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Review
. 2013 Oct-Dec;753(2):131-146.
doi: 10.1016/j.mrrev.2013.08.001. Epub 2013 Aug 19.

ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics

Peter D Simone  1 , Youri I Pavlov  2 , Gloria E O Borgstahl  3
Affiliations
Review

ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics

Peter D Simone et al. Mutat Res. 2013 Oct-Dec.

Abstract

Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.

Keywords: Base analogs; DSB; Dominant negative; HAM1; HAP; HGPRT; ITP; ITPA; ITPA gene polymorphism; MEF; Mercaptopurines; NUDT16; Nucleotide pool; Pharmacogenetics; Protein stability; SSB; Saccharomyces cerevisiae; TPMT; XTP; base analog 6-hydroxylaminopurine; double-strand break; homolog RdgB E. coli ITPA homolog (Rec-dependent growth B); hypoxanthine-guanine phosphoribosyltransferase; inosine triphosphate; inosine triphosphate pyrophosphatase; mouse embryonic fibroblasts; nudix (nucleoside diphosphate linked moiety X)-type motif 16; single-strand break; thiopurine S-methyltransferase; xanthosine triphosphate.

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Conflict of interest statement

Conflict of interest statement

None of the contributors to this article has any conflicting interests.

Figures

Fig. 1
Fig. 1
The structure of human ITPA. (A) The structure of ITPA (PDB code 2CAR [17]) without substrate. The protein is composed of a central β-sheet (yellow) with the active site flanked by two predominantly α-helical lobes: an upper lobe in light blue and a lower lobe in red. (B) In the ITPA dimer the monomers are related by a 2-fold symmetry axis. The majority of the dimerization interactions occur in the upper lobe (light blue). Residues in the interface are colored as follows: glutamic acid = green, glutamine = purple, lysine = gray, leucine = brown, phenylalanine = red, tryptophan = orange, tyrosine = dark blue. This interface buries 1080 Å2 of surface area and involves numerous hydrophobic contains and hydrogen bonds. (C) Stereopair of the structure of ITPA with ITP bound (PDB code 2J4E [17]), shown in purple, pink, and green, reveals several structural changes upon substrate binding. The most prominent is in the lower lobe with the closing of the central α-helix towards ITP, indicated by the arrow. The α-carbons of the two structures align with an r.m.s.d. of 1.87 Å. Ribbon figures were generated with PyMOL [134].
Fig. 2
Fig. 2
Substrate binding and catalysis. (A) Stereopair of ITPA with ITP bound (PDB code 2J4E [17]) is shown with relevant residues involved in substrate binding and discrimination shown in stick form. A Mg2+ cation is shown as a green sphere. (B) Potential reaction mechanisms for ITPA are shown schematically. Due to the low resolution of the structure, a single catalytic mechanism cannot be determined. Asp 72 is in position to coordinate a water molecule for attack on the α phosphate (circled on the top), while Asn-16 is positioned to coordinate a water for attack on the β phosphate (circled on the bottom). Either would result in the scission of the α-β phosphoanhydride bond. The positively charged ε-amino groups of Lys 19 and Lys 89 are positioned to stabilize the negatively charged intermediate.
Fig. 3
Fig. 3
Purine metabolism and the formation of ITP and XTP. IMP is the first purine nucleotide formed in the de novo synthesis pathway. IMP is converted to either AMP or GMP, the latter through XMP as an intermediate. IMP and GMP can also be made from the free bases hypoxanthine and guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in the salvage pathway. Similarly, AMP can be synthesized from adenine by adenine phosphoribosyltransferase (APRT). AMP and GMP are sequentially phosphorylated to ATP and GTP by nucleoside monophosphate kinases and nucleoside diphosphate kinase. These enzymes may also work on IMP and XMP, providing one means of generating ITP and XTP. Highlighted in green are the functional groups that differ between ATP and ITP and between GTP and XTP. ATP and GTP contain amino groups, while ITP and XTP contain keto groups. The amino groups can undergo deamination to keto groups. Hence, this deamination of ATP and GTP is another mechanism that can produce ITP and XTP.
Fig. 4
Fig. 4
Problems caused by non-canonical NTPs. If ITP or XTP accumulate in cells, they can be inserted into ribosomal RNA, potentially leading to structural alterations in the ribosome (indicated by *), or mRNA (indicated by I). Either of these could lead to mistranslation and altered proteins. Alternatively, they can inhibit enzymes that normally use ATP or GTP. If dITP or dXTP accumulate, they could be inserted into DNA (indicated by I). If not repaired, this could lead to mutations (indicated by *). Alternatively, excision of the abnormal base creates a SSB, which can lead to DSBs if repair is not completed before replication.
Fig. 5
Fig. 5
E. coli rdgB mutant, synthetic lethality, and rescue. Wild type E. coli do not accumulate dITP or dXTP because RdgB catalyzes their conversion to dIMP and dXMP. In rdgB mutant E. coli, dITP and dXTP accumulate, leading to their incorporation into DNA (indicated by I/X). Endonuclease V recognizes these lesions and creates SSBs. If the RecA protein is present, the DNA can be repaired, and so rdgB mutants are viable. If recA is also mutated, then DSBs can accumulate, leading to the inviability of rdgB recA double mutants. However, mutating the nfi gene, which codes for endonuclease V, prevents SSBs from forming and thus rescues the synthetic lethality of rdgB recA double mutants. Based on [59].
Fig. 6
Fig. 6
Disruption of the hydrophobic core in Pro32Thr ITPA. (A) The surface of Pro32Thr ITPA shows a hole in the protein structure due to Phe 31 and Thr 32 moving from the hydrophobic core out into the solvent. (B) An inverse view generated by HOLLOW [135] clearly exhibits the large cavity (gray) left in the protein’s core. The volume of the cavity is 887 Å3 as determined by VOIDOO [136]. (C) By comparison, the surface of wild type ITPA does not reveal any holes. (D) Only small cavities (gray) exist in the core of wild type ITPA (volume = 159 Å3). Wall-eyed stereo pairs were generated with PyMOL [134].
Fig. 7
Fig. 7
Mercaptopurine metabolism and mechanisms of cytotoxicity. After being taken up into cells, 6-mercaptopurine is converted to 6-thioIMP. (Azathioprine is a prodrug of 6-mercaptopurine.) 6-thioIMP can be converted to 6-thioGMP, which can be phosphorylated to 6-thioGTP. 6-thioGTP is known to cause DNA strand breaks by being incorporated into DNA and to induce T-cell apoptosis by Rac1 inhibition. 6-thioIMP can also be phosphorylated to 6-thioITP. ITPA can catalyze the conversion of this back to 6-thioIMP. TPMT can methylate both 6-thioIMP and 6-thioITP. These methylated forms inhibit de novo purine synthesis. In ITPA deficient patients, 6-thioITP and 6-methylthioITP can accumulate and lead to adverse reactions. Based on [108, 116, 117].
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