Bioluminescent succinate detection monitors dioxygenases and JMJC demethylases

December 14, 2017, Society for Laboratory Automation and Screening
Demethylation of DNA and Histone in the Nucleosome by Demethylases. Credit: Promega Corporation

A new (and freely available) original research article published ahead-of-print at SLAS Discovery Online describes a new methodology that enables the investigation of a large number of structurally conserved enzymes belonging to the Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily. This superfamily comprises approximately 60 enzymes that are responsible for regulating a wide number of biological processes including epigenetic regulation, DNA/RNA repair and oxygen sensing. Of these, JumonjiC histone lysine demethylases (JMJCs) and prolyl hydroxylases are potential drug targets because of their relevance to human diseases.

Succinate is a common product to all Fe(II)/2-oxoglutarate-dependent reactions. A new homogeneous assay by Alves et al. of Promega Corporation (Madison, WI) detects succinate using luminescence. This assay enables scientists to characterize the biochemical activity of virtually any enzyme that produces succinate, regardless of the chemical structure. This demonstrates the universality of the assay and its advantages over assays that usually depend on the modification or labeling of either the substrate or the reagent needed to capture or monitor the product.

The authors present an array of applications for their method. Using a miniaturized format, they characterize the enzymatic activity of several JumonjiC histone demethylases and other dioxygenases, for which high-throughput screening methods are currently unavailable. Using this simple succinate detection assay, the authors investigate substrate specificities and determine apparent kinetic parameters for different enzymes. They also validate the method as a screening tool for inhibitors using the LOPAC 1280 compound library and study the mode of action of selected hits against members of this family.

Bioluminescent succinate detection is resistant to chemical interference and it is effective for characterizing multiple Fe(II)/2-oxoglutarate-dependent dioxygenases with diverse substrate structures in a single format. By investigating a large number of these enzymes, this could have a significant impact on the field of dioxygenase research.

Explore further: Fluorescent probes to study cellular activity

More information: Juliano Alves et al, Bioluminescent High-Throughput Succinate Detection Method for Monitoring the Activity of JMJC Histone Demethylases and Fe(II)/2-Oxoglutarate-Dependent Dioxygenases, SLAS DISCOVERY: Advancing Life Sciences R&D (2017). DOI: 10.1177/2472555217745657

Related Stories

Spotting the invisible

May 31, 2017

Chemists at UmeƄ University have succeeded in mapping structures and functions of a transient enzyme state. By modifying the enzyme adenylate kinase, researchers were able to isolate the molecule and study it using the quantitative ...

Recommended for you

EPA adviser is promoting harmful ideas, scientists say

March 22, 2019

The Trump administration's reliance on industry-funded environmental specialists is again coming under fire, this time by researchers who say that Louis Anthony "Tony" Cox Jr., who leads a key Environmental Protection Agency ...

Coffee-based colloids for direct solar absorption

March 22, 2019

Solar energy is one of the most promising resources to help reduce fossil fuel consumption and mitigate greenhouse gas emissions to power a sustainable future. Devices presently in use to convert solar energy into thermal ...


Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.