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1.  Structural and functional characterization of a noncanonical nucleoside triphosphate pyrophosphatase from Thermotoga maritima  
A 2.15 Å resolution crystal structure of TM0159 with bound IMP and enzyme-kinetic data are presented. This noncanonical nucleoside triphosphatase from T. maritima helps to maintain a correct pool of DNA and RNA precursor molecules.
The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k cat/K m values determined at 323 and 353 K fall between 1.31 × 104 and 7.80 × 104  M −1 s−1. ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg2+ as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 Å resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase).
doi:10.1107/S0907444912044630
PMCID: PMC3565439  PMID: 23385455
enzymes; Thermotoga maritima; noncanonical nucleoside triphosphate pyrophosphatase; nucleotide metabolism; dimer interface
2.  Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans 
Journal of molecular biology  2009;392(3):602-613.
Sanitization of the cellular nucleotide pools from mutagenic base analogs is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic and toxic incorporation of purine base analogs (i.e. ITP, dITP, XTP, dXTP or 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele 94C->A encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analog drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by western blot is severely diminished in a human fibroblast cell line with the 94C->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability and availability.
doi:10.1016/j.jmb.2009.07.051
PMCID: PMC2745931  PMID: 19631656
3.  Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant 
Journal of Nucleic Acids  2010;2010:872180.
Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA) hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs.
doi:10.4061/2010/872180
PMCID: PMC2948936  PMID: 20936128
4.  Quantitative in vitro and in vivo characterization of the human P32T mutant ITPase 
Biochimica et biophysica acta  2009;1802(2):269.
Human ITPase, encoded by the ITPA gene, and its orthologs (RdgB in Escherichia coli and HAM1 in Saccharomyces cerevisiae) exclude noncanonical nucleoside triphosphates (NTPs) from NTP pools. Deoxyinosine triphosphate (dITP) and 2′-deoxy-N-6-hydroxylaminopurine triphosphate are both hydrolyzed by ITPase to yield the corresponding deoxynucleoside monophosphate and pyrophosphate. In addition, metabolites of thiopurine drugs such as azathioprine have been shown to be substrates for ITPase. The ITPA 94C>A [P32T] variant is one of two polymorphisms associated with decreased ITPase activity. Furthermore, the ITPA 94C>A [P32T] variant is associated with an increased risk of adverse drug reactions for patients treated with azathioprine. The nature of the observed phenotypes for ITPA 94C>A [P32T] variant individuals is currently unclear. Our biochemical assays indicate the P32T ITPase has 55% activity with dITP compared to wild-type ITPase. Complementation experiments at 37°C show that N-6-hydroxylaminopurine sensitivity of E. coli rdgB mutants is reduced with a plasmid bearing the ITPA 94C>A [P32T] gene approximately 50% less than with a plasmid bearing the wild-type ITPA gene. The reduction in sensitivity is less at 42°C. Experiments with synthetic lethal E. coli recA(ts) rdgB mutants show that the ITPA 94C>A [P32T] gene also complements the recA(ts) rdgB growth deficiency at 42°C approximately 40% lower than wild-type ITPA gene. Western blot analysis indicates the expression level of P32T ITPase is reduced in these cells relative to wild-type. Our data support the idea that P32T ITPase is a functional protein, albeit with a reduced rate of noncanonical NTP pyrophosphohydrolase activity and reduced protein stability.
doi:10.1016/j.bbadis.2009.11.002
PMCID: PMC2818573  PMID: 19914375
ITPA; ITPase; RdgB; noncanonical purines; dITP; N-6-hydroxylaminopurine
5.  The human ITPA polymorphic variant P32T is destabilized by the unpacking of the hydrophobic core 
Journal of structural biology  2013;182(3):197-208.
Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients’ response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.
doi:10.1016/j.jsb.2013.03.007
PMCID: PMC4212276  PMID: 23528839
Inosine triphosphate pyrophosphatase; Nucleotide pool; X-ray crystallography; Protein stability; Hydrophobic surfaces; Genomic instability
6.  Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii 
Nucleic Acids Research  2001;29(14):3099-3107.
A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized. Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein. Under optimal reaction conditions the kcat /Km value for these substrates was ∼10 000 times that with dATP. Neither endonuclease nor 3′-exonuclease activities were detected in this protein. Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I. Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified. These have catalytic activities similar to Mj0226 protein under optimal conditions. The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms. It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA. This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.
PMCID: PMC55802  PMID: 11452035
7.  Erythrocyte Inosine Triphosphatase Activity Is Decreased in HIV-Seropositive Individuals 
PLoS ONE  2012;7(1):e30175.
Background
Inosine triphosphatase (ITPase) is encoded by the polymorphic gene ITPA and maintains low intracellular levels of the inosine nucleotides ITP and dITP. The most frequently reported polymorphisms are ITPA c.94C>A (rs 1127354) and ITPA c. 124+21 A>C (rs7270101). Some nucleoside-analogues used in the treatment of HIV-seropositive (HIV+) patients are potential substrates for ITPase. Therefore, the frequency of ITPA SNPs and ITPase activity were studied in a population of HIV+-patients.
Methods
The study population consisted of 222 patients, predominantly Caucasian males, >95% using HAART. Erythrocyte ITPase activity was determined by measuring the formation of IMP from ITP. ITPA genotype was determined by sequencing genomic DNA. Distribution of ITPase activity, genotype-phenotype correlation and allele frequencies were compared to 198 control subjects. The effect of nucleoside analogues on ITPase activity was studied using lymphoblastic T-cell cultures and human recombinant ITPase. Enzyme kinetic experiments were performed on erythrocyte ITPase from HIV+ patients and controls.
Results
No difference was observed in the allele frequencies between the HIV+-cohort (± HAART) and the control population. HIV+ carriers of the wild type and ITPA c.94C>A had significantly lower ITPase activities than control subjects with the same genotype (p<0.005). This was not observed in ITPA c. 124+21 A>C carriers. Nucleoside analogues did not affect ITPase activity in cell culture and human recombinant ITPase. Conclusion: ITPA population genetics were identical in HIV+ and control populations. However, the majority of HIV+-patients had decreased erythrocyte ITPase activity compared to controls, probably due to decreased amounts of ITPase protein. It seems unlikely that ITPase activity is decreased due to nucleoside analogues (HAART). Long-term effects of HIV-infection altering ITPase protein expression or stability may explain the phenomenon observed.
doi:10.1371/journal.pone.0030175
PMCID: PMC3260219  PMID: 22272297
8.  Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of Apoptosis in Human Cells 
PLoS ONE  2012;7(2):e32313.
Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.
doi:10.1371/journal.pone.0032313
PMCID: PMC3288088  PMID: 22384212
9.  Inosine Triphosphate Pyrophosphohydrolase (ITPA) polymorphic sequence variants in adult hematological malignancy patients and possible association with mitochondrial DNA defects 
Background
Inosine triphosphate pyrophosphohydrolase (ITPase) is a ‘house-cleaning’ enzyme that degrades non-canonical (‘rogue’) nucleotides. Complete deficiency is fatal in knockout mice, but a mutant polymorphism resulting in low enzyme activity with an accumulation of ITP and other non-canonical nucleotides, appears benign in humans. We hypothesised that reduced ITPase activity may cause acquired mitochondrial DNA (mtDNA) defects. Furthermore, we investigated whether accumulating mtDNA defects may then be a risk factor for cell transformation, in adult haematological malignancy (AHM).
Methods
DNA was extracted from peripheral blood and bone marrow samples. Microarray-based sequencing of mtDNA was performed on 13 AHM patients confirmed as carrying the ITPA 94C>A mutation causing low ITPase activity, and 4 AHM patients with wildtype ITPA. The frequencies of ITPA 94C>A and IVS2+21A>C polymorphisms were studied from 85 available AHM patients.
Results
ITPA 94C>A was associated with a significant increase in total heteroplasmic/homoplasmic mtDNA mutations (p<0.009) compared with wildtype ITPA, following exclusion of haplogroup variants. This suggested that low ITPase activity may induce mitochondrial abnormalities. Compared to the normal population, frequencies for the 94C>A and IVS2+21A>C mutant alleles among the AHM patients were higher for myelodyplastic syndrome (MDS) - but below significance; were approximately equivalent for chronic lymphoblastic leukemia; and were lower for acute myeloid leukemia.
Conclusions
This study invokes a new paradigm for the evolution of MDS, where nucleotide imbalances produced by defects in ‘house-cleaning’ genes may induce mitochondrial dysfunction, compromising cell integrity. It supports recent studies which point towards an important role for ITPase in cellular surveillance of rogue nucleotides.
doi:10.1186/1756-8722-6-24
PMCID: PMC3765497  PMID: 23547827
ITPA; Mitochondria; Haematological malignancy; Microarray; N-call
10.  NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals 
Nucleic Acids Research  2010;38(9):2891-2903.
Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa− mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa− embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa− primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa− MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa− embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa− embryos. However, immortalized Itpa− MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa− MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.
doi:10.1093/nar/gkp1250
PMCID: PMC2875033  PMID: 20081199
11.  The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis  
The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described.
Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuber­culosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophos­phatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.
doi:10.1107/S0907444908007105
PMCID: PMC2631106  PMID: 18560150
histidine biosynthesis; pyrophosphatase; tuberculosis; actinobacteria; catalytic mechanism
12.  ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics 
Mutation research  2013;753(2):10.1016/j.mrrev.2013.08.001.
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.
doi:10.1016/j.mrrev.2013.08.001
PMCID: PMC3827912  PMID: 23969025
nucleotide pool; ITPA gene polymorphism; pharmacogenetics; base analogs; mercaptopurines; protein stability; dominant negative
13.  Structure of a putative NTP pyrophosphohydrolase: YP_001813558.1 from Exiguobacterium sibiricum 255-15 
The crystal structure of a putative NTP pyrophosphohydrolase, YP_001813558.1 from E. sibiricum, reveals a novel segment-swapped linked-dimer assembly.
The crystal structure of a putative NTPase, YP_001813558.1 from Exiguo­bacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a ‘linked dimer’ that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.
doi:10.1107/S1744309110025534
PMCID: PMC2954211  PMID: 20944217
structural genomics; putative NTP pyrophosphohydrolase; MazG nucleotide pyrophosphohydrolase; dUTPases
14.  ITPA Gene Polymorphisms Significantly Affect Hemoglobin Decline and Treatment Outcomes in Patients Coinfected With HIV and HCV 
Journal of medical virology  2012;84(7):1106-1114.
Published studies have described a strong association with a single-nucleotide polymorphism (SNP) in the inosine triphosphate pyrophosphatase (ITPA) gene and ribavirin (RBV)-induced hemolytic anemia in HCV-infected patients receiving pegylated interferon (pegIFN) and RBV. This study sought to evaluate the effect of these polymorphisms on anemia, hemoglobin reduction, HCV kinetics, and treatment outcomes. Sixty-three patients coinfected with HIV and HCV and 58 patients infected with HCV only were treated with pegIFN/RBV were genotyped using the ABI Taq-Man allelic discrimination kit for the 2 ITPA SNP variants rs1127354 and rs7270101. A composite variable of ITPA deficiency using both SNPs was created as previously reported. Statistical analysis was performed using Mann-Whitney test or Chi square/Fishers exact test for categorical data and mixed model analysis for multiple variables. Thirty-five patients (30%) were predicted to have reduced ITPA activity. ITPA deficiency was found to be protective against the development of hemoglobin reduction >3 g/dl over the course of treatment. The rates of hemoglobin reduction >3 g/dl decreased in correlation with the severity of ITPA deficiency. ITPA deficiency was associated with slower hemoglobin decline early in treatment (week 4, P = 0.020) and rapid virologic response (RVR) at week 4 (P = 0.017) in patients coinfected with HIV and HCV. ITPA polymorphisms are associated with hemoglobin decline and in patients coinfected with HIV and HCV it is also associated with early virologic outcomes. Determination of ITPA polymorphisms may allow prediction of RBV-induced anemia and earlier initiation of supportive care to ensure optimal therapeutic outcomes.
doi:10.1002/jmv.23302
PMCID: PMC3518921  PMID: 22585729
ribavirin-induced hemolytic anemia; ITPA; HIV/HCV; pharmacogenomics
15.  Pre-treatment role of inosine triphosphate pyrophosphatase polymorphism for predicting anemia in Egyptian hepatitis C virus patients 
AIM: To investigate and clarify, for the first time, the role of inosine triphosphate pyrophosphatase (ITPA) polymorphism in Egyptian chronic hepatitis C virus (HCV) patients.
METHODS:The human genomic DNA of all patients was extracted from peripheral blood cells in order to determine the single nucleotide polymorphism (SNP) of ITPA (rs1127354). SNP genotyping was performed by real time polymerase chain reaction (PCR, ABI TaqMan allelic discrimination kit) for 102 treatment-naive Egyptian patients with chronic HCV. All patients had no evidence of cardiovascular or renal diseases. They received a combination treatment of pegylated interferon α (PEG-IFNα) as a weekly subcutaneous dose plus an oral weight-adjusted dose of ribavirin (RBV). The majority received PEG-IFNα2a (70.6%) while 29.4% received PEG-IFNα2b. The planned duration of treatment was 24-48 wk according to the viral kinetics throughout the course of treatment. Pre-treatment liver biopsy was done for each patient for evaluation of fibrosis stage and liver disease activity. The basal viral load level was detected quantitatively by real time PCR while viral load throughout the treatment course was performed qualitatively by COBAS TaqMan assay.
RESULTS: Ninety-three patients (91.2%) had ITPA SNP CC genotype and 9 (8.8%) had non-CC genotype (CA and AA). The percentage of hemoglobin (Hb) decline was higher for CC patients than for non-CC patients, particularly at weeks 4 and 8 (P = 0.047 and 0.034, respectively). During the first 12 wk of treatment, CC patients had significantly more Hb decline > 3 g/dL than non-CC patients: 64.5% vs 22.2% at weeks 8 and 12, respectively, (P = 0.024 and 0.038). Reduction of the amount of the planned RBV dose was significantly higher for CC patients than non-CC patients during the first 12 wk (18% ± 12.1% vs 8.5% ± 10.2%, P = 0.021). The percentage of CC patients with RBV dose reduction was significantly greater than that of non-CC patients (77.4% vs 44.4%, P = 0.044). Multivariate analysis identified only the percentage of RBV dose as a predictor for Hb decline. Platelet decline was significantly higher in non-CC patients than CC patients at weeks 12, 24 and 48 (P = 0.018, 0.009 and 0.026, respectively).
CONCLUSION: Rs1127354 ITPA polymorphism plays a decisive role in protecting against treatment-induced anemia and the need for RBV dose reduction in Egyptian HCV patients.
doi:10.3748/wjg.v19.i9.1387
PMCID: PMC3602498  PMID: 23538996
Anemia; Dose reduction; Hepatitis C; Inosine triphosphate; Ribavirin; Rs1127354
16.  Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii  
Recombinant nucleoside triphosphate hydrolases from N. caninum and T. gondii have been purified and crystallized for X-ray structure analysis.
The nucleoside triphosphate hydrolases that are produced by Neospora caninum (NcNTPase) and Toxoplasma gondii (TgNTPase-I) have a different physio­logical function from the ubiquitous ecto-ATPases. The recombinant enzymes were crystallized at 293 K using polyethylene glycol 3350 as a precipitant and X-­ray diffraction data sets were collected for NcNTPase (to 2.8 Å resolution) and TgNTPase-I (to 3.1 Å resolution) at 100 K using synchrotron radiation. The crystals of NcNTPase and TgNTPase-I belonged to the orthorhombic space group I222 (unit-cell parameters a = 93.6, b = 140.8, c = 301.1 Å) and the monoclinic space group P21 (unit-cell parameters a = 87.1, b = 123.5, c = 120.2 Å, β = 96.6°), respectively, with two NcNTPase (V M = 3.7 Å3 Da−1) and four TgNTPase-I (V M = 2.7 Å3 Da−1) molecules per asymmetric unit. SAD phasing trials using a data set (λ = 0.97904 Å) collected from a crystal of seleno­methionylated NcNTPase gave an initial electron-density map of sufficient quality to build a molecular model of NcNTPase.
doi:10.1107/S1744309110032136
PMCID: PMC3001644  PMID: 21045291
nucleoside triphosphate hydrolases; Neospora caninum; Toxoplasma gondii
17.  NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest 
Nucleic Acids Research  2010;38(14):4834-4843.
Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest kcat/Km values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.
doi:10.1093/nar/gkq249
PMCID: PMC2919730  PMID: 20385596
18.  Genetic Polymorphism of Inosine Triphosphate Pyrophosphatase Is a Determinant of Mercaptopurine Metabolism and Toxicity During Treatment for Acute Lymphoblastic Leukemia 
The influence of genetic polymorphism in inosine triphosphate pyrophosphatase (ITPA) on thiopurine-induced adverse events has not been investigated in the context of combination chemotherapy for acute lymphoblastic leukemia (ALL). This study investigated the effects of a common ITPA variant allele (rs41320251) on mercaptopurine metabolism and toxicity during treatment of children with ALL. Significantly higher concentrations of methyl mercaptopurine nucleotides were found in patients with the nonfunctional ITPA allele. Moreover, there was a significantly higher probability of severe febrile neutropenia in patients with a variant ITPA allele among patients whose dose of mercaptopurine had been adjusted for TPMT genotype. In a cohort of patients whose mercaptopurine dose was not adjusted for TPMT phenotype, the TPMT genotype had a greater effect than the ITPA genotype. In conclusion, genetic polymorphism of ITPA is a significant determinant of mercaptopurine metabolism and of severe febrile neutropenia, after combination chemotherapy for ALL in which mercaptopurine doses are individualized on the basis of TPMT genotype.
doi:10.1038/clpt.2008.154
PMCID: PMC2762405  PMID: 18685564
19.  Imperfect pseudo-merohedral twinning in crystals of fungal fatty acid synthase 
A case of imperfect pseudo-merohedral twinning in monoclinic crystals of fungal fatty acid synthase is discussed. A space-group transition during crystal dehydration resulted in a Moiré pattern-like interference of the twinned diffraction patterns.
The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6 MDa fungal FAS was initially solved to 5 Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model.
doi:10.1107/S0907444909000778
PMCID: PMC2631638  PMID: 19171964
imperfect pseudo-merohedral twinning; fungal fatty acid synthase
20.  Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRζ fragments 
P212121 crystals of SIV Nef core domain bound to a peptide fragment of the T-cell receptor ζ subunit exhibited noncrystallographic symmetry and nearly perfect pseudo-merohedral twinning simulating tetragonal symmetry. For a different peptide fragment, nontwinned tetragonal crystals were observed but diffracted to lower resolution. The structure was determined after assignment of the top molecular-replacement solutions to various twin or NCS domains followed by refinement under the appropriate twin law.
HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (R work = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (R work = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning.
doi:10.1107/S090744490904880X
PMCID: PMC2815668  PMID: 20124696
pseudo-merohedral twinning; noncrystallographic symmetry; pseudosymmetry; human immunodeficiency virus; Nef; T-cell receptor
21.  Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis  
The production and purification of recombinant SoGST3 and SoGST6, two GST-like proteins from S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented.
Genome analysis of Shewanella oneidensis, a Gram-negative bacterium with an unusual repertoire of respiratory and redox capabilities, revealed the presence of six glutathione S-transferase-like genes (sogst1–sogst6). Glutathione S-­transferases (GSTs; EC 2.5.1.18) are found in all kingdoms of life and are involved in phase II detoxification processes by catalyzing the nucleophilic attack of reduced glutathione on diverse electrophilic substrates, thereby decreasing their reactivity. Structure–function studies of prokaryotic GST-like proteins are surprisingly underrepresented in the scientific literature when compared with eukaryotic GSTs. Here, the production and purification of recombinant SoGST3 (SO_1576) and SoGST6 (SO_4697), two of the six GST-like proteins in S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented. SoGST3 was crystallized in two different crystal forms in the presence of GSH and DTT that diffracted to high resolution: a primitive trigonal form in space group P31 that exhibited merohedral twinning with a high twin fraction and a primitive monoclinic form in space group P21. SoGST6 yielded primitive orthorhombic crystals in space group P212121 from which diffraction data could be collected to medium resolution after application of cryo-annealing protocols. Crystal structures of both SoGST3 and SoGST6 have been determined based on marginal search models by maximum-likelihood molecular replacement as implemented in the program Phaser.
doi:10.1107/S1744309108014589
PMCID: PMC2496851  PMID: 18540073
glutathione S-transferases; Shewanella oneidensis
22.  Inosine triphosphate pyrophosphohydrolase (ITPA) polymorphic sequence variants in Chinese ALL children and possible association with mercaptopurine related toxicity 
Genetic polymorphisms are important factors in effects and toxicity of chemotherapeutics. This study aimed to investigate whether there was a correlation between genotype or haplotype of inosine triphosph pyrophosphohydrolase(ITPA) and toxicities during maintenance therapy with mercaptopurine (6-MP) in Chinese patients with acute lymphoblastic leukemia (ALL). 95 ALL children who hospitalized between October 2004 and September 2007,were retrospectively analyzed. 6-MP toxicity was documented according to Common Toxicity Criteria, Version 2.0. ITPA sequencing was undertaken. Correlation between genotype/haplotype and 6-MP toxicity was analyzed. The results indicated that 50 cases (52.6%) had grade III-IV of bone marrow inhibition. These children had long-term disease-free survival (DFS), without hepatic and other organs’ dysfunction and secondary tumors. Three variations were observed in ITPA exon 2 (94 C → A), exon 3 (138 G → A), and exon 8 (561 G → A), the 94A carriers (CA and AA) had a lower risk of developing 6-MP toxicity when compared with carriers of the CC genotype (odds ratio [OR] 0.34, 95% confidence interval [CI] 0.12-0.98, P = 0.039). The risk of 6-MP intolerance was decreased in patients with 138 allele and 561 allele polymorphism, but with no significant difference. Patients carrying the haplotype 94A-138A-561A was tolerance compared to those with wild-type haplotype 94C-138G-561G (OR: 0.26, 95% CI 0.07-0.94 P = 0.043). In conclusion, the risk of 6-MP intolerance was decreased in patients with 138 allele and 561 allele polymorphism, but without significant difference. Patients carrying the haplotype 94A-138A-561A was tolerance compared to those with the wild-type haplotype 94C-138G-561G.
PMCID: PMC4129087  PMID: 25120852
ITPA; ALL children; mercaptopurine related toxicity
23.  Monoclinic crystal form of Aspergillus niger α-­amylase in complex with maltose at 1.8 Å resolution 
Two new crystal structures of A. niger α-amylase are reported, one of which reveals two hitherto unobserved maltose-binding sites.
Aspergillus niger α-amylase catalyses the hydrolysis of α-1,4-glucosidic bonds in starch. It shows 100% sequence identity to the A. oryzae homologue (also called TAKA-amylase), three crystal structures of which have been published to date. Two of them belong to the orthorhombic space group P212121 with one molecule per asymmetric unit and one belongs to the monoclinic space group P21 with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger α-amylase crystallized in the monoclinic space group P21 with two molecules per asymmetric unit in complex with maltose at 1.8 Å resolution is reported. Furthermore, a novel 1.6 Å resolution orthorhombic crystal form (space group P21212) of the native enzyme is presented. Four maltose molecules are observed in the maltose–α-amylase complex. Three of these occupy active-site subsites −2 and −1, +1 and +2 and the hitherto unobserved subsites +4 (Asp233, Gly234) and +5 (Asp235). The fourth maltose molecule binds at the distant binding sites d1 (Tyr382) and d2 (Trp385), also previously unobserved. Furthermore, it is shown that the active-site groove permits different binding modes of sugar units at subsites +1 and +2. This flexibility of the active-site cleft close to the catalytic centre might be needed for a productive binding of substrate chains and/or release of products.
doi:10.1107/S1744309106024729
PMCID: PMC2242925  PMID: 16880540
α-amylase; Aspergillus niger; maltose; Aspergillus oryzae; TAKA-amylase
24.  Test of the potential of a dATP surrogate for sequencing via MALDI-MS. 
Nucleic Acids Research  1997;25(24):5072-5076.
1-(2'-Deoxy-beta-d-ribofuranosyl)-3-nitropyrrole phosphate was incorporated into a DNA decamer and analyzed via matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The extent and composition of the various fragment peaks were compared with those in the MALDI-MS spectrum of dT4AT5. The nitropyrrole-containing oligomer proved to be more robust. Two different DNA template assays were then used to attempt to identify DNA replicating enzymes that would incorporate the corresponding triphosphate, i.e. 1-(2'-deoxy-beta-d-ribofuranosyl)-3-nitropyrrole triphosphate (dXTP). It was shown that dXTP was not incorporated by some enzymes and it inhibited others. However, DNA polymerase I Klenow fragment and avian myeloblastosis virus reverse transcriptase incorporated dXTP in place of dATP and then replicated the template overhang in the usual way. The potential of dXTP as a surrogate for dATP in DNA sequencing with MALDI-MS analysis is discussed.
PMCID: PMC147155  PMID: 9396818
25.  Prevalence of TPMT and ITPA gene polymorphisms and effect on mercaptopurine dosage in Chilean children with acute lymphoblastic leukemia 
BMC Cancer  2014;14:299.
Background
Mercaptopurine (6-MP) plays a pivotal role in treatment of childhood acute lymphoblastic leukemia (ALL); however, interindividual variability in toxicity of this drug due to genetic polymorphism in 6-MP metabolizing enzymes has been described. We determined the prevalence of the major genetic polymorphisms in 6-MP metabolizing enzymes in Chilean children with ALL.
Methods
103 Chilean pediatric patients with a confirmed diagnosis of ALL were enrolled. DNA was isolated from whole blood and genetic polymorphism in thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) coding genes were detected by polymorphism chain reaction-restriction fragment length (PCR-RFLP) assay.
Results
The total frequency of variant TPMT alleles was 8%. TPMT*2, TPMT*3A and TPMT*3B alleles were found in 0%, 7%, and 1% of patients, respectively. For ITPA, the frequency of P32T allele was 3%. We did not observe any homozygous variant for TPMT and ITPA alleles. We also analyzed a subgroup of 40 patients who completed the maintenance phase of ALL treatment, and we found that patients carrying a TPMT gene variant allele required a significantly lower median cumulative dosage and median daily dosage of 6-MP than patients carrying wild type alleles.
Conclusion
TMPT genotyping appears an important tool to further optimize 6-MP treatment design in Chilean patients with ALL.
doi:10.1186/1471-2407-14-299
PMCID: PMC4012712  PMID: 24774509
Genetic polymorphism; Acute lymphoblastic leukemia (ALL); 6-Mercaptopurine; TPMT

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