Biochemical and molecular characterization of the homocysteine S-methyltransferase from broccoli ( Brassica oleracea var. italica)

• Published in: Phytochemistry (Oxford) 2007-04, Vol.68 (8), p.1112-1119
• Main Authors: Lyi, Sangbom M., Zhou, Xin, Kochian, Leon V., Li, Li
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Biological and medical sciences; Brassica - enzymology; Brassica - genetics; Brassica - metabolism; Brassica oleracea var. italica; Brassicaceae; Broccoli; Cadmium - pharmacology; Cloning, Molecular; complementary DNA; DNA, Complementary - chemistry; enzyme activity; Enzymes; Fundamental and applied biological sciences. Psychology; Gene expression; gene expression regulation; Gene Expression Regulation, Plant - drug effects; homocysteine; homocysteine S-methyltransferase; Homocysteine S-Methyltransferase - genetics; Homocysteine S-Methyltransferase - isolation & purification; Homocysteine S-Methyltransferase - metabolism; messenger RNA; Metabolism; methionine; Methionine - pharmacology; methyltransferases; molecular cloning; Molecular Sequence Data; Phylogeny; Plant physiology and development; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; RNA, Messenger - metabolism; S-methylmethionine; selenium; Selenium - pharmacology; selenocysteine methyltransferase; Sequence Alignment; SMM:homocysteine S-methyltransferase; Substrate Specificity; Sulfates - pharmacology; sulfur; Sulfur - metabolism; tissue distribution
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2007.02.007

Two cDNAs encoding proteins with homocysteine S-methyltransferase (HMT) activity were isolated from broccoli and functionally characterized. While one gene product exhibits only HMT activity, the other displayed both HMT and selenocysteine Se-methyltransferase (SMT) activities, indicating the gene has evolved to include an additional function in sulfur/selenium accumulating species. Plants are known for their unique ability to synthesize methionine from S-methylmethionine (SMM) and homocysteine using the enzyme SMM: homocysteine S-methyltransferase (HMT) in the SMM cycle. Two cDNAs exhibiting HMT activity were cloned from broccoli and functionally expressed in E. coli. One cDNA, that encodes an enzyme with high substrate specificity for homocysteine, was designated as BoHMT1. The other cDNA was the BoSMT gene that we previously characterized and encodes a selenocysteine methyltransferase (Lyi, S.M., Heller, L.I., Rutzke, M., Welch, R.M., Kochian, L.V., Li, L., 2005. Molecular and biochemical characterization of the selenocysteine Se-methyltransferase gene and Se-methylselenocysteine synthesis in broccoli. Plant Physiol. 138, 409–420). Both exist as single gene sequences in the broccoli genome. While BoSMT expression was extremely low or undetectable in broccoli plants unless the plants were exposed to selenium, the BoHMT1 mRNA accumulated in most tissues of the plant except older leaves. In contrast to BoSMT whose expression was dramatically upregulated by treating plants with selenate, the transcript levels of BoHMT1 were not markedly affected in plants exposed to selenium. BoHMT1 expression responded significantly to changes in plant sulfur status. However, its expression was not dramatically affected in plants treated with methionine, SMM, homocysteine, or the heavy metal, cadmium. The differences in the substrate specificity and gene expression in response to changes in plant sulfur and selenium status between BoHMT1 and BoSMT suggest that the enzymes encoded by these two genes play distinct roles in sulfur and selenium metabolism in broccoli.