Characterizing Enzymes of the Diphosphoinositol Polyphosphate Phosphohydrolase (DIPP) Family

ABSTRACT The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5′-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k cat/K m ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-β-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K m values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.

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Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5′,5'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations

Biochemical Journal ◽

10.1042/bj20020733 ◽

2003 ◽

Vol 369 (3) ◽

pp. 519-528 ◽

Cited By ~ 27

Author(s):

Stephen W. INGRAM ◽

Stephen T. SAFRANY ◽

Larry D. BARNES

Keyword(s):

Growth Rate ◽

Schizosaccharomyces Pombe ◽

Morphological Changes ◽

Enzymic Activity ◽

Detectable Change ◽

Rich Media ◽

Intracellular Concentrations ◽

Diphosphoinositol Polyphosphate

Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (ApnA, n = 5 or 6) and diphosphoinositol polyphosphates {diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)} in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap5A, PP-InsP5, [PP]2-InsP4 and a novel diphosphoinositol polyphosphate ([PP]x-InsPx) in S. pombe using HPLC methods. Ap5A was present at 0.06pmol/mg of protein (approx. 4nM). PP-InsP5, [PP]x-InsPx and [PP]2-InsP4 were present at 15pmol/mg (approx. 1.1μM), 15pmol/mg (approx. 1.1μM) and 30pmol/mg (approx. 2.2μM) respectively, while the intracellular concentration of InsP6 was 0.5nmol/mg of protein (approx. 36μM). Disruption of aps1 resulted in a 52% decrease in Ap6A hydrolase activity in vitro, no detectable change in the intracellular Ap5A concentration, and 3-fold increased intracellular concentrations of PP-InsP5 and [PP]x-InsPx. Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30°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-InsP4, [PP]x-InsPx or PP-InsP5, but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap5A 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 Ap5A occurred upon overexpression of aps1E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP5 and [PP]x-InsPxin vivo.

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