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BMC Biochemistry Biomed Central BMC Biochemistry BioMed Central Research3 BMC2002, Biochemistry article x Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases Nick R Leslie1, Alexander G McLennan2 and Stephen T Safrany*1 Address: 1Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK and 2School of Biological Sciences, Life Sciences Building, University of Liverpool, PO Box 147, Liverpool, L69 7ZB, UK E-mail: Nick R Leslie - [email protected]; Alexander G McLennan - [email protected]; Stephen T Safrany* - [email protected] *Corresponding author Published: 16 July 2002 Received: 17 May 2002 Accepted: 16 July 2002 BMC Biochemistry 2002, 3:20 This article is available from: http://www.biomedcentral.com/1471-2091/3/20 © 2002 Leslie et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any purpose, provided this notice is preserved along with the article's original URL. Abstract Background: The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised. Results: We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate × 3 -1 -1 (kcat/Km = 11, 8 and 16 10 M s respectively for hAps1, hAps1-39N and hAps2) and diadenosine × 3 -1 -1 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 10 M s respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell. Conclusions: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers. Background ported to contain high concentrations of Ap5A and Ap6A In addition to the canonical ribonucleoside and deoxyri- in addition to Ap3A and Ap4A, all of which can be exocy- bonucleoside phosphates and cofactors, cells contain a tosed following appropriate stimuli and bind to target cell large number of minor nucleotides. Among these are the purinoceptors causing a variety of physiological responses diadenosine polyphosphates (ApnA, where n = 2–7 [1]). in the cardiovascular and central and peripheral nervous Ap3A and Ap4A are the most intensively studied of these systems [1,3–5]. However, although Ap6A has been de- and are generally present in the soluble fraction of eukary- tected in erythrocytes [6], there are no substantiated meas- otic and prokaryotic cells at concentrations between 10 urements of Ap5A and Ap6A in the soluble fraction of nM and 5 µM [2]. Platelet dense granules, adrenal chro- nucleated cells, and it is likely that they are typically maffin granules and certain synaptic vesicles have been re- present at concentrations much lower than those of Ap3A Page 1 of 13 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/20 µ and Ap4A. The estimated 7 M Ap5A reported in cardiac to the others. The intracellular levels of diphosphoinositol muscle [7] may also be confined to granules. polyphosphates appear to be lower than those of the nu- cleotide substrates. PP-InsP5 levels are often between 1 Whilst the extracellular diadenosine polyphosphates have [19] and 3% [20,21] of the levels of InsP6, which has been partially characterised signalling properties, the possible estimated as being between 15 and 100 µM (see [22]) and functions of the soluble, intracellular compounds remain [PP]2-InsP4 levels are an order of magnitude lower unclear. On the one hand, they may simply be by-prod- [20,21], and as such are virtually undetectable in many ucts of several enzymic reactions (e.g. those catalysed by cell types. PP-InsP5 turnover is regulated by the tumour aminoacyl-tRNA synthetases and other ligases [8]). On promoter thapsigargin [19], while [PP]2-InsP4 turnover is the other, they may have one or more of a number of im- regulated by cyclic AMP- and cyclic GMP-dependent proc- portant regulatory roles, including involvements in DNA esses via an undefined mechanism [23]. However, as for replication/repair, metabolic stress responses, the deter- the ApnAs, no clear functions have yet been found for the mination of cellular fate, and the regulation of enzyme ac- diphosphoinositol polyphosphates, although they have tivities and ion channels (see [1]). For example, Ap5A is a been implicated in vesicle trafficking [24], apoptosis [25] potent inhibitor of adenylate kinase [9]. and DNA repair [26]. It is clear that cells have a variety of relatively specific en- We describe here two further members of the human zymes able to degrade these compounds. These include DIPP subfamily with novel properties. These proteins, symmetrically-cleaving Ap4A hydrolases (in prokaryotes), hAps1 and hAps2, products of the NUDT11 and NUDT10 members of the histidine triad protein family such as the genes respectively, closely resemble the other DIPPs in FHIT tumour suppresser protein and (in lower eukaryo- primary structure, but show a selectivity towards Ap5A tes) the related Ap4A phosphorylases, and most wide- and Ap6A and reduced activity towards PP-InsP5 and spread of all, members of the Nudix hydrolase family (see [PP]2-InsP4. They also display a novel pattern of tissue- [10,11] for reviews). Nudix hydrolases cleave predomi- specific expression. nantly the diphosphate linkage in compounds of general structure: nucleoside diphosphate linked to another moi- Results ety, X. Some family members have a relatively broad sub- Sequence alignment strate specificity, while others have a much more restricted An initial BLAST search of the GenBank expressed se- range. They all possess the Nudix sequence signature mo- quence tag (EST) database with the hDIPP-1 sequence tif Gx5Ex5 [UA]xREx2EexGU (where U is an aliphatic hy- identified two closely related but previously uncharacter- drophobic amino acid), which represents the catalytic site ised predicted proteins, which we have called hAps1 and of the enzyme. Animal and plant Ap4A hydrolases (EC hAps2 (human ApsixA hydrolases 1 and 2). Alignment of 3.1.6.17) degrade Ap4A, Ap5A and Ap6A, always produc- the cDNA sequences with the human genome indicated ing ATP as one of the products. They are most active to- that the genomic sequences encoding these proteins, wards Ap4A; however, the related enzymes from FLJ10628 and LOC139770, lie about 150 kb apart on the Escherichia coli [12] and Rickettsia prowazekii [13] show a X chromosome at Xp11.23 and are transcribed in oppo- marked preference for Ap5A. site directions (Fig. 1). In accordance with the guidelines for the Nudix protein family, the genes for hAps1 and More recently, distinct members of the Nudix family that hAps2 have been designated NUDT11 and NUDT10 re- prefer Ap6A have been purified and characterised. The spectively by the HUGO Gene Nomenclature Committee three human enzymes termed diphosphoinositol (see [http://www.gene.ucl.ac.uk/nomenclature/gene- polyphosphate phosphohydrolases 1 [14,15], 2α and 2β family/npym.html]). These proteins are each 164 amino [16] (hDIPP-1, -2α and -2β) as well as Schizosaccharomyces acids long and are most similar to hDIPP-2β, hAps1 show- pombe Aps1 [17] and Saccharomyces cerevisiae Ddp1p [18] ing 76, 90 and 91% identity at the amino acid level to α β all favour Ap6A over Ap5A. Activity with Ap4A is lower, hDIPP-1, 2 and 2 , respectively. In particular, they both and is in fact lacking in the case of Ddp1p. Remarkably possess the additional Gln residue (Q86 in DIPP-2β) that β α these Ap6A hydrolases are also able to utilise the structur- distinguishes hDIPP-2 from hDIPP-2 and hDIPP-1 and ally unrelated non-nucleotide substrates, diphosphoi- which is responsible for the reduced activity of hDIPP-2β nositol pentakisphosphate (PP-InsP5) and bis- towards diphosphoinositol polyphosphates compared to diphosphoinositol tetrakisphosphate ([PP]2-InsP4), the other DIPPs. hAps1 and hAps2 are also identical to the hence they are commonly referred to as the DIPP subfami- hDIPP-2 enzymes throughout the Nudix motif (Fig. 2). ly. These substrates are generally favoured in vitro, with These observations led us to anticipate that hAps1 and kcat/Km ratios being 50–500-fold higher for PP-InsP5 hAps2 would be potent dinucleoside polyphosphate hy- β compared to Ap6A, except for DIPP-2 , which has a signif- drolases with reduced activity towards diphosphoinositol icantly reduced relative activity with PP-InsP5 compared polyphosphates. The existence of two such similar ex- Page 2 of 13 (page number not for citation purposes) BMC Biochemistry 2002, 3 http://www.biomedcentral.com/1471-2091/3/20 5 kb Telomere Centromere 150kB NUDT11 NUDT10 Figure 1 The NUDT11/NUDT10 region of chromosome Xp11.23. The two known exons of NUDT11, encoding hAps1, and NUDT10, encoding hAps2 are shown represented as boxes. Introns are represented by angled connecting lines. The coding region of each gene is shaded. Solid arrows indicate the direction of transcription of each gene. pressed gene products was surprising.
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