title: 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.
authors.affiliation: Department of Comparative Biochemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Anting
authors.initials: A
authors.lastname: Xu
authors.forename: Anna M
authors.initials: AM
authors.lastname: Desai
authors.forename: Steven E
authors.initials: SE
authors.lastname: Brenner
authors.forename: Jack F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D003374:Coumarins; D034301:Deinococcus / Q000201:enzymology / Q000235:genetics; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D017020:Methanosarcina / Q000201:enzymology / Q000235:genetics; D005810:Multigene Family; D028044:Phosphate-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D000096964:Nudix Hydrolases
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011755:Pyrophosphatases; D000096964:Nudix Hydrolases; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide