Insertional Inactivation of the Methionine S-Methyltransferase Gene Eliminates the S-Methylmethionine Cycle and Increases the Methylation Ratio

Abstract

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met(Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.S-Methyl-Met (SMM) synthesis is a unique feature of plant sulfur and one-carbon metabolism (Pokorny et al., 1970; Mudd and Datko, 1990; Ranocha et al., 2001). SMM is formed by the S-adenosyl-Met (AdoMet)-dependent methylation of Met, catalyzed by Met S-methyltransferase (MMT; Bourgis et al., 1999). SMM can be reconverted to Met by transferring a methyl group to homo-Cys in a reaction mediated by homo-Cys S-methyltransferase (HMT; Ranocha et al., 2000). The tandem action of MMT and HMT, together with that of Ado-Met synthetase and S-adenoyslhomo-Cys (AdoHcy) hydrolase, sets up a futile cycle (the SMM cycle) in which Met is converted to SMM, and SMM is reconverted to Met (Mudd and Datko, 1990). This cycle in effect short-circuits the activated methyl cycle (Fig. 1), and each of its turns hydrolyzes a molecule of ATP to adenosine, pyrophosphate, and phosphate. The SMM cycle operates throughout the plant, and consumes one-half the Ado-Met produced in Arabidopsis leaves (Ranocha et al., 2001).The functions of SMM and its seemingly wasteful cycle are for the most part unknown. The only established role of SMM is in transporting reduced sulfur in the phloem, for which there is qualitative evidence in a range of plants including Arabidopsis (Bourgis et al., 1999 Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

Improving the Content of Essential Amino Acids in Crop Plants: Goals and Opportunities

“…SMM is synthesized from Met by Met S-methyltransferase (MMT) and is recycled back to Met by homo-Cys methyltransferase. However, despite the efficient transport capability of SMM, Arabidopsis and maize mmt mutants, which are unable to synthesize SMM, grow and reproduce normally, suggesting a minimal regulatory role for SMM in sulfur transport of at least these two species (Kocsis et al, 2003). Notwithstanding, a recent report (Lee et al, 2008) showed that a mutant Arabidopsis plant overaccumulating Met in its seeds is due to a mutation eliminating the activity of HMT2, one of the three Arabidopsis HMT isozymes that recycle SMM into Met (Lee et al, 2008).…”

BRANCHED-CHAIN AMINOTRANSFERASE4 Is Part of the Chain Elongation Pathway in the Biosynthesis of Methionine-Derived Glucosinolates inArabidopsis

“…Here, the flux into Met metabolism has not or has only inefficiently been shut down, resulting in an enhanced generation of SMM, which is undetectable in seeds of wild-type plants (Bourgis et al, 1999). This might also keep AdoMet levels low (Kocsis et al, 2003), which is also required for SMM formation explaining the almost unaffected Thr level. The latter might also be kept low by the increased Ile formation.…”

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

“…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). 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).…”