Members of the Nudix family typically contain a amino
Members of the Nudix family typically contain a 23-amino-acid sequence (Nudix box) of GxExREUxEExGU, where U is usually Ile, Leu, or Val and x represents any amino acid. The EUxEE core residues serve as anchors for an Mg ligand that associates with a characteristic pyrophosphate linkage that is common to nearly all Nudix substrates .
Although several of the Nudix proteins’ experimentally defined functional assignments are secured by genetic evidence or by high-quality kinetic characterization , , the vast majority of them have been made by crude enzyme screens. In many cases, the reported values of / are too low to inspire confidence that the natural substrate was identified.
Many of the Nudix hydrolases catalyze the hydrolysis of aberrant deoxynucleoside triphosphates efficiently, thereby reducing the extent of incorporation of the corresponding undesired Fmoc-L-Arg(Aloc)2-OH mg into DNA. The function of these enzymes is to “sanitize the nucleotide pool”, thereby serving to prevent incorporation of non-canonical bases into DNA . The best-characterized member of this group is MutT from . This gene product exhibits a nearly diffusion controlled / value of approximately 10Ms for the hydrolysis of mutagenic 8-oxo-dGTP; however, it also catalyzes the hydrolysis of the canonical dGTP with a / value of approximately 10Ms. This last figure likely represents unavoidable collateral damage due to the structural similarity of the substrates.
The standard assay for those Nudix hydrolases that expose a product phosphate ester or P is a variant of the classical Fiske–SubbaRow method following treatment of the product, if necessary, with alkaline phosphatase (APase) . This discontinuous assay necessitates removing aliquots during the time course from the reaction mixture. Thus, it is slow, labor intensive, and subject to pipetting error, and it is not optimal for rapid screening of candidate substrates.
We describe here a continuous assay that employs the fluorescently tagged phosphate binding protein (PBP) . It is free of the described limitations of the Fiske–SubbaRow assay and is suitable for substrate and inhibitor screening in a moderate throughput format using 96-well plates.
The enzymes characterized here via this assay are MutT (), NudD (), DR_1025 (), and MM_0920 (). X-ray or nuclear magnetic resonance (NMR) structures are available for all four enzymes. The first two were well characterized by the Fiske–SubbaRow assay and were selected to validate the new assay. DR_1025 was chosen because , which is highly resistant to radiation damage, has 26 putative Nudix enzymes that likely are responsible for some of the protection . Some substrate screening of the DR_1025 activity has been reported . MM_0920 is a putative Nudix enzyme. A phylogenetic analysis of MM_0920 with 142 biochemically or structurally characterized Nudix proteins has been performed (J.R. Srouji, unpublished work). It was determined that MM_0920 is located in a clade where protein function appears to change frequently. The biochemical characterization of MM_0920 would allow us to infer the function of many other uncharacterized Nudix hydrolases, and it would greatly aid in more accurate function predictions for the entire Nudix family.
Materials and methods
Acknowledgments We thank Albert Mildvan, Maurice Bessman, Martin Webb, and the New York Structural Genomics Research Center for the research materials specified in Materials and Methods. We also thank A. Iavarone and Ulla Andersen for performing the mass spectrometer analyses. This research was supported by the National Institutes of Health (NIH, R01 GM071749 and R01 GM071749-03S2).
Introduction Uracil residues are introduced into genomic DNA through incorporation of dUMP in place of dTMP by DNA polymerases during replication, and by deamination of existing dCMP residues , . In bacteria such as Escherichia coli and Salmonella typhimurium, dUTP biosynthesis is an obligatory intermediate in the de novo synthesis of dTTP , , . Since DNA polymerases can utilize dUTP in place of dTTP , , some incorporation of dUMP into the bacterial chromosome is unavoidable. Studies performed in vivo or with cell-free systems have shown that the frequency of dUMP incorporation depends largely on the relative sizes of the intracellular dUTP and dTTP pools , . The intracellular concentration of dUTP in E. coli is governed by deoxycytidine triphosphate deaminase (dcd), which converts dCTP directly to dUTP , and by dUTP pyrophosphatase (dut) activity, which hydrolyzes dUTP to dUMP and pyrophosphate , . Together, these reactions generate about 75% of the dUTP pool . The balance of the dUTP pool is produced by the reduction of UDP to dUDP by ribonucleotide reductase, followed by conversion to dUTP by nucleoside diphosphate kinase .