An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation.

Analytical Biochemistry
Lusong LuoRobert P Hausinger

Abstract

The Fe(II)/2-oxoglutarate-dependent dioxygenases are a catalytically diverse family of nonheme iron enzymes that oxidize their primary substrates while decomposing the 2-oxoglutarate cosubstrate to form succinate and CO(2). We report a generic assay for these enzymes that uses succinyl-coenzyme A synthetase, pyruvate kinase, and lactate dehydrogenase to couple the formation of the product succinate to the conversion of reduced nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide. We demonstrate the utility of this new method by measuring the kinetic parameters of two bacterial Fe(II)/2-oxoglutarate-dependent dioxygenases. Significantly, this method can be used to investigate both the productive turnover reactions and the nonproductive "uncoupled" decarboxylation reactions of this enzyme family, as demonstrated by using wild-type and variant forms of 2-oxoglutarate-dependent taurine dioxygenase. This assay is amenable to miniaturization and easily adapted to a format suitable for high-throughput screening; thus, it will be a valuable tool to study Fe(II)/2-oxoglutarate-dependent dioxygenases.

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Citations

Nov 10, 2010·Proceedings of the National Academy of Sciences of the United States of America·Terry R WrightZhanyuan Zhang
Jul 4, 2012·Acta Crystallographica. Section D, Biological Crystallography·Michael A JoyceMarie E Fraser
May 19, 2012·Analytical Biochemistry·Motoki Matsuda, Yasuhisa Asano
Dec 30, 2008·Analytical Biochemistry·Kelly L Gorres, Ronald T Raines
Mar 28, 2012·FEMS Microbiology Letters·Sergey V SmirnovJun Ogawa
Feb 22, 2017·Archives of Biochemistry and Biophysics·Hou-Fu GuoJonathan M Kurie
Feb 29, 2020·The Journal of Biological Chemistry·Lennart BrewitzChristopher J Schofield
Jun 2, 2020·Chembiochem : a European Journal of Chemical Biology·Amjad KhanTimothy D W Claridge
Mar 11, 2011·Chemical Society Reviews·Nathan R RoseChristopher J Schofield
Sep 11, 2012·Chembiochem : a European Journal of Chemical Biology·Khaled H AlmabrukTaifo Mahmud
Dec 24, 2021·Nature Chemical Biology·Monica E NeugebauerMichelle C Y Chang

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