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Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases.

Leslie NR, McLennan AG, Safrany ST - BMC Biochem. (2002)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK. n.r.leslie@dundee.ac.uk

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 (kcat/Km = 11, 8 and 16 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 10(3) M(-1) x s(-1) 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.

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Properties of hAps1 and hAps2. (a) Mn2+-dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM). (b) pH dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM).
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Figure 5: Properties of hAps1 and hAps2. (a) Mn2+-dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM). (b) pH dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM).

Mentions: Initially, the ability of hAps1 and hAps2 to metabolise Ap5A was determined at 37°C in the presence of 50 mM HEPES, pH 7.6 and 100 μM substrate. A divalent cation was essential for activity, with Mn2+ by far the most effective between 2 and 6 mM (Fig. 5a). Cu2+ supported less than 30% and Zn2+ and Co2+ each less than 3% of the maximum activity. Ni2+, Ca2+ and, surprisingly, Mg2+ were unable to activate hAps1 or hAps2. When assayed in the presence of 1 mM MnCl2, hAps1 and hAps2 showed alkaline pH optima of pH 8.5 (Fig. 5b), a feature common among Nudix hydrolases. Activity increased from undetectable at pH 6 to over 70% maximum between 7.5 and 9. Previous characterisation of hDIPP-1, 2α and 2β, as well as S. pombe Aps1 was performed in a buffer containing 1 mM MnCl2 at pH 7.6. In order to compare results for hAps1 and hAps2 directly with hDIPP-1, hDIPP-2 and S. pombe Aps1, we have performed assays using identical conditions.


Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases.

Leslie NR, McLennan AG, Safrany ST - BMC Biochem. (2002)

Properties of hAps1 and hAps2. (a) Mn2+-dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM). (b) pH dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC117780&req=5

Figure 5: Properties of hAps1 and hAps2. (a) Mn2+-dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM). (b) pH dependency of hAps1 (filled circles) and hAps2 (open circles) activity towards Ap5A (100 μM).
Mentions: Initially, the ability of hAps1 and hAps2 to metabolise Ap5A was determined at 37°C in the presence of 50 mM HEPES, pH 7.6 and 100 μM substrate. A divalent cation was essential for activity, with Mn2+ by far the most effective between 2 and 6 mM (Fig. 5a). Cu2+ supported less than 30% and Zn2+ and Co2+ each less than 3% of the maximum activity. Ni2+, Ca2+ and, surprisingly, Mg2+ were unable to activate hAps1 or hAps2. When assayed in the presence of 1 mM MnCl2, hAps1 and hAps2 showed alkaline pH optima of pH 8.5 (Fig. 5b), a feature common among Nudix hydrolases. Activity increased from undetectable at pH 6 to over 70% maximum between 7.5 and 9. Previous characterisation of hDIPP-1, 2α and 2β, as well as S. pombe Aps1 was performed in a buffer containing 1 mM MnCl2 at pH 7.6. In order to compare results for hAps1 and hAps2 directly with hDIPP-1, hDIPP-2 and S. pombe Aps1, we have performed assays using identical conditions.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK. n.r.leslie@dundee.ac.uk

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 (kcat/Km = 11, 8 and 16 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 10(3) M(-1) x s(-1) 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.

Show MeSH