(Last modification: 05.
Apr. 2009) A new type of S-Methyltransferase (SMT) Publication: Coiner et al., 2006 In our studies we found a cDNA which by homology to known OMTs clearly belonged to the
type 1 OMT family. We expressed a recombinant protein in E. coli and tested all sorts of hydroxyl compounds that might have been substrates, but no activity was detectable. However, some unusual properties made us suspect that it might be capable of methylating DTT, and actually that could be confirmed. It was definitely a surprise, however, that the methylation was not at a hydroxyl group, but at a sulfhydryl group (R-SH zu R-S-CH3) !!!
Its identification raises an interesting question: Are there other S-methyltransferases hidden in the large family of proteins usually considered to be
type 1 O-methyltransferases? Or even enzymes with still other methyl group acceptors?
Yes there are: a N-methyltransferase
(more...), and another case, early 2008:
more... Pages describing the most interesting results The first evidence for S-methyltransferase (SMT) activity: more... Position of the SMT in a relationship tree of
type 1 OMTs: more...(from
end of 2005, PDF-file, 70 KB)
-> and here is a more up to date version of a relationship tree:
more...(PDF-File, about 45 KB)
The C. roseus SMT (CrSMT1) contains all residues typical for
type 1 O-methyltransferases:
more... Substrate preferences of the S-methyltransferase: more... Comparison of a CrSMT1 model with the 3D structure of a caffeic acid OMT (COMT): more...
In this context: artificial SMT activity of an OMT Based on our results, the possibility of DTT methylation was subsequently tested with an enzyme cloned from R. graveolens. This was a bona fide OMT, with methoxyphenols as best substrates (Burga et al., 2005). Indeed, that enzyme was also capable of methylating DTT, and the methyl transfer was again to one of the sulfhydryl groups! However, this activity was not noticeable under standard assay conditions, and it required the addition of zinc. All of the evidence indicated that DTT in this case had to be considered as a pseudosubstrate under artificial in vitro conditions. The effect of zinc is actually quite interesting: zinc is in general a powerful inhibitor of all O-methyltransferase (OMT) reactions. Something special happens in the case of DTT: Many people are not aware of that, but DTT is a zinc chelator. The activity of that enzyme with DTT actually required stoichiometric relations of DTT and zinc. This clearly indicated: a) DTT complexed the zinc (thus blocking the inhibitory effect of the metal), and b) it is the DTT/zinc complex that is the substrate of the OMT, presumably because the complexation increased the ratio between R-S- and R-SH in DTT, i.e. the number of the reactive species for the methyl transfer! Return to page top
References Coiner, H., Schröder, G., Wehinger, E., Liu, C. J., Noel, J. P., Schwab, W. and Schröder, J.:
Methylation of sulfhydryl groups: a new function for a family of small molecule plant O-methyltransferases. Plant Journal 46, 193-205 (2006), available online: 22.02.2006
In plants, type I and II SAM-dependent O-methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology based RT-PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56-58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated DTT, the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol (Km 220 µM) and furfuryl thiol (Km 60 µM) were the best substrates (6-7 fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least fivefold lower than with thiols. The enzyme was named CrSMT1 (C. roseus S-methyltransferase 1). Models based on the COMT crystal structure suggest that S-methylation is mechanistically identical to O-methylation. CrSMT1 is the first recognized example of an S-methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a SMT. Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups.
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Burga, L., Wellmann, F., Lukacin, R., Schwab, W., Schröder, J., Matern, U.: Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L. Archives of Biochemistry and Biophysics
440, 54-64 (2005)
A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-L-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 µM for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 µM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols.
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