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Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination.

ABSTRACT: We investigated the potential (d)NDP/(d)NTP discrimination mechanisms in nucleotide pyrophosphatases. Here, we report that dUTPase, an essential nucleotide pyrophosphatase, uses a C-terminal P-loop-like sequence in a unique mechanism for substrate discrimination and efficient hydrolysis. Our spectroscopy and transient kinetics results on human dUTPase mutants combined with previous structural studies indicate that (i) H-bond interactions between the ?-phosphate and the P-loop-like motif V promote the catalytically competent conformation of the reaction center at the ?-phosphate group; (ii) these interactions accelerate the chemical step of the kinetic cycle and that (iii) hydrolysis occurs very slowly or not at all in the absence of the ?-phosphate--motif V interactions, i.e., in dUDP, dUDP.BeFx, or in the motif V-deleted mutant. The physiological role of dUTPase is to set cellular dUTPdTTP ratios and prevent injurious uracil incorporation into DNA. Based upon comparison with related pyrophosphate generating (d)NTPases, we propose that the unusual use of a P-loop-like motif enables dUTPases to achieve efficient catalysis of dUTP hydrolysis and efficient discrimination against dUDP at the same time. These specifics might have been advantageous on the appearance of uracil-DNA repair. The similarities and differences between dUTPase motif V and the P-loop (or Walker A sequence) commonly featured by ATP- and GTPases offer insight into functional adaptation to various nucleotide hydrolysis tasks.

SUBMITTER: Pecsi I

PROVIDER: S-EPMC3167503 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination.

Pécsi Ildikó I Szabó Judit E JE Adams Scott D SD Simon István I Sellers James R JR Vértessy Beáta G BG Tóth Judit J

Proceedings of the National Academy of Sciences of the United States of America 20110810 35

We investigated the potential (d)NDP/(d)NTP discrimination mechanisms in nucleotide pyrophosphatases. Here, we report that dUTPase, an essential nucleotide pyrophosphatase, uses a C-terminal P-loop-like sequence in a unique mechanism for substrate discrimination and efficient hydrolysis. Our spectroscopy and transient kinetics results on human dUTPase mutants combined with previous structural studies indicate that (i) H-bond interactions between the γ-phosphate and the P-loop-like motif V promot ...[more]

PMID: 21831832

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Project description:Molecular marker technology is used widely in plant variety discrimination, molecular breeding, and other fields. To lower the cost of testing and improve the efficiency of data analysis, molecular marker screening is very important. Screening usually involves two phases: the first to control loci quality and the second to reduce loci quantity. To reduce loci quantity, an appraisal index that is very sensitive to a specific scenario is necessary to select loci combinations. In this study, we focused on loci combination screening for plant variety discrimination. A loci combination appraisal index, variety discrimination power (VDP), is proposed, and three statistical methods, probability-based VDP (P-VDP), comparison-based VDP (C-VDP), and ratio-based VDP (R-VDP), are described and compared. The results using the simulated data showed that VDP was sensitive to statistical populations with convergence toward the same variety, and the total probability of discrimination power (TDP) method was effective only for partial populations. R-VDP was more sensitive to statistical populations with convergence toward various varieties than P-VDP and C-VDP, which both had the same sensitivity; TDP was not sensitive at all. With the real data, R-VDP values for sorghum, wheat, maize and rice data begin to show downward tendency when the number of loci is 20, 7, 100, 100 respectively, while in the case of P-VDP and C-VDP (which have the same results), the number is 6, 4, 9, 19 respectively and in the case of TDP, the number is 6, 4, 4, 11 respectively. For the variety threshold setting, R-VDP values of loci combinations with different numbers of loci responded evenly to different thresholds. C-VDP values responded unevenly to different thresholds, and the extent of the response increased as the number of loci decreased. All the methods gave underestimations when data were missing, with systematic errors for TDP, C-VDP, and R-VDP going from smallest to biggest. We concluded that VDP was a better loci combination appraisal index than TDP for plant variety discrimination and the three VDP methods have different applications. We developed the software called VDPtools, which can calculate the values of TDP, P-VDP, C-VDP, and R-VDP. VDPtools is publicly available at https://github.com/caurwx1/VDPtools.git.

Dataset Information

Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination.

Publications

Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination.

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