Biochemical characterization of a novel halide/bisulfide methyltransferase purified from Brassica oleracea L.
Abstract (summary)
Biogenic emissions of halomethanes and reduced sulfur gases are important because of the impact of these gases on the global environment; halomethanes affect the integrity of stratospheric ozone and the sulfur gases contribute to the formation of acid precipitation. Although these effects have been well documented, the biosynthetic pathways leading to the formation of these gases are still the subject of much scientific debate. The results reported in this thesis advanced the previous findings through purification of several isoforms of the enzyme to homogeneity, determination of the subcellular localization of the enzyme, studying its substrate specificity, and placing it within a physiological context.
The enzyme was originally named as S-adenosyl- L-methionine:halide/bisulfide methyltransferase (H/BMT) (EC 2.1.1.-), because it catalyzed the methylation of iodide, bromide, and chloride to the respective monohalomethanes, and of bisulfide to methanethiol. H/BMT was purified from leaves of red cabbage through (NH4)2SO4 precipitation, followed by a combination of conventional and high performance chromatography steps, including gel filtration, affinity chromatography, and anion exchange. Anion exchange chromatography resolved multiple peaks of H/BMT activity suggesting the presence of isoforms, of which the first isoform was initially obtained in homogeneous form. Subsequently, a total of five isoforms of H/BMT were purified to homogeneity from the same tissue using a variation of the above protocol. The proteins had distinct molecular masses, ranging from 26 to 31 kDa, and all functioned as monomers. Characterization of the three most abundant proteins indicated that they had different pH optima, covering the pH range from 5 to 9. Substrate interaction studies for the three isoforms indicated that they shared a sequential substrate binding mechanism.
The results of this study show that halide and bisulfide methylations are indeed catalyzed at the same active site of a single enzyme, H/BMT, but that the natural role of the enzyme is unlikely to be halide methylation. Instead, weight of the evidence indicates that H/BMT is a cytosolic thiol methyltransferase that is probably involved in the detoxification of reactive thiol groups produced upon glucosinolate degradation. The methylation of thiocyanate and of bisulfide by H/BMT strongly suggests that this enzyme is part of the defense machinery in glucosinolate-containing plants. (Abstract shortened by UMI.)
Indexing (details)
Environmental science;
Biochemistry
0487: Biochemistry
0479: Plant sciences