Developing a colorimetric assay for Fe(II)/2-oxoglutarate-dependent dioxygenase

Abstract

The Fe(II)/2-oxoglutarate-dependent dioxygenases (2-OGDs) catalyze the oxidation of substrates ranging from small molecules to large biomolecules with concomitant oxidation of co-substrate (2-oxoglutarate) into succinate. In the present study, we reported a coupled colorimetric assay that can be generally applied to measure the activities of all members of 2-OGDs family. Succinyl-CoA synthetase is employed as the coupling enzyme to transform succinate produced from 2-OGDs catalysis to form succinyl-CoA with concomitant hydrolysis of ATP to form ADP and orthophosphate. Orthophosphate can be quantitated by reacting it with molybdic acid forming a blue pigment. As a proof of concept, kinetic parameters of ectoine hydroxylase obtained using this method are compared to a traditional time- and labor-consuming HPLC based method. As 2-OGDs family enzymes are important drug targets due to their impressive versatility in catalyzing numerous oxidative reactions that are still very challenging using synthetic chemistry, colorimetric method detailed in the manuscript has the potential to enable the practice of high throughput drug screening for 2-OGDs.

Keywords: Ectoine hydroxylase; Enzyme kinetics; Enzyme-coupled colorimetric assay; Fe(II)/2-oxoglutarate-dependent dioxygenase; High throughput drug screening; Succinyl-CoA synthetase.