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.

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.

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.