Characterization and functional expression of cDNAs encoding methionine-sensitive and -insensitive homocysteine S-methyltransferases from Arabidopsis.
Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (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 --> 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 zinc-binding 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.
Ranocha
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P
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA.
Bourgis
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F
Ziemak
M J
MJ
Rhodes
D
D
Gage
D A
DA
Hanson
A D
AD
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D050904:Homocysteine S-Methyltransferase; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D013058:Mass Spectrometry; D008715:Methionine / Q000494:pharmacology; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D008715:Methionine; D008780:Methyltransferases; D050904:Homocysteine S-Methyltransferase; C494408:mmuM protein, E coli; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase