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.The aging process, as well as several human diseases, has been linked to the accumulation of spontaneously damaged biomolecules. Cells have evolved several ways of dealing with these altered molecules, including degradation, excretion, and repair pathways (1-6). The balance between the formation of age-altered molecules and the pathways that limit their cellular accumulation has been described as a battle between chemistry and biochemistry, where chemistry ultimately wins (2).Although 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. Of the large number of metabolites that are produced and used by biological systems, many are unstable, degrading into forms that may have reduced function or that may be toxic. One pathway of small molecule degradation and cellular recognition has been described recently. Here, trans-aconitate formed spontaneously from the citric acid cycle intermediate cis-aconitate results in the inhibition of at least two steps in the cycle (7, 8). trans-Aconitate is then recognized by a specific yeast methyltransferase; the methyl ester formed has reduced inhibitory properties (9).One of the crucial small molecule metabolites in all organisms is S-adenosyl-L-methionine (AdoMet) 2 (10 -12). Second to ATP, it is probably the most widely used cofactor in nature (12, 13). Not only does it serve as the primary methyl donor, but it also functions as an amino, adenosyl, and ribosyl donor (11). It also plays a role in the formation of adenosyl radicals (14) and as a precursor of polyamines (15). AdoMet has been shown to...

The biological methyl donor S-adenosyl-L-methionine (AdoMet) is spontaneously degraded by inversion of its sulfonium center to form the R,S diastereomer. Unlike its precursor, (S,S)-AdoMet, (R,S)-AdoMet has no known cellular function and may have some toxicity. Although the rate of (R,S)-AdoMet formation under physiological conditions is significant, it has not been detected at substantial levels in vivo in a wide range of organisms. These observations imply that there are mechanisms that either dispose of (R,S)-AdoMet or convert it back to (S,S)-AdoMet. Previously, we identified two homocysteine methyltransferases (Mht1 and Sam4) in yeast capable of recognizing and metabolizing (R,S)-AdoMet. We found similar activities in worms, plants, and flies. However, it was not established whether these activities could prevent R,S accumulation. In this work, we show that both the Mht1 and Sam4 enzymes are capable of preventing R,S accumulation in Saccharomyces cerevisiae grown to stationary phase; deletion of both genes results in significant (R,S)-AdoMet accumulation. To our knowledge, this is the first time that such an accumulation of (R,S)-AdoMet has been reported in any organism. We show that yeast cells can take up (R,S)-AdoMet from the medium using the same transporter (Sam3) used to import (S,S)-AdoMet. Our results suggest that yeast cells have evolved efficient mechanisms not only for dealing with the spontaneous intracellular generation of the (R,S)-AdoMet degradation product but for utilizing environmental sources as a nutrient.Aging can be seen as the accumulation of damaged biomolecules over time (1-3). As such, understanding the mechanisms by which organisms can slow such accumulation, as well as how these mechanisms may themselves eventually break down and fail, is crucial to an understanding of the aging process. Repair pathways for damaged DNA have been well established (4); damaged proteins can be removed by a combination of proteolytic and repair pathways (3,(5)(6)(7)(8). However, we only are beginning to understand how cells can prevent the accumulation of damaged small molecules.To date, there are only a few pathways known for metabolizing damaged or unwanted small molecules. trans-Aconitate, the spontaneous degradation product of the citric acid cycle intermediate cis-aconitate, can be detoxified by the action of a specific methyltransferase (9). L-2-Hydroxyglutarate is formed as an abnormal byproduct when L-malate dehydrogenase uses ␣-ketoglutarate rather than oxalacetate as a substrate. The accumulation of the toxic L-2-hydroxyglutarate product is prevented, however, by the action of an enzyme that converts it back to ␣-ketoglutarate.

Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the “affinity maturation” step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold.

The biological methyl donor S-adenosyl-l-methionine [(S,S)-AdoMet] can spontaneously break down under physiological conditions to form the inactive diastereomer (R,S)-AdoMet, which may interfere with cell function. Although several lower organisms metabolize (R,S)-AdoMet via homocysteine methyltransferases, it is unclear how mammals deal with it. In this paper, we show that the mouse liver extracts, containing the BHMT-2 homocysteine methyltransferase candidate for a similar activity, recognizes (S,S)-AdoMet but not (R,S)-AdoMet. We find no evidence for the enzymatic breakdown of (R,S)-AdoMet in these extracts. Thus, mammals may metabolize (R,S)-AdoMet using a different strategy than other organisms.