|
(Last modification:
21. Mar. 2008)
Cycle of Active Methyl Groups and AdoMet Decarboxylase
(Supported by grants from the Deutsche Forschungsgemeinschaft to Gudrun and Joachim Schröder)
Organisation of this page
Abbreviations:
There are two different commonly used abbreviations of
S-adenosyl-L-methionine;
this is pretty confusing, I admit:
-> S-Adenosyl-L-Methionine =
AdoMet (Ado = adenosyl residue)
->
S-Adenosyl-L-Methionine =
SAM
The same applies to S-adenosyl-L-homocysteine:
->
S-Adenosyl-L-Homocysteine =
AdoHcy (Hcy = homocysteine)
->
S-Adenosyl-L-Homocysteine =
SAH
Enzymes and reactions
A large number of substances are methylated in all organisms by transfer of a methyl group from AdoMet (SAM) [MetSyn is an interesting exception, see below], e.g. nucleic acids, proteins, various small metabolites, and especially in plants: cell wall components and secondary metabolites. Disturbances in methylation processes have serious consequences for the organisms.
The cycle of active methyl groups provides, utilizes, and regenerates the supply of AdoMet (= SAM). Several enzymes are involved:
-
S-Adenosyl-L-Methionine Synthetase (AdoMetSyn, SAM-Syn): synthesizes AdoMet from methionine and ATP.
-
Methyltransferases: They transfer the methyl groups to acceptor molecules,
-
S-Adenosyl-L-Homocysteine Hydrolase
(AdoHcyHy, SAH-hydrolase): cleaves the demethylated AdoMet [= AdoHcy = SAH] into homocysteine and adenosine.
-
Methionine Synthase (MetSyn): re-methylates homocysteine to methionine, with 5-methyltetrahydrofolate as methyl group donor.
-
S-Adenosyl-L-Methionine-Decarboxylase (AdoMetDC): Is not directly part of the cycle, but is very important for the biosynthesis of polyamines, e.g. spermidine and spermine
(more...).
We have cloned all these enzymes from Catharanthus roseus and investigated the molecular biology, the regulation, and the properties of the enzymes after heterologous expression.
Simple overview of the methyl cycle, its enzymes, and the branch-off to spermidine biosynthesis.
The enzymes: AdoMetSyn = S-Adenosyl-L-Methionine Synthetase;
AdoHcyHy = S-Adenosyl-L-Homocysteine Hydrolase;
MetSyn = Methioninsynthase (THF = Tetrahydrofolate);
AdoMetDC = S-Adenosyl-L-Methionin-Decarboxylase
Page top
Our publications in this field
AdoMet-Synthetase
(AdoMetSyn)
This enzyme activity raises an interesting question: why does
C. roseus (and also many other plants) contain three closely related isoenzymes with different regulation of expression? The three proteins expressed in
E. coli have no detectable differences in their properties.
Take a look at the work:
-
Schröder, G., Eichel, J.,
Breinig, S. and Schröder, J.: Three differentially expressed
S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization.
Plant Molecular Biology 33, 211-222 (1997).
We describe the molecular and functional characterization of three closely related
S-adenosyl-L-methionine synthetase (here abbreviated as SAMS; otherwise often as AdoMetSyn) isozymes from
Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl). Seedlings revealed organ-specific expression and a differential gene response after salt stress. A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid differences. SAMS1 and SAMS2 are of type I and SAMS3 is of type II. A possible functional role of the exchanges was investigated with the individual enzymes expressed in
Escherichia coli. No significant differences were detected in a) optima for temperature (37 to 45oC) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, NH4+, Na+); c) Kms for ATP and L-methionine; d) inhibition by reaction products (SAM, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography). The results represent the first characterization of plant SAMS isozyme properties.
Accession for DNA sequences: SAMS1, SAMS2, SAMS3
Request a reprint
Page top
Methyltransferases
See the pages on O-Methyltransferases (more...) and
S-Methyltransferases (more...).
S-Adenosyl-L-Homocysteine Hydrolase (AdoHcyHy, SAH-Hydrolase)
The enzyme is one of the interesting proteins requiring NAD+ as 'catalytic cofactor'.
-
Schröder, G., Waitz, A., Hotze, M. and Schröder, J.: cDNA for S-adenosyl-L-homocysteine hydrolase from
Catharanthus roseus. Plant Physiology 104, 1099-1100 (1994).
This short publication describes the full-length cDNA and some of the properties of the protein. The enzyme is one of the interesting proteins requiring NAD+ as 'catalytic' cofactor.
Accession: Z26881
Request a reprint
Methionine synthase (MetSyn)
This enzyme is of particular interest, because nature evolved two entirely different types of 5-methyltetrahydrofolate-dependent enzymes for this reaction: a) vitamin B12-dependent, and b) vitamin B12-independent.
-> We isolated and characterized the first MetSyn cDNA from a plant, and demonstrated with the protein expressed in
E. coli that the enzyme is indeed B12-independent (Eichel
et al., 1995), consistent with the widely unknown fact that plants cannot synthesize and do not contain vitamin B12. Recent studies actually indicate that
children of strict vegetarians may seriously suffer from vitamin B12 deficiency.
-> Much work has been done on the human enzyme (cobalamin-dependent) and the two proteins in
E. coli (contains both forms!), but little had been carried out on the properties of the plant MetSyn: the few available data were mostly about 30 years old, and had simply been repeated for decades in reviews. This is why we carried out a detailed study on the biochemical properties of the plant enzyme: Eckermann
et al. 2000, see below. Now we have 2005, but these data are still uptodate because nothing new or comparable was published in the last five years. The work is not only a critical re-investigation of the previous data, but also reports new and interesting findings: the plant enzyme contains zinc, like the bacterial and animal proteins! The paper also reports some data on the tissue-specific expression of the enzyme.
Thanks for invaluable help: We would like to stress here that the enzymatic characterization of the plant methionine synthase was only possible because of the generous help by
Rowena Matthews at the University of Michigan (USA).
-
Eichel, J., Gonzįlez, J.C., Hotze, M., Matthews, R.G. and Schröder, J.: Vitamin B12-independent L-methionine synthase from a higher plant (Catharanthus
roseus): molecular characterization, regulation, heterologous expression, and enzyme properties.
European Journal of Biochemistry 230, 1053-1058 (1995).
Accession: X83499
Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in L-methionine biosynthesis, and it also serves to regenerate the methyl group of S-adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant
Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50% identity with the cobalamin-independent methionine synthase from
Escherichia coli (MetE) and 41% identity with a partial sequence of a yeast homolog of MetE. The
C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require S-adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14). In contrast to the
E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plant extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the
C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.
Request a reprint
-
Eckermann, C., Eichel, J. and Schröder, J.
: Plant methionine synthase: new insights into properties and expression.
Biological Chemistry 381, 695-703 (2000) .
We investigated methionine synthase (MSY) in
Catharanthus roseus. The properties were characterized with purified protein isolated either from plant cell cultures or after heterologous expression in
Escherichia coli. The protein was a monomer and accepted both the triglutamate (CH3-H4PteGlu3, apparent Km 80 µM) and the monoglutamate (CH3-H4PteGlu1, apparent Km 350 µM) of methyl-5,6,7,8-tetrahydropteroate as methyl donor, with a ratio of approximately 90 : 1 in favor of the triglutamate. Both activities required inorganic phosphate, but with different kinetics, and both were dependent on reducing agents. The activity required
zinc, as shown by depletion and reconstitution experiments. Mg2+
had no effect on the activity. Two MSY isoforms purified from parsley cell cultures revealed the same properties as the
C. roseus enzyme, but the parsley proteins had no detectable activity with the monoglutamate substrate. The second part of the work compared the expression of the three enzymes of the methyl cycle (MSY, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase). In cell cultures, all three enzymes were present under all conditions investigated, with small changes at the protein level and more pronounced changes at the RNA level. Studies with seedlings revealed a low expression of all three enzymes in cotyledons, when compared to hypocotyls and radiculas. Immunohistochemical experiments indicated that MSY expression in cotyledons is cell-type specific, with the strongest signals detected in the upper epidermis.
Request a reprint
Other interesting findings on cobalamin-independent MetSyn:
-
The crystal structures of the B12-independent methionine synthase from
Arabidopsis
(Ferrer et al., 2004) and from a bacterium (Pejchal and Ludwig, 2005) have been published. Some fascinating questions remain, however:
More...
-
Plastid-located enzyme. There was a long-standing puzzle with respect to the biosynthesis of methionine: all of the enzymes were located to the plastids (chloroplasts), except for the final enzyme, the methionine synthase. Off and on there have been claims that there is MetSyn activity in plastids. This would really make sense because it would separate the
de novo biosynthesis from the regeneration, but the evidence was not very convincing. Recent data, however, are much more convincing: the plastids of
Arabidopsis contain a MetSyn isoform that apparently is responsible specifically for the last step in the
de novo biosynthesis:
Ravanel et al. (2004)!
Page top
S-Adenosyl-L-Methionine decarboxylase (AdoMetDC)
AdoMet is also used by the enzyme that catalyzes the rate-limiting step in the biosynthesis of the polyamines spermidine and spermine: these are essential cell components in all higher organisms, and in plants they are suspected to possess hormone properties. The protein is interesting because it is synthesized as proenzyme which autocatalytically processes itself into the active enzyme by cleavage at a serine residue, creating from the serine a pyruvate residue that is the active site of the enzyme (pyruvoyl enzyme). We cloned the plant cDNA from
C. roseus, expressed the protein in E. coli, and identified by site-directed mutagenesis the serine producing the pyruvate residue (Schröder and Schröder, 1995, see below).
-> An interesting feature of the plant AdoMetDC mRNA is that its very long 5'-untranslated leader contains an open reading frame (uORF) for a highly conserved peptide of 51 aminoacids:
more...
-
Schröder, G. and Schröder, J.: cDNAs for
S-adenosyl-L-methionine decarboxylase from Catharanthus roseus, heterologous expression, identification of the proenzyme processing site, evidence for the presence of both subunits in the active enzyme, and a conserved region in the 5' mRNA leader.
European Journal of Biochemistry 228, 74-78 (1995).
S-Adenosyl-L-methionine decarboxylases (AdoMetDC) are pyruvoyl-dependent enzymes producing the aminopropyl group for spermidine biosynthesis, and this reaction is the rate-limiting step in polyamine biosynthesis. We characterized cDNAs from
Catharanthus roseus (Madagascar periwinkle) and investigated the enzyme after heterologous expression. The largest cDNA (1864 bp) had an 5' leader of 469 bp and encoded a protein of 357 residues and 30-35% identity with mammalian AdoMetDC. The proenzyme expressed in
Escherichia coli was processed into active enzyme, and the processing site was identified by site-directed mutagenesis as the second Ser in the sequence Leu-Ser-Glu-Ser-Ser. The analysis of affinity-purified proteins indicated that the active enzyme contained both subunits. The Km for
S-adenosyl-L-methionine was 35-40 µM, and the enzyme activity was not stimulated by putrescine. The 5' leader of the mRNA contained start and stop codons for a polypeptide of 51 amino acids, and this region was conserved in the 5' leaders of other plant AdoMetDC mRNAs. The putative polypeptide had no similarity with the hexapeptide responsible for modulation of AdoMetDC mRNA translation in mammals. The possibility is discussed that plants evolved a different type of translational regulation.
Accession: U12573
more...
Reprint request
Page top
Return to Overview of
Catharanthus roseus projects
Visits since March 10, 2007:
Idaho Web Design Tools |
