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.

Figure 1

Construction of plasmid pETMCT. The expression plasmid pETMCT was prepared by inserting the NdeI/BamHI-digested PCR-amplified MCT gene into pET 22b (Novagen) that also had been digested with NdeI/BamHI.

Figure 2

SDS/20% PAGE analysis of recombinant MCT. (Lane 1) Protein molecular mass markers (Novex) in kDa. (Lane 2) Total cellular proteins. (Lane 3) Recombinant MCT after separation from the soluble fraction and overnight dialysis. (Lane 4) Soluble fraction of cellular proteins.

Figure 3

The effect of salt on the activity of recombinant (R) and wild-type (WT) MCT and malate dehydrogenases from A. spongiosa (AS) and Halobacteriaum spp. (HB). The methyl iodide assay was used for analyzing the activity of native MCT, and the methyl chloride assay was used for recombinant MCT. The data for the MDH activity of A. spongiosa (9) is replotted from the original data by using the maximum activity obtained at 0.05 M NaCl as 100% activity. The data for the Halobacterium MDH (10) is replotted from the original by using the results from 0–2M NaCl.

Figure 4

The nucleotide sequence of MCT genomic DNA. Nucleotide position 1 is assigned to the A of the ATG initiation codon. The deduced amino acid sequence for each exon is indicated. The intron sequences are underlinded.