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Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (Ap(n)A, n =5 or 6) and diphosphoinositol polyphosphates [diphosphoinositol pentakisphosphate (PP-InsP(5)) and bisdiphosphoinositol tetrakisphosphate ([PP](2)-InsP(4))] in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap(5)A, PP-InsP(5), [PP](2)-InsP(4) and a novel diphosphoinositol polyphosphate ([PP](x)-InsP(x)) in S. pombe using HPLC methods. Ap(5)A was present at 0.06 pmol/mg of protein (approx. 4 nM). PP-InsP(5), [PP](x)-InsP(x) and [PP](2)-InsP(4) were present at 15 pmol/mg (approx. 1.1 microM), 15 pmol/mg (approx. 1.1 microM) and 30 pmol/mg (approx. 2.2 microM) respectively, while the intracellular concentration of InsP(6) was 0.5 nmol/mg of protein (approx. 36 microM). Disruption of aps1 resulted in a 52% decrease in Ap(6)A hydrolase activity in vitro, no detectable change in the intracellular Ap(5)A concentration, and 3-fold increased intracellular concentrations of PP-Ins P(5) and [PP](x)-InsP(x). Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30 degrees C. Overexpression of aps1 via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity in vitro caused no detectable changes in the intracellular concentrations of [PP](2)-InsP(4), [PP](x)-InsP(x) or PP-InsP(5), but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap(5)A concentration. Overexpression of aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells. No phenotypic changes or changes in intracellular Ap(5)A occurred upon overexpression of aps1 E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP(5) and [PP](x)-InsP(x) in vivo.

Diadenosine oligophosphates are ubiquitous compounds that were discovered over 30 years ago. Diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A) is the most studied member of this family, and its function in yeast is unknown. To investigate possible functions, we changed the intracellular Ap(4)A concentration in Schizosaccharomyces pombe via disruption and overexpression of the aph1 gene, which encodes an Ap(4)A hydrolase (Aph1). S. pombe Aph1 is 52% identical with a human tumour suppressor protein, Fhit, in a core region of 109 amino acids. Disruption of aph1 resulted in an 85% decrease in Ap(4)A hydrolase activity and a 290-fold increase in the intracellular Ap(4)A concentration. The disruption and subsequent increase in intracellular Ap(4)A concentration had no significant effect on the growth of S. pombe. Overexpression of the S. pombe aph1 gene, resulting in 17- and 84-fold increases in Ap(4)A hydrolase activity above wild-type levels, resulted in 60 and 80% decreases respectively in the intracellular Ap(4)A concentration. This represents the first report of a decrease in the intracellular Ap(4)A concentration in response to overexpression of a degradative enzyme in any eukaryotic organism. We describe a new S. pombe expression plasmid, pPOX, which was used to achieve the largest increase in expression of aph1. Overexpression of aph1 at the highest level resulted in a 46% increase in generation time in comparison with the control strain. Neither overexpression nor disruption had any effect on the intracellular ATP or ADP concentrations. This is the first report of ADP and ATP concentrations in S. pombe. These data also indicate that Aph1 functions in vivo to degrade Ap(4)A, and that high-level overexpression of this enzyme reduces the growth rate.

Aps1 from Schizosaccharomyces pombe (Ingram, S. W., Stratemann, S. A. , and Barnes, L. D. (1999) Biochemistry 38, 3649-3655) and YOR163w from Saccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604-8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap(6)A and Ap(5)A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap(6)A and Ap(5)A, in preference to other diadenosine polyphosphates. The emergence of Ap(6)A and Ap(5)A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.

The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E. The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture. Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation. The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography. Aps1, whose expression was detected in S. pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates. The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A. Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively. The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH. Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP. Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2. Aps1 is the first nudix hydrolase isolated from S. pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.

Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively. Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.