cDNA cloning of Batis maritima methyl chloride transferase and purification of the enzyme

Abstract

Methyl chloride transferase catalyzes the synthesis of methyl chloride from S-adenosine-l-methionine and chloride ion. This enzyme has been purified 2,700-fold to homogeneity from Batis maritima, a halophytic plant that grows abundantly in salt marshes. The purification of the enzyme was accomplished by a combination of ammonium sulfate fractionation, column chromatography on Sephadex G100 and adenosine-agarose, and TSK-250 size-exclusion HPLC. The purified enzyme exhibits a single band on SDS/PAGE with a molecular mass of approximately 22.5 kDa. The molecular mass of the purified enzyme was 22,474 Da as determined by matrix-associated laser desorption ionization mass spectrometry. The methylase can function in either a monomeric or oligomeric form. A 32-aa sequence of an internal fragment of the methylase was determined (GLVPGCGGGYDVVAMANPER FMVGLDIXENAL, where X represents unknown residue) by Edman degradation, and a full-length cDNA of the enzyme was obtained by rapid amplification of cDNA ends–PCR amplification of cDNA oligonucleotides. The cDNA gene contains an ORF of 690 bp encoding an enzyme of 230 aa residues having a predicted molecular mass of 25,761 Da. The disparity between the observed and calculated molecular mass suggests that the methylase undergoes posttranslational cleavage, possibly during purification. Sequence homologies suggest that the B. maritima methylase defines a new family of plant methyl transferases. A possible function for this novel methylase in halophytic plants is discussed.

It is estimated that the production of methyl chloride, the most abundant halohydrocarbon species (1) in the upper atmosphere, is 5 × 10⁶ tons per year (2), and that most of the CH₃Cl comes from biological sources (3). Hutchinson (4) first reported the presence of methyl chloride in the gas phase above fungal cultures of the genus Fomes. A survey by Cowan et al. (5) found that 6 of 35 different species of Fomes produced methyl chloride. The synthesis of methyl chloride by cultures of wood root fungi has been studied in vivo (3, 6). There also are reports that methyl chloride and other volatile halogenated organic compounds are produced by marine macroalgae and phytoplankton (7, 8). More recently, Wuosmaa and Hager (9) discovered a methyl chloride transferase (MCT) that catalyzes the formation of methyl chloride via the reaction of S-adenosine-l-methionine (AdoMet) with chloride ion. This enzyme was found in cell-free extracts of Phellinus promaceus (a fungus), Endocladia muricata (a marine algae), and Messembryanthemum crystallium (ice plant, a halophytic plant). Wuosmaa (10) also found that 20 of 31 marine algae collected in Pacific coastal waters near Monterey, CA, produce methyl chloride and other methyl halides. In contrast to the established peroxidative enzymatic mechanisms (11, 12) for the biosynthesis of halometabolites, which generate an electrophilic halogenating species, the halide anion functions as a nucleophile in the MCT reaction.

CH₃Cl and other halogenated hydrocarbons in the upper atmosphere (7) are implicated in affecting the integrity of the stratospheric ozone layer (13). The biological production of CH₃Cl and other volatile halogenated organic compounds in ocean waters is commonly viewed as a major source of these halohydrocarbons (14). However, the finding of CH₃Cl production by halophytic plants, which grow in great abundance in coastal soils and salt marshes, indicate that halophytic plants also may be major contributors to the formation of the 5 million tons of methyl chloride that are produced each year. Batis maritima, a succulent halophytic plant of the Batidaceae family, commonly grows in low-lying coastal areas that frequently are flooded with sea water. In our laboratory, we found that Batis maritima has high MCT activity.

To study more closely the function and mechanism of MCT, this enzyme was isolated from B. maritima, a partial amino acid sequence was obtained, and cDNA clones of the MCT gene were prepared. The complete amino acid sequence of this protein has been deduced from the cloned gene. It contains two motifs that are conserved among small-molecule and protein methyl transferases (15–19) and one motif that is conserved among DNA N-6- and N-4-methyltransferases (20–21).

MATERIALS AND METHODS

Preparation of B. maritima Crude Extracts.

Crude plant extracts were prepared from B. maritima plants that were collected in Puerto Penasco, Sonora, Mexico, during the winter season. All isolation steps were performed at 4°C unless otherwise mentioned. Usually, 300 g of plant leaves were frozen with liquid nitrogen, ground with a mortar and pestle, transferred to a Waring blender with 200–250 ml of buffer A (0.25 M Tris/0.2 M Na₂B₂O₇/0.02 M NaS₂O₅/0.05 M EDTA, pH 6.8), and homogenized at high speed with two 30-sec pulses. The homogenate was squeezed through an eight-layer cheesecloth and centrifuged at 17,000 × g for 30 min. The supernatant fraction was transferred to a 1-liter beaker, and the proteins were precipitated with solid [NH4]₂SO₄, which was added to a final concentration of 70% saturation. After standing at 4°C for 20 min the precipitated proteins were collected by centrifugation at 10,000 × g for 10 min. The precipitated proteins were resuspended in 20–25 ml of buffer B (50 mM Tris/20 mM Na₂S₂O₅/0.05 mM EDTA, pH 6.8) and centrifuged at 17,000 × g for 15 min. The supernatant fraction was used for further purification. Typical protein concentrations in this supernatant fraction were 9–10 mg/ml, as estimated by the method of Bradford (22), with BSA as the comparison standard.

Assay for the Methyl Chloride Transferase Activity.

MCT activity was measured in both a methyl iodide and methyl chloride assay (10). Routine measurements used iodide as the acceptor halide because the enzyme was most active with iodide ion. Suitable enzyme aliquots in a total volume of 400 μl of 20 mM Tris buffer, pH 6.8, were incubated with 200 μl of AdoMet (1 mg/ml)/200 μl of KI (1.2 M)/20 μl of DTT (0.1 mM) in a 5-ml vial, which then was sealed with a rubber stopper and an aluminum cap. Usually, the reaction was carried out at room temperature for 1 hr. Methyl iodide formation was analyzed by gas chromatography by using a flame-ionization detector. One milliliter of headspace gas was injected into a 6-ft Chromosorb 101 column (Varian) installed on a Varian 3700 Aerograph Gas Chromatograph. The column temperature was 134°C, and the flow rate of the nitrogen carrier gas was 30 ml/min. Chromatographic peaks were calibrated with the appropriate methyl halide standard.

Isolation of Methyl Chloride Transferase.

MCT was purified using three chromatography steps. The supernatant fraction from the ammonium sulfate step was loaded onto a Sephadex G 100 (2 × 60 cm) column that had been equilibrated with buffer B. The enzyme was eluted by using the same buffer B. Three-milliliter fractions were collected, and the active fractions containing the enzyme (tubes 24–40) were pooled and concentrated to a volume of 2–3 ml by using a Centricon 10 apparatus (Amicon). The concentrated fractions were loaded on a 5-ml adenosine-agarose column that had been equilibrated with buffer B. The adenosine-agarose column was prepared by removing the phosphate group from AMP-agarose (Sigma) by using alkaline phosphatase at 37°C for 24 hr. The adenosine-agarose column was washed with 20 ml of buffer B, followed by 20 ml of 0.2 M NaCl in buffer B, and MCT was eluted with 20 ml of 1 mM AdoMet and 0.2 M NaCl in buffer B. One-milliliter fractions were collected. The active fractions (fractions 23–27) were pooled, concentrated to 200–300 μl, and stored in 30% glycerol at −20°C for at least 3 weeks. After storage, the MCT fractions were loaded onto an HPLC size-exclusion column (TSK-250, Bio-Rad) equilibrated with buffer C (20 mM Tris/0.5 mM DTT, pH 6.8). The purified MCT was eluted from the size-exclusion column with buffer C by using a constant flow rate of 1 ml/min. The active fractions (fractions eluting at 11 and 12 min) were concentrated to 10 μl. Each fraction was analyzed by SDS/PAGE under denaturing conditions by using 20% polyacrylamide gels in the Phastsystem (Pharmacia). The purified MCT also was analyzed by matrix-associated laser desorption ionization (MALDI) mass spectrometry to check for purity and molecular mass.

Amino Acid Sequencing.

MCT was digested with endopeptidase Lys-C following the protocol of Matsudaira (23). Briefly, 40 μg (≈2 nmol) methyl chloride transferase was dissolved in 25 μl of 8 M urea and 0.4 M ammonium bicarbonate. Five microliters of 45 mM DTT was added and the reaction mixture was incubated at 50°C for 15 min. In the next step the enzyme was carboxyamidomethylated by using 5 μl of 100 mM iodoacetamide at room temperature for 15 min and dialyzed. Finally, 60 μl of H₂O and 2 μg of Lys-C (Boehringer Mannheim) were added, and the solution was incubated at 37°C for 24 hr. The resulting peptides were separated by reverse-phase chromatography on a microbore C18 HPLC column (2.1 × 50 mm i.d.). The peptides first were eluted with 0.1% trifluoroacetic acid (TFA) for 10 min, then with a gradient from 0 to 60% CH₃CN in 0.1% TFA for 60 min, and finally with 80% CH₃CN in 0.1% TFA for 20 min. The flow rate was 0.15 ml/min, and the elution profile was monitored at 214 nm. Selected fractions were subjected to MALDI mass spectrometric analysis to determine purity and molecular mass. Peptide L26 was selected for sequencing.

RNA Isolation and cDNA Synthesis.

Poly(A)⁺ RNA was isolated from greenhouse-grown B. maritima by using the Straight A’s mRNA Isolation System (Novagen). The first-strand cDNA was synthesized by reverse transcription using the SuperScript Preamplification System (BRL). The double-stranded cDNA used for rapid amplification of cDNA ends (RACE)-PCR amplification of the 3′ and 5′ ends of the cDNA was synthesized by using the Marothon cDNA Amplification Kit (CLONTECH).

Reverse Transcription–PCR and Cloning of a Partial MCT cDNA.

A number of oligonucleotides were synthesized for use as hybridization probes and/or primers. Most of the oligonucleotides were synthesized at the University of Illinois Biotechnology Center Genetic Engineering Laboratory; some were furnished in specific cloning kits. The oligonucleotide sequences, all written in the 5′ → 3′ direction, are: P1-CGGAATTCGGNTTRGTNCCNGGNT-GYGGNGGNGG; P2-CGGGATCCYAANGCMTTYTCAAADATRTCYAANCCNA-CC; GSPa-GGAGGTCCACCCTCATGGGTGATCG; GSPb-ATGGCGAACCCCG-AGAGATTCATGG; RACE 1-CGGGCAGGTTTCTAGAATTCAGCG; AP1-CCA-TCCCTAATACGACTCACTATAGGGC; AP2-ACTCACTATAGGGCTCGAG-CGGC; and SP-TATATCCAATCCGACCATGAATCTCTCG. In oligonucleotides P1 and P2, N represents an A, T, G, or C; D represents an A, T, or G; Y represents a C or T; R represents an A or G; and M represents an A or C.

PCR and Cloning of a Partial MCT cDNA.

Two oligonucleotide primers, P1 and P2, were used to generate a partial cDNA clone. The sequences of P1, a 2¹⁴-fold degenerate oligonucleotide primer (sense direction), and P2, a 3 × 2¹¹-fold degenerate oligonucleotide primer (antisense direction), were based on amino acid sequences 1–9 and 22–32, respectively, of peptide L26. An EcoRI site and a BamHI site were designed into the 5′ ends of P1 and P2, respectively. Two rounds of PCR were carried out to amplify the partial cDNA by using these two primers. The first round of PCR amplification was carried out with first-strand cDNA as the template. The second round of PCR was carried out with the first-round product as template. The PCR was performed by using a DNA Thermal Cycler (Perkin–Elmer) with cycling conditions as follows: denaturation at 94°C for 30 sec, extension at 72°C for 30 sec, followed by annealing for 15 sec at 67°C for 2 cycles, 64°C for 2 cycles, 61°C for 3 cycles, 58°C for 5 cycles, 55°C for 40 cycles, and finally a 7-min extension at 72°C. The reaction mixture included 4 mM MgCl₂, 1× Taq polymerase buffer, 0.2 mM of each dNTP, 0.2 mM of P1 and P2, and 1 unit of Taq polymerase (BRL). The ≈110-bp fragment obtained in the second-round PCR was purified by gel electrophoresis using a Qiaex II Gel Extraction Kit (Qiagen). The purified oligonucleotide was ligated into an EcoRI-digested and a BamHI-digested pTZ 19b plasmid (Pharmacia) and used to transform Escherichia coli to ampicillin resistance. Independent clones containing inserts of the proper size were isolated, and their DNA sequences were determined.

RACE-PCR and Cloning of 3′ and 5′ End cDNA of MCT.

Oligonucleotides 3′-GSPa and 3′-GSPb, both containing sequences in the sense direction, were based on the cDNA sequence of the partial MCT clone obtained above. Oligonucleotide RACE 1 was based the DNA adapter sequence. The 3′ end cDNA of MCT was generated by two rounds of PCR with the primer pair 3′-GSPa and RACE 1(first round) and 3′-GSPb and RACE 1 (second round). The first-round 3′ end RACE-PCR was carried out by using double-stranded (ds) cDNA as template, and the second-round PCR was carried out by using the first-round PCR products as template. Both rounds of PCR were carried out under the following conditions: 94°C for 1–1/2 min, 72°C for 2 min for 5 cycles, 94°C for 30 sec, 68°C for 2 min for 5 cycles, 94°C for 30 sec, 68°C for 2 min for 25 cycles, and, finally, 72°C for 7 min. Oligonucleotides 5′-GSPa and 5′-GSPb were based on the 3′ end cDNA sequence obtained above. The 5′ end cDNA of MCT was generated by two rounds of PCR with the primer pair 5′-GSPa and AP1 (first round) and 5′-GSPb and AP2 (second round). Both AP1 and AP2 were provided by CLONTECH. The first round of 5′ end RACE-PCR was carried out by using ds cDNA as template, and the second round of PCR was carried out by using the first-round PCR products as template. The PCR was carried out under the same conditions used for 3′ end cDNA amplification. Southern blot analysis was carried out on the 3′ end and 5′ end RACE-PCR products by using a ³²P-labeled probe, SP. The SP probe DNA sequence was based on the partial MCT cDNA sequence and was prepared as described in Ausubel et al. (24). The desired fragments in the 3′ end and 5′ end PCR products were gel-purified by electrophoresis, ligated to pGEM-T vector (Promega), and used to transform E. coli cells to ampicillin resistance. Independent clones containing inserts of the proper size were isolated, and their DNA sequences were determined.

RESULTS

Isolation of MCT from B. maritima.

The purification of MCT from B. maritima is summarized in Table 1 and Fig. 1. It was essential to include sodium tetraborate and sodium metabisulfite in the extraction buffer in the preparation of the crude extract. In their absence MCT activity was lost rapidly presumably because of the reaction of the enzyme with the large quantities of phenolic compounds in the crude extract. Fig. 2 shows the SDS/PAGE analysis of each fraction during the isolation of MCT. After the final chromatography step, MCT moved as a 22- to 23-kDa single band on SDS/PAGE. The key step in the procedure is the adenosine-agarose affinity column, which gives a 600-fold purification (Fig. 1b). The last step, size-exclusion chromatography, took advantage of the fact that upon storage at −20°C, MCT forms an aggregate having a molecular mass of approximately 500 kDa. Thus, the size-exclusion HPLC step readily separates MCT from contaminating proteins (Fig. 1c). The aggregated species of MCT retains full activity.

Table 1.

Purification of methyl chloride transferase

Fraction	Total protein, μg	Total activity, units*	Specific activity, units/mg protein	Yield, %	Purification, -fold
Crude extract	242 × 103	26.7	0.11	100	—
Sephadex G100	43 × 103	6.8	0.17	25	1.5
Adenosine column	34	3.4	99.5	12	900
TSK-HPLC	6	1.7	300	5	2,700

^*One unit of activity is defined as 1 nmol of methyl iodide formed per minute in the methyl iodide assay described in Methods and Materials. 

Figure 1.

Purification of methyl chloride transferase. The elution profile of MCT (dotted line) from the Sephadex G100 (a) and TSK-250 columns is shown. (b) The elution profile of MCT (dotted line) and the protein concentrations (solid line) in mg/ml in the eluates from the adenosine-agarose column. The insert in c identifies the molecular mass markers used for calibration purposes. The protein markers are cytochrome c [log molecular weight (MW_r), 12.1; retention time (Rt), 26.6 min], myoglobin (MW_r, 18.8; Rt, 25.2 min), BSA (MW_r, 66; Rt, 19.2 min), alcohol dehydrogenase (MW_r, 150; Rt, 17.9 min), and β-amylase (MW_r, 200; Rt, 16.2 min). The retention time for MCT was 12 min. The procedures used for purification are given in Methods and Materials.

Figure 2.

SDS/PAGE analysis of MCT fractions. The procedures used in the SDS/PAGE analyses are given in Methods and Materials. Lane 1, protein markers; lane 2, crude plant extract; lane 3, ammonium sulfate fraction; lane 4, Sephadexa G100 fraction; lane 5, adenosine-agarose fraction; lane 6, TSK-250 column fraction. Proteins were visualized with Coomassie brilliant blue stain.

Halide vs. Bisulfide Methyltransferase Activity.

Attieh et al. purified and characterized a novel methyltransferase from Brassica oleracea (25). This cabbage enzyme uses both halides and bisulfide as methyl group acceptors. In contrast, purified MCT from B. maritima is unable to use bisulfide (HS-) as an acceptor.

cDNA cloning of MCT.

The purified MCT was subjected to Lys-C digestion, and the peptides were fractionated by reverse-phase HPLC. Several fractions were subjected to MALDI mass spectrometric analysis for purity and molecular weight. Peptide L26 was sequenced by Edman degradation (32 cycles). All 32 residues except amino acid residue 28 were identified. The amino acid sequence for L26 is: GLVPGCGGGYDVVAMANPERFMVGLDIXENAL. The X at position 28 represents the unidentified residue, which was later identified as serine.

Messenger RNA was isolated from B. maritima, reverse-transcribed into single-stranded cDNA, and converted to double-stranded cDNA and adapters were added to both ends. Fig. 3 shows the strategy for the cDNA cloning of MCT. In the first step, single-stranded (ss) cDNA was used in a reverse transcription–PCR experiment with oligonucleotides P1 and P2. This step yielded a 110-bp cDNA fragment, which was cloned into pTZ 19b and sequenced. Two independent clones gave DNA sequences that coded correctly for the amino acid sequence of L26. A 57-nt-long consensus sequence was obtained by comparing these two cDNA sequences. In the second step, this cDNA sequence was used to design two gene-specific primers, 3′-GSPa and 3′-GSPb, and a Southern blot probe, SP. The 3′ end cDNA of MCT was generated by two rounds of PCR amplification by using primer pairs of 3′-GSPa and RACE 1 and 3′-GSPb and RACE 1. The product was cloned into the pGEM-T Easy vector and sequenced. A 768-bp cDNA fragment including a poly(A) tail sequence was obtained. This sequence was used to design two new gene specific primers, 5′-GSPa and 5′-GSPb. The 5′ end cDNA of MCT also was generated by two rounds of PCR with primer pairs, 5′-GSPa and AP1 and 5′-GSPb and AP2, cloned into pGEM-T Easy vector and sequenced. A 556-bp fragment was obtained. The cDNA from the 3′ end and 5′ end had a common 267-nt overlapping sequence. Fig. 4 shows the sequence of the full-length cDNA of MCT obtained by the combining the 3′ end and 5′ end of the methylase cDNA. This sequence contains a 690-bp ORF terminated by a TAA stop codon and encodes a protein of 230 aa residues having a calculated molecular mass of 25,761 Da and a pI of 5.12. The L26 amino acid sequences were found exactly in place in the deduced sequence (residues 71–102, underlined) and established the identity of residue 28 as serine. The 3′ end of the cDNA consists of a 313-bp noncoding sequence. The consensus polyadenylation sequence, AAUAAA, was not identified, but sequences AAUAUA and AAAUAA are observed 24 and 142 nt upstream of the poly(A) tail.

Figure 3.

Schematic for cloning the MCT gene. The arrows indicate the primers and their direction. The underlined region identifies the hybridization site for the ³²P-labeled probe used in the Southern blot analysis.

Figure 4.

The nucleotide sequence of the cDNA of the MCT gene and the predicted amino acid sequence of the enzyme. The adenosine of the start codon ATG is numbered 1, the downstream nucleotide bases have positive numbers, and the upstream bases have negative numbers. The amino acid codons for peptide L26 are underlined. Conserved motifs I, II, and III are in boldface type. The GenBank accession no. for this nucleotide sequence is BankIt217095 AF084829.

Molecular Mass Differences.

The molecular mass of the purified MCT, as estimated by SDS/PAGE analysis, was in the 22- to 23-kDa range. A more accurate molecular mass value of 22,438 Da was obtained by mass spectrometry. In contrast, the MCT gene codes for an enzyme having a molecular mass of 25,761 Da. N-terminal amino acid sequence data suggest that the purified native enzyme has been processed at the N terminus. Edman degradation of the purified MCT indicated that the enzyme, as isolated, contains a mixed, variable N terminus. A unique amino acid at position 1 in the native enzyme could not be identified, and multiple residues also appeared in positions 2–9. However, one major Edman N-terminal sequence contained prolines at positions 2 and 4. In the predicted amino acid sequence no proline-X-proline sequences occur within the first 18 residues. The first proline-X-proline sequence occurs at positions 19–21 and another, at 53–55. Although neither the 19–21 nor the 53–55 site precisely account for the molecular mass differences between the isolated enzyme and the sequence deduced from the MCT gene, these preliminary data suggest that processing has occurred at the N terminus of the enzyme.

Sequence Homology.

A blast (26) search for sequence homology indicated that MCT has little sequence homology with other known methyltransferases, implying that MCT defines a new family of methyltransferases. However, recent blast data (January 1998) identify two Arabidopsis thaliana ORFs having coding sequences very similar to MCT. S. D. Roundsley, S. Kaul, X. Lin, K. A. Ketchum, M. L. Crosby, et al. (unpublished data) report genomic data for A. thaliana chromosome II which show that A. thaliana unknown protein 1 (accession no. 3212851, gene “F6E13.7”) has 134 of 190 residues that are identical to the amino acid sequences in MCT (70% identity and 84% overall similarity). A. thaliana unknown protein 2 (accession no. 3212850, gene “F6E13.5”) shows 67% identity and 82% similarity to MCT. It would appear safe to conclude that these two unknown proteins are methylases.

MCT does share three conserved motifs with other methyl transferases. Two conserved mammalian small-molecule methylase sequences can be identified when the amino acid sequence of MCT is compared with other small-molecule methyl transferases (15, 16). These MCT sequences, identified in Fig. 4, are LVPGCGGG (motif I) and LKPDGEL (motif II). These two MCT sequences fit with the methylase-conserved sequences L(D/E)oGjGjG and L(R/K)PGGuL, which are found in catechol-O-methyltransferease (COMT) and several small-molecule methylases (15, 16). In these conserved sequences the o represents a hydrophobic residue, j represents a small amino acid residue, and u stands for any amino acid residue. Motif I is involved in the AdoMet binding pocket, as revealed in the crystal structure of COMT (27). Motif II is not part of the active site, a fact that is confirmed in the crystal structure of COMT. Similar motifs also are found among the protein methyltransferases (15). A third motif GPPF (motif III) in MCT is very close to the (D/N/S)PP(Y/F) that is found among DNA N-6-methyl-transferases (17–20). Motif III is involved in the AdoMet binding pocket in the M. TaqI DNA methyltransferase (28).

DISCUSSION

Methyl chloride is the most abundant halohydrocarbon species in the upper atmosphere, and, although biological sources serve as a principle source of this volatile gas, the major biogenetic contributors remain to be established. In 1981, our laboratory (11, 12) demonstrated the biosynthesis of dibromomethane and tribromomethane via haloperoxidase-catalyzed reactions. In these reactions an electrophilic halogenating species first is formed. This electrophilic intermediate is capable of reacting with a broad spectrum of organic compounds and can produce a variety of halohydrocarbon species. However, in the haloperoxidase reactions, methyl chloride (the monohalomethane) was never detected. This led to a search for an alternative route for the biosynthesis of monohalomethanes. In 1990, Wousmaa and Hager (9) discovered a novel route for the biosynthesis of methyl halides (CH₃Cl, CH₃Br, and CH₃I). Methylases, capable of catalyzing the formation of methyl chloride were found in fungi, marine algae, and halophytic plants (9). In the methylase reaction, the halogen anion acts as a nucleophilic acceptor for the reaction with the electrophilic methyl group of S-adenosine-l-methionine. This discovery suggested that methylase activity in halophytic plants and marine algae may make a major contribution to the annual production of 5 million tons of methyl chloride (9).

The lack of catalytic activity of the Batis maritima enzyme in catalyzing the formation of methanethiol is in sharp contrast to the report that a purified methylase from cabbage has both MCT and methanethiol activity (25). The cabbage enzyme, an S-adenosyl-l-methionine:halide/bisulfide methyl transferase, has been purified to homogeneity from Brassica oleracea, and the halide and bisulfide activities copurify at a constant ratio throughout the purification procedure. Based on the low rates of methyl chloride synthesis and the high K_m for chloride, Attieh et al. (25) postulate that the cabbage enzyme functions in sulfur rather than halide metabolism. In addition, it has been reported that extracts of many diverse species of higher plants produce both halomethanes and methanethiol when supplied with an appropriate halide or with bisulfide (29). However, these studies were carried out with whole-plant extracts. Thus, in this latter case it is not clear whether a single enzyme or multiple enzymes are involved in product formation. Since the methylase of B. maritima lacks bisulfide methyltransferase activity, it appears reasonable to conclude that the halophytic methylase functions in halide metabolism.

An obvious function for a halophytic methylase would be the maintenance of homeostatic levels of cytoplasmic chloride ion. The secretion of excess chloride into the soil could not greatly benefit a halophytic plant. On the other hand, the synthesis and distillation of a volatile gas, methyl chloride, into the atmosphere could be a useful mechanism for disposing of excess chloride.

ABBREVIATIONS

MCT

methyl chloride transferase

AdoMet

S-adenosyl-L-methionine

MALDI

matrix-assisted laser desorption ionization

RACE

rapid amplification of cDNA ends

TFA

trifluoroacetic acid