Nudix hydrolases are a family of proteins that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Twenty-four genes of the Nudix hydrolase homologues (AtNUDTs) with predicted localizations in the cytosol, chloroplasts, and mitochondria exist in Arabidopsis thaliana. Here, we demonstrated the comprehensive analysis of nine types of cytosolic AtNUDT proteins (AtNUDT1, -2, -4, -5, -6, -7, -9, -10, and -11). The recombinant proteins of AtNUDT2, -6, -7, and -10 showed both ADP-ribose and NADH pyrophosphatase activities with significantly high affinities compared with those of animal and yeast enzymes. The expression of each AtNUDT is individually regulated in different tissues. These findings suggest that most cytosolic AtNUDTs may substantially function in the sanitization of potentially hazardous ADP-ribose and the regulation of the cellular NADH/NAD ؉ ratio in plant cells. On the other hand, the AtNUDT1 protein had the ability to hydrolyze 8-oxo-dGTP with a K m value of 6.8 M and completely suppress the increased frequency of spontaneous mutations in the Escherichia coli mutT ؊ strain, indicating that AtNUDT1 is a functional homologue of E. coli MutT in A. thaliana and is involved in the prevention of spontaneous mutation. The results obtained here suggest that the plant Nudix family has evolved in a specific manner that differs from that of yeast and humans.

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its K m value was approximately 100-fold lower for ADP-ribose (13.0 6 0.7 mM) than for ADP-glucose (1,235 6 65 mM). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 6 3.4 mM) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 6 5.4 mM) and NADPH (36.9 6 3.5 mM). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 6 0.9 mM). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4-5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13,, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes.

Here, we assessed modulation of the poly(ADP-ribosyl)ation (PAR) reaction by an Arabidopsis (Arabidopsis thaliana) ADPribose (Rib)/NADH pyrophosphohydrolase, AtNUDX7 (for Arabidopsis Nudix hydrolase 7), in AtNUDX7-overexpressed (Pro 35S :AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused by paraquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro 35S :AtNUDX7 plants but increased in the KOnudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7 hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro 35S :AtNUDX7 and KO-nudx7 plants showed increased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib) in the Pro 35S :AtNUDX7 and KO-nudx7 plants were markedly higher and lower, respectively, than those in the control plants. Depletion of NAD + and ATP resulting from the activation of the PAR reaction under oxidative stress was completely suppressed in the Pro 35S :AtNUDX7 plants. Accumulation of NAD + and ATP was observed in the KO-nudx7-and 3-aminobenzamide-treated plants, in which the PAR reaction was suppressed. The expression levels of DNA repair factors, AtXRCC1 and AtXRCC2 (for x-ray repair cross-complementing factors 1 and 2), paralleled that of AtNUDX7 under both normal conditions and oxidative stress, although an inverse correlation was observed between the levels of AtXRCC3, AtRAD51 (for Escherichia coli RecA homolog), AtDMC1 (for disrupted meiotic cDNA), and AtMND1 (for meiotic nuclear divisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD + by NADH turnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD + levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.