Jump to main content
Jump to site search

Issue 11, 2011
Previous Article Next Article

Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors

Author affiliations

Abstract

Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z′ values of 0.83–0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8 × 5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM, are inert toward the panel of human PMTs. These findings therefore provide the useful structure–activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues.

Graphical abstract: Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors

Back to tab navigation

Publication details

The article was received on 09 Jun 2011, accepted on 02 Aug 2011 and first published on 24 Aug 2011


Article type: Paper
DOI: 10.1039/C1MB05230F
Citation: Mol. BioSyst., 2011,7, 2970-2981
  •   Request permissions

    Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors

    R. Wang, G. Ibáñez, K. Islam, W. Zheng, G. Blum, C. Sengelaub and M. Luo, Mol. BioSyst., 2011, 7, 2970
    DOI: 10.1039/C1MB05230F

Search articles by author

Spotlight

Advertisements