A continuous fluorescence assay for the characterization of Nudix hydrolases

Abstract

The common substrate structure for the functionally diverse Nudix protein superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The substrate specificity is known for less than 1% of the 29,400 known members. Most activities result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. Reactions have typically been monitored by a modification of the discontinuous Fiske–SubbaRow assay, which is relatively insensitive and slow. We report here the development of a continuous fluorescence assay that enables the rapid and accurate determination of substrate specificities in a 96-well format. We used this novel assay to confirm the reported substrate characterizations of MutT and NudD of Escherichia coli and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of Methanosarcina mazei. Novel findings enabled by the new assay include the following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP at the α–β position, MutT cleaves at the β–γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α–β position. Second, MutT also catalyzes the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best substrate for DR_1025 of the 41 compounds screened.

A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III

“…For that purpose, we used a continuous fluorescence assay (Xu et al 2013), that employs fluorescently tagged E. coli phosphate binding protein (PBP) as a phosphate sensor. We screened PpND and AtND with substrates of Nudix hydrolases, with the common structure nucleoside–diphosphate-X.…”

“…Nudix hydrolase activity was determined by a continuous fluorescence assay (Xu et al 2013), using fluorescently tagged E. coli phosphate binding protein (PBP) as a phosphate sensor. The phosphate sensor was produced and purified using the methods described by Xu et al (2013).…”

“…The phosphate sensor was produced and purified using the methods described by Xu et al (2013). The assay contained 10 mM Tris HCl pH 7.6, 1 mM MgCl 2 , and 5–10 µM phosphate sensor.…”

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

“…Fluorescently labeled phosphate binding protein (PBP) was expressed and purified as described in Xu et al …”

“…The most reactive compounds were assayed carefully to determine the kinetic parameters. The medium throughput assays were carried out with a phosphate sensor fluorescence assay …”