Recognition of Age-damaged (R,S)-Adenosyl-L-methionine by Two Methyltransferases in the Yeast Saccharomyces cerevisiae

Chris R Vinci,Steven G Clarke

doi:10.1074/jbc.m610029200

Abstract

The biological methyl donor S-adenosylmethionine (AdoMet) can exist in two diastereoisomeric states with respect to its sulfonium ion. The S configuration, (S,S)-AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the R form, producing (R,S)-AdoMet. As of yet, (R,S)-AdoMet has no known physiological function and may inhibit cellular reactions. In this study, we found two Saccharomyces cerevisiae enzymes that are capable of recognizing (R,S)-AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, identified previously as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine, respectively. We found here that Sam4 recognizes both (S,S)- and (R,S)-AdoMet, but that its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet, whereas no activity is seen with the S,S form. R,S-Specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)-AdoMet in these organisms.

Highlights

Enzymes that recognize damaged DNA [3] and proteins [1, 2, 5] have been well characterized, this is not yet the case for spontaneously altered small molecules
We demonstrate that Sam4 and Mht1 in S. cerevisiae are both capable of using (R,S)-AdoMet as a methyl donor
Our data suggest that Sam4 and Mht1 are responsible for the (R,S)-AdoMet-dependent homocysteine methyltransferase activity seen in S. cerevisiae and that Sam4 is responsible for the activity with (S,S)-AdoMet

Summary

EXPERIMENTAL PROCEDURES

Preparation and Purification of (R,S)- and (S,S)-[3H]AdoMet— S-Adenosyl-L-[methyl-3H]methionine (referred to throughout as [3H]AdoMet; 1 mCi/ml, 79.0 Ci/mmol, in dilute HCl/ethanol (9:1, v/v) at pH 2–2.5; GE Healthcare) was diluted 10-fold in 0.1 M HCl and divided into two aliquots. An aliquot (100 ␮l) of each preparation was fractionated with a cationexchange HPLC column (Whatman Partisil SCX, 10-␮m bead diameter, 4.6 mm, inner diameter, ϫ 250 mm) using a method similar to that described previously [18]. The column was equilibrated and eluted with 80% buffer A (0.7 ml of concentrated NH4OH added to 1000 ml of 20% acetonitrile in H2O and brought to pH 3.0 with 88% acetic acid) and 20% buffer B (buffer A adjusted to 50 mM (NH4)2SO4 and to pH 3.0 with concentrated sulfuric acid). The column was eluted at room temperature at a flow rate of 1 ml/min. The fractions corresponding to the R,S peak of the incubated sample and to the S,S peak of the non-incubated sample were each

SGDPa SGDPa SGDPa SGDPa SGDPa

RESULTS

DISCUSSION

Methyl donor