Characterization and Functional Expression of cDNAs Encoding Methionine-sensitive and -insensitive Homocysteine S-Methyltransferases from Arabidopsis

Abstract

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 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.

Dynamics of methionine biosynthesis

“…Two methionine molecules are then regenerated from SMM by transfer of a methyl group from SMM to homocysteine. This second reaction is catalysed by homocysteine S-methytransferase (EC 2.1.1.10) (Giovanelli et al 1980;Ranocha et al 2000). SMM was identified as a major component in the phloem of several higher plant species (Bourgis et al 1999), suggesting that it has a role in the transport of methionine in plant tissues.…”

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

“…At5g49810, a non-essential gene encodes methionine methyltransferase (EC 2.1.1.12) in A. thaliana (Tagmount et al, 2002;Kocsis et al, 2003). Extensive biochemical characterization, including in vitro enzyme assays and complementation of S. cerevisiae and E. coli mutations (Ranocha et al, 2000;Ranocha et al, 2001), demonstrated that homocysteine methyltransferase (EC 2.1.1.10) is encoded by three A. thaliana genes, At3g25900 (HMT1), At3g63250 (HMT2), and At3g22740 (HMT3). Whereas HMT1 is feedback-inhibited by methionine, HMT2 and HMT3 are not.…”

“…Whereas homocysteine methyltransferase forms two molecules of methionine from S-methylmethionine and homocysteine, methionine methyltransferase uses S-adenosylmethionine as a methyl group donor to form S-methylmethionine from methionine. Although homocysteine methyltransferase is also present in bacteria, fungi, and animals, methionine methyltransferase has been found only in plants (Ranocha et al, 2000). At5g49810, a non-essential gene encodes methionine methyltransferase (EC 2.1.1.12) in A. thaliana (Tagmount et al, 2002;Kocsis et al, 2003).…”

“…Whereas HMT1 is feedback-inhibited by methionine, HMT2 and HMT3 are not. All three A. thaliana HMT enzymes use (S,S)-S-adenosylmethionine as a methyl group donor much less efficiently than S-methylmethionine (Ranocha et al, 2000;Ranocha et al, 2001). However, in natural systems, biologically active (S,S)-S-adenosylmethionine can spontaneously racemize at the sulfonium ion to form toxic (R,S)-S-adenosylmethionine (de la Haba et al, 1959), which is removed via a salvage pathway.…”

Selenium Metabolism in Plants: Molecular Approaches

“…It shares significant sequence homology with homocysteine Smethyltransferases (HMT). Despite the fact that both SMT and HMT enzymes catalyze methyl transfer using S-methylmethionine as the methyl donor, they exhibit significant Secontaining (for SMT) and S-containing (for HMT) substrate choice as a methyl acceptor in vitro (Neuhierl and Bock, 1996;Ranocha et al, 2000). SMT was found to be constitutively expressed in roots and leaves of A. bisulcatus, and appear to be not affected by Se induction (Pickering et al, 2003).…”