Characterization and Functional Expression of cDNAs Encoding Methionine-sensitive and -insensitive HomocysteineS-Methyltransferases fromArabidopsis

Philippe Ranocha, Fabienne Bourgis, Michael J. Ziemak, David Rhodes, Douglas A. Gage, Andrew D. Hanson
2000 Journal of Biological Chemistry  
Plants synthesize S-methylmethionine (SMM) from Sadenosylmethionine (AdoMet), and methionine (Met) by a unique reaction and, like other organisms, use SMM as a methyl donor for Met synthesis from homocysteine (Hcy). These reactions comprise the SMM cycle. Two Arabidopsis cDNAs specifying enzymes that mediate the SMM 3 Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by homology and authenticated by complementing an Escherichia coli yagD mutant and by detecting HMT activity in
more » ... HMT activity in complemented cells. Gel blot analyses indicate that these enzymes, AtHMT-1 and -2, are encoded by single copy genes. The deduced polypeptides are similar in size (36 kDa), share a zincbinding motif, lack obvious targeting sequences, and are 55% identical to each other. The recombinant enzymes exist as monomers. AtHMT-1 and -2 both utilize L-SMM or (S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM. Both enzymes also use either methyl donor in vivo because both restore the ability to utilize AdoMet or SMM to a yeast HMT mutant. However, AtHMT-1 is strongly inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to the control of flux through the HMT reaction and the SMM cycle. Plant HMT is known to transfer the pro-R methyl group of SMM. This enabled us to use recombinant AtHMT-1 to establish that the other enzyme of the SMM cycle, AdoMet:Met S-methyltransferase, introduces the pro-S methyl group. These opposing stereoselectivities suggest a way to measure in vivo flux through the SMM cycle. Unlike other organisms, plants synthesize L-S-methylmethionine (SMM) 1 from Met and S-adenosylmethionine (AdoMet) in
doi:10.1074/jbc.m001116200 pmid:10747987 fatcat:pix2e3e2g5echhmqgndfq7ppm4