Rad) or EAH-Sepharose (Pharmacia) failed to bind the enzyme.


Figure 4: Competition between the alternative substrates of the halide/bisulfide methyltransferase. A, inhibition of iodide methylation by different fixed concentrations of bisulfide. B, inhibition of bisulfide methylation by different fixed concentrations of iodide. The concentration of AdoMet was kept constant at 0.5 m M. nkat, nanokatals.



High performance liquid chromatography on Protein Pak Q anion exchange column resolved multiple peaks of halide/bisulfide methyltransferase activity (Fig. 3). An enzyme preparation processed through a procedure in which salt precipitation was replaced with polyethylene glycol precipitation also gave multiple peaks in this anion exchange step (not shown). These observations suggest that the enzyme may exist in multiple charge isoforms, although this possibility remains to be confirmed through further experiments. Interestingly, rat liver thiol methyltransferase, with which the halide/bisulfide methyltransferase shares many properties, was reported in five different cellular compartments (26) . The harsh freeze-thaw treatment needed to maximize the extraction of halide/bisulfide methyltransferase was also similar to the procedure used to extract the compartmented rat liver enzyme (18) .


Figure 3: Elution of halide/bisulfide methyltransferase from a Protein Pak Q column upon anion exchange high performance liquid chromatography. Bound proteins were eluted in a 0-200 m M and 200-500 m M two-step linear gradient of NaCl in buffer C. Halide- () and bisulfide-methylating () activities were assayed in each fraction. Proteins were monitored by absorbance at 280 nm ( solid line). nkat, nanokatals.



The protein from a fraction containing peak halide/bisulfide methyltransferase activity from the Superdex 75 step migrated as a single band of 28,000 Da on SDS-PAGE (Fig. 1). Moreover, when consecutive fractions across the first peak of activity after anion exchange HPLC were applied to SDS-PAGE, the Coomassie Blue staining intensity of the 28,000-Da protein band peaked in the fraction containing peak enzyme activity (not shown). These observations confirmed that the molecular mass of the denatured halide/bisulfide methyltransferase was 28,000 Da. Since the molecular weight of the enzyme, calculated from gel filtration chromatography, was 29,500, the native protein probably functions as a monomer. The molecular weight of this enzyme was similar to the algal methyl chloride transferase (15) and mammalian thiol methyltransferase (18, 26) .

The halide/bisulfide methyltransferase exhibited different pH optimum values for its alternative halide (Fig. 5 A) and bisulfide (Fig. 5 B) substrates. A likely reason for this difference is the pH dependence of the HSion concentration. Since HS has a p K1 of 7.02 (27) , [HS] would be expected to fall sharply as the pH drops below 7. The halide- and bisulfide-methylating activities had the same pI value at pH 4.8, which was consistent with the observation that both activities existed on a single protein.


Figure 5: Ef