Se-Adenosyl-l-selenomethionine Cofactor Analogue as a Reporter of Protein Methylation

Abstract

Posttranslational methylation by S-adenosyl-L-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-L-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM’s utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

“…Their study shows that the decomposition rate of SeAdoMet is 10‐fold higher than that of AdoMet, but the protein‐MTase‐catalyzed transalkylation reaction is only 3–5‐fold faster for the SeAdoMet analogue. Hence, the authors suggest that the protein‐MTase‐catalyzed reaction rates are not determined solely by the strength of the chalcogen–carbon bond, but are likely caused by other factors 28, 30. Interestingly, the study also suggests that the β‐sp 2 carbon atom, which is essential for activity with S‐alkyl AdoMet analogues, is likely not required for some protein MTases when Se‐alkyl AdoMet analogues are used 30…”

“…Comparative decay studies with the propargylic SeAdoYn ( 9 , Figure 1) and ProSAM ( 20 , Table S2) analogues showed that decomposition of the ProSAM compound first results in the hydrated product (keto byproduct 28 , Table S2), which further decomposes to the thioether. However, SeAdoYn follows a different mechanism and directly forms the selenoether 20h, 28. SeAdoYn is used as a substrate for transalkylation by a large variety of wild‐type MTases, which is in contrast with many of the AdoMet analogues with larger transferable groups, which are only active with mutated MTases 9b, 19a, 20h, 28, 29…”

“…However, SeAdoYn follows a different mechanism and directly forms the selenoether 20h, 28. SeAdoYn is used as a substrate for transalkylation by a large variety of wild‐type MTases, which is in contrast with many of the AdoMet analogues with larger transferable groups, which are only active with mutated MTases 9b, 19a, 20h, 28, 29…”

“…Enzyme‐mediated transalkylation reactions using an azido‐modified AdoMet analogue with engineered G9a and GLP121, 60 and an alkyne‐modified AdoMet analogue with engineered G9a20c and PRMT1 have been demonstrated 20g. Additionally, a selenium‐based SAM analogue with an alkyne linker was effectively employed in transalkyation reactions directed and catalyzed by the native protein MTases GLP1, G9a and SUV39H2 28, 60, 61…”

“…This approach has been used by the Luo group for the specific targeted capturing of proteins using MTase‐based labeling (Figure 3). 20a,20b, 28, 64 …”