Expression of Batis maritima methyl chloride transferase in Escherichia coli

Abstract

Methyl chloride transferase, a novel enzyme found in several fungi, marine algae, and halophytic plants, is a biological catalyst responsible for the production of atmospheric methyl chloride. A previous paper reports the purification of this methylase from Batis maritima and the isolation of a cDNA clone of the gene for this enzyme. In this paper, we describe the isolation of a genomic clone of the methylase gene and the expression of recombinant methyl chloride transferase in Escherichia coli and compare the kinetic behavior of the wild-type and recombinant enzyme. The recombinant enzyme is active and promotes the production of methyl chloride by E. coli under in vivo conditions. The kinetic data indicate that the recombinant and wild-type enzymes have similar halide (Cl ؊ , Br ؊ , and I ؊ )-binding capacities. Both the recombinant and wild-type enzymes were found to function well in high NaCl concentrations. This high salt tolerance resembles the activity of halobacterial enzymes rather than halophytic plant enzymes. These findings support the hypothesis that this enzyme functions in the control and regulation of the internal concentration of chloride ions in halophytic plant cells.It is estimated that methyl chloride, the most abundant halohydrocarbon species in the upper atmosphere, is produced at a level of 5 ϫ 10 6 tons per year (1). The biological production of CH 3 Cl and other volatile halogenated organic compounds in ocean waters has been commonly viewed as one of the major source of chlorinated halohydrocarbons in the upper atmosphere (2). In 1990, Wuosmaa and Hager (3) discovered the presence of methyl chloride transferase (MCT) in fungi, marine algae, and a halophytic plant. MCT catalyzes the conversion of chloride to methyl chloride by using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Wuosmaa and Hager showed that cell-free extracts of Phellinus pomaceus (a white rot fungus), Endocladia muricata (a marine red algae), and Mesembryanthemum crystallium (ice plant, a halophytic plant) contain MCT activity. Wuosmaa (4) also found that 20 of 31 marine algae collected along the Pacific coastal water near Monterey, CA produced methyl chloride. The production of methyl choride by halophytic plants suggests that halophytic plants may be an additional major source of atmospheric methyl chloride (3, 4). Methyl chloride and other halogenated methanes pollute the atmosphere (5) and affect the integrity of the stratospheric ozone layer (6).Previous work led to the isolation of a homogenous MCT preparation and allowed the cDNA cloning of the MCT gene (7). The isolated native enzyme has a M r of 22,434 as determined by matrix-assisted laser-desorption ionization (MALDI) MS, whereas the ORF in the cDNA clone has 230 codons coding for an enzyme having a calculated M r of 25,761. Thus, there is a Ϸ3.3-kDa difference in the molecular masses of the isolated enzyme and the deduced amino acid sequence.It is unclear whether this difference is due to proteolytic cleavage during isolation of the native enz...

Rice OsHOL1 and OsHOL2 proteins have S-adenosyl-L-methionine-dependent methyltransferase activities toward iodide ions

“…Plant proteins in which amino acid sequences showed high similarities to Arabidopsis HOL were classi ed as HOL family proteins, including independently characterized methyl chloride transferase (MCT) Hager 1998, 1999), thiol methyltransferase (TMT) (Attieh et al , 2000(Attieh et al , 2002, and halide/thiol methyltransferase (HTMT) (Itoh et al 2009). MCT, TMT, and HTMT proteins are also shown to possess SAM-dependent methyltransferase activity toward not only iodide ions, but also toward ions such as chloride, bromide, thiocyanate, and hydrosul de (Ni and Hager 1999;Attieh et al 1995Attieh et al , 2000Itoh et al 2009). All kinetic analyses of plant HOL family proteins reported to date have been performed using proteins from Brassicales plants.…”

“…MCT, TMT, and HTMT proteins are also shown to possess SAM-dependent methyltransferase activity toward not only iodide ions, but also toward ions such as chloride, bromide, thiocyanate, and hydrosul de (Ni and Short Communication DOI: 10.5511/plantbiotechnology.12.0207a Abbreviations: GC-MS, Gas chromatography-mass spectrometry; HPLC, High performance liquid chromatography; MALDI-TOF-MS, Matrixassisted laser desorption/ionization time-of-ight mass spectrometry; SAH, S-Adenosyl-L-homocysteine; SAM, S-Adenosyl-L-methionine; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1999;Attieh et al 1995Attieh et al , 2000Itoh et al 2009). All kinetic analyses of plant HOL family proteins reported to date have been performed using proteins from Brassicales plants.…”

Arabidopsis HARMLESS TO OZONE LAYER Protein Methylates a Glucosinolate Breakdown Product and Functions in Resistance to Pseudomonas syringae pv. maculicola

“…As high concentrations of ions such as Cl Ϫ are often detrimental to plants, halophytic plants are considered to possess various salt tolerance mechanisms. MCT was hypothesized to control and regulate the internal concentration of Cl Ϫ , rich in the habitat in which halophytic plant grows (10,11).…”

Characterization of three halide methyltransferases in Arabidopsis thaliana

“…The hypotheses concerning the physiological meaning of these enzymes include their involvement in salt tolerance (Ni and Hager 1999) or in metabolizing glucosinolate hydrolysis products such as thiols and thiocyanate (Attieh et al 2000a). The AtHOL1 gene was isolated from Arabidopsis as the MCT homolog, and the T-DNA insertion mutant analyses revealed that AtHOL1 was involved in the synthesis of methyl halides including methyl chloride, methyl bromide, and methyl iodide (Rhew et al 2003).…”