Alexander G. McLennan

The Nudix hydrolase superfamily

Abstract.Nudix hydrolases are found in all classes of organism and hydrolyse a wide range of organic pyrophosphates, including nucleoside di- and triphosphates, dinucleoside and diphosphoinositol polyphosphates, nucleotide sugars and RNA caps, with varying degrees of substrate specificity. Some superfamily members, such as Escherichia coli MutT, have the ability to degrade potentially mutagenic, oxidised nucleotides while others control the levels of metabolic intermediates and signalling compounds. In prokaryotes and simple eukaryo tes, the number of Nudix genes varies from 0 to over 30, reflecting the metabolic complexity and adaptability of the organism. Mammals have around 24 Nudix genes, several of which encode more than one variant. This review integrates the sizeable recent literature on these proteins with information from global functional genomic studies to provide some insights into the possible roles of different superfamily members in cellular metabolism and homeostasis and to stimulate discussion and further research into this ubiquitous protein family.

Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells.

The ygdP and apaH genes of Salmonella enterica serovar Typhimurium (S. Typhimurium) encode two unrelated dinucleoside polyphosphate (NpnN) hydrolases. For example, YgdP cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, while ApaH cleaves Ap4A producing 2ADP. Disruption of ygdP, apaH individually, and disruption of both genes together reduced intracellular invasion of human HEp-2 epithelial cells by S. Typhimurium by 9-, 250-, and 3000-fold, respectively. Adhesion of the mutants was also greatly reduced compared with the wild type. Invasive capacity of both single mutants was restored by transcomplementation with the ygdP gene, suggesting that loss of invasion was due to increased intracellular NpnN. The normal level of 3 μm adenylated NpnN (ApnN) was increased 1.5-, 3.5-, and 10-fold in the ygdP, apaH and double mutants, respectively. Expression of the putative ptsP virulence gene downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. The increase in intracellular NpnN in the mutants appears to cause changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells.

The Crystal Structure of Diadenosine Tetraphosphate Hydrolase from Caenorhabditis elegans in Free and Binary Complex Forms

The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 A and 1.8 A, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.

THE SACCHAROMYCES CEREVISIAE YOR163W GENE ENCODES A DIADENOSINE 5', 5-P1, P6-HEXAPHOSPHATE (AP6A) HYDROLASE MEMBER OF THE MUTT MOTIF (NUDIX HYDROLASE) FAMILY

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes ofM r 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5′,5‴-P 1,P 6-hexaphosphate is the preferred substrate, and hydrolysis in H2 18O shows that ADP and adenosine 5′-tetraphosphate are produced by attack at Pβ and AMP and adenosine 5′-pentaphosphate are produced by attack at Pα with a K m of 56 μmand k cat of 0.4 s−1. Diadenosine 5′,5‴-P 1,P 5-pentaphosphate, adenosine 5′-pentaphosphate, and adenosine 5′-tetraphosphate are also substrates, but not diadenosine 5′,5‴-P 1,P 4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 °C, and 4–10 mm Mg2+ (or 200 μm Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mm), and is inhibited by Ca2+ with an IC50 of 3.3 mm and F− (noncompetitively) with aK i of 80 μm. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.

The Saccharomyces cerevisiae PCD1 Gene Encodes a Peroxisomal Nudix Hydrolase Active toward Coenzyme A and Its Derivatives

The PCD1 nudix hydrolase gene ofSaccharomyces cerevisiae has been cloned and the Pcd1p protein characterized as a diphosphatase (pyrophosphatase) with specificity for coenzyme A and CoA derivatives. Oxidized CoA disulfide is preferred over CoA as a substrate with K m andk cat values of 24 μm and 5.0 s− 1, respectively, compared with values for CoA of 280 μm and 4.6 s− 1respectively. The products of CoA hydrolysis were 3′-phosphoadenosine 5′-monophosphate and 4′-phosphopantetheine. F− ions inhibited the activity with an IC50 of 22 μm. The sequence of Pcd1p contains a potential PTS2 peroxisomal targeting signal. When fused to the N terminus of yeast-enhanced green fluorescent protein, Pcd1p was shown to locate to peroxisomes by confocal microscopy. It was also shown to co-localize with peroxisomal thiolase by immunofluorescence microscopy. N-terminal sequence analysis of the expressed protein revealed the loss of 7 or 8 amino acids, suggesting processing of the proposed PTS2 signal after import. The function of Pcd1p may be to remove potentially toxic oxidized CoA disulfide from peroxisomes in order to maintain the capacity for β-oxidation of fatty acids.

Ap4A induces apoptosis in human cultured cells

Diadenosine oligophosphates (ApnA) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5′,5‴‐P1,P3‐triphosphate to diadenosine 5′,5‴‐P1,P4‐tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of ApnA into cells was achieved by cold shock. Ap4A at 10 μM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis‐resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12‐deoxyphorbol‐13‐O‐phenylacetate and 12‐deoxyphorbol‐13‐O‐phenylacetate‐20‐acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co‐inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.

The repair of ultraviolet light-induced DNA damage in plant cells

Abstract In comparison with other eucaryotic systems, our understanding of the repair of ultraviolet light-induced DNA damage in plants is still rather limited. Morphological features and pigments afford considerable protection and attenuate the dose of ultraviolet light reaching the plant cell DNA. Nevertheless, the available evidence supports the additional existence of enzyme-mediated photoreactivation and excision-repair mechanisms in a variety of systems including pollen, whole seedlings and plants and protoplasts derived from leaves and cultured cells. The properties of these mechanisms appear similar to those of other eucaryotes. The action spectra of photolyases isolated from beans and maize are similar to those of the flavin-containing enzymes from bacteria and yeast. In the absence of visible light, pyrimidine dimers are excised from the DNA of whole plants and isolated protoplasts at rates typical of animal cells i.e. 25–30 000 dimers/cell/h. UV-stimulated repair replication has been demonstrated in non-replicating pollen and leaf protoplasts and also in dividing cultured protoplasts in the presence of a novel replicational inhibitor, compactin. This process appears to be initiated by a high-molecular-weight UV-endonuclease which has been partially purified and which recognises bulky lesions in DNA, including pyrimidine dimers.

Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both?

Many members of the nudix hydrolase family exhibit considerable substrate multispecificity and ambiguity, which raises significant issues when assessing their functions in vivo and gives rise to errors in database annotation. Several display low antimutator activity when expressed in bacterial tester strains as well as some degree of activity in vitro towards mutagenic, oxidized nucleotides such as 8-oxo-dGTP. However, many of these show greater activity towards other nucleotides such as ADP-ribose or diadenosine tetraphosphate (Ap4A). The antimutator activities have tended to gain prominence in the literature, whereas they may in fact represent the residual activity of an ancestral antimutator enzyme that has become secondary to the more recently evolved major activity after gene duplication. Whether any meaningful antimutagenic function has also been retained in vivo requires very careful assessment. Then again, other examples of substrate ambiguity may indicate as yet unexplored regulatory systems. For example, bacterial Ap4A hydrolases also efficiently remove pyrophosphate from the 5′ termini of mRNAs, suggesting a potential role for Ap4A in the control of bacterial mRNA turnover, while the ability of some eukaryotic mRNA decapping enzymes to degrade IDP and dIDP or diphosphoinositol polyphosphates (DIPs) may also be indicative of new regulatory networks in RNA metabolism. DIP phosphohydrolases also degrade diadenosine polyphosphates and inorganic polyphosphates, suggesting further avenues for investigation. This article uses these and other examples to highlight the need for a greater awareness of the possible significance of substrate ambiguity among the nudix hydrolases as well as the need to exert caution when interpreting incomplete analyses.

Adenine dinucleotide-mediated cytosolic free Ca2+ oscillations in single hepatocytes

Single rat hepatocytes microinjected with aequorin respond to Ca2+‐mobilizing agonists, including ADP and ATP, with oscillations in cytosolic free Ca2+. We show here that single rat hepatocytes also respond to the adenine dinucleotides Ap3A and Ap4A with Ca2+ oscillations which resemble those induced by ADP and ATP.

The heat shock response of the cryptobiotic brine shrimp Artemia — II. Heat shock proteins

Abstract 1. 1.The synthesis of heat shock protein (hsps) and heat shock-related proteins has been examined in cysts and nauplius larvae of Artemia exposed to different temperatures. 2. 2.One dimensional SDS-PAGE analysis of proteins from larvae of between 24 and 72 h of development shows a temperature-dependent increase in the synthesis of two proteins, p68 and p89 between 28 and 40°C. A 5 min heat shock at 40°C is sufficient to enhance the subsequent expression of p68 and p89 while 45 min to 1 h at this temperature causes the strong repression of non-hsp synthesis. 3. 3.Cysts synthesise p68 and p89 appreciably at 28°C but enhance this synthesis up to 40°C. However normal protein synthesis is not repressed relative to hsp synthesis until 47°C, in agreement with the known thermotolerance of the cysts. 4. 4.Two-dimensional IEF/SDS-PAGE analysis coupled to immunoblotting with an anti-chick hsp70 antibody shows p68 to consist of inducible hsp68 and hsp70 and constitutively synthesised heat shock cognate (hsc70) forms. The hsp68 isoforms are absent from cysts but are induced upon heat shock. Larvae appear constitutively to express some hsp68 isoforms but these are enhanced considerably by heat. The p70 proteins show a variable pattern of stage-specific and heat activation. 5. 5.Further heat-inducible species, p74 and p45 isoforms probably arise through proteolytic degradation of p89 and p68/p70 respectively. The p45 proteins cross-react with anti-chick hsp70. 6. 6.A low molecular weight hsp, hsp31, is constitutively synthesised by cysts but is strictly heat inducible in larvae. Its behaviour thus mirrors the constitutive and inducible thermotolerance of cysts and larvae respectively and it may therefore have a role to play in this phenomenon. It may also be related to artemin, the abundant cyst-specific 19S protein particle which appears to characterise the crytobiotic state.