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.