Human Inosine Research Articles

Structure-function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum.

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.

Dynamics of nucleoplasm in human leukemia cells: A thrust towards designing anti-leukemic agents

The human inosine monophosphate dehydrogenase (hIMPDH) is a metabolic enzyme that possesses a unique ability to self-assemble into higher-order structures, forming cytoophidia. The hIMPDH II isoform is more active in chronic myeloid leukemia (CML) cancer cells, making it a promising target for anti-leukemic therapy. However, the structural details and molecular mechanisms of the dynamics of hIMPDHcytoophidia assembly in vitro need to be better understood, and it is crucial to reconstitute the computational nucleoplasm model with cytophilic-like polymers in vitro to characterize their structure and function. Finally, a computational model and its dynamics of the nucleoplasm for CML cells have been proposed in this short review. This research on nucleoplasm aims to aid the scientific community's understanding of how metabolic enzymes like hIMPDH function in cancer and normal cells.However, validating and justifying the computational results from modeling and simulation with experimental data is essential. The new insights gained from this research could explain the structure/topology, geometrical, and electronic consequences of hIMPDH inhibitors on leukemic and normal cells. They could lead to further advancements in the knowledge of nucleoplasmic chemical reaction dynamics.

Targeting human inosine 5’ monophosphate dehydrogenase type 2 for anti-dengue lead identification – a computational approach

Dengue is a rapidly evolving arboviral disease that mainly affects tropical and subtropical regions of the world. The lack of therapeutic drugs and effective vaccines suggests that further resources need to be investigated. The effectiveness of the existing dengue vaccine is improbable as its efficacy depends on prior exposure to the dengue virus(DENV). Although the mechanism underlying the action of bioactive compounds to limit viral replication is less studied and still needs to be further explored, medicinal plants are excellent alternatives to combat DENV infection. In the current study, an in silico screening of phytochemicals from Annona reticulata Linn. against human Impdh2 was performed using Autodock Vina. Daucosterol (−9.0 kcal/mol) and Kaurenoic acid (−8.5 kcal/mol) were chosen as the top hits based on molecular interaction analysis. The hits were further exposed to pharmacokinetics and toxicity properties to determine their drug-like parameters. Molecular dynamics simulation studies of the Impdh2-top hits were carried out to investigate their kinetic behaviour and structural stabilities. The binding free energies of the Impdh2-hit complexes were determined using MM-PBSA analysis. According to the overall conclusions of the study, Daucosterol showed good binding affinity and high structural stability to the binding site residues of the target, therefore it is recommended as a lead compound against dengue. Communicated by Ramaswamy H. Sarma

Triple in silico targeting of IMPDH enzyme and RNA-dependent RNA polymerase of both SARS-CoV-2 and Rhizopus oryzae.

Aim: Mucormycosis has been associated with SARS-CoV-2 infections during the last year. The aim of this study was to triple-hit viral and fungal RNA-dependent RNA polymerases (RdRps) and human inosine monophosphate dehydrogenase (IMPDH). Materials & methods: Molecular docking and molecular dynamics simulationwere usedto test nucleotide inhibitors (NIs) against the RdRps of SARS-CoV-2 and Rhizopus oryzae RdRp. These same inhibitors targeted IMPDH. Results: Four NIs revealed a comparable binding affinity to the two drugs, remdesivir and sofosbuvir. Binding energies were calculated using the most abundant conformations of the RdRps after 100-ns molecular dynamics simulation. Conclusion: We suggest the triple-inhibition potential of four NIs against pathogenic RdRps and IMPDH, which is worth experimental validation.

Triple targeting of human IMPDH, SARS-CoV-2 RdRp, and Rhizopus oryzae RdRp: An in silico perspective

Objectives: Mucormycosis has been reported associated with SARS-CoV-2 infections during the last year. The viral RNA-dependent RNA polymerase (RdRp) was proved to be a critical protein target in viral and fungal pathogens. The human inosine monophosphate dehydrogenase (IMPDH) is an evolved antiviral and antimicrobial therapeutic target. The aim is to triple-hit the viral and fungal RdRps and the human IMPDH. Methods: In the current study, molecular docking combined with molecular dynamics simulation (MDS) is utilized to test nucleotide inhibitors against the RdRps of SARS-CoV-2 and Rhizopus oryzae (the main causing agent of mucormycosis) RdRp. Additionally, the same inhibitors targeted the human Inosine monophosphate dehydrogenase (IMPDH). Results: The results reveal a comparable binding affinity of four nucleotide derivatives compared to remdesivir and sofosbuvir against both IMPDH and the RdRps of SARS-CoV-2 and Rhizopus oryzae. The binding affinities are calculated using different conformations of the RdRps after 100 ns MDS and trajectories clustering. Conclusions: The current study suggests the triple inhibition potential of four nucleotide inhibitors against SARS-CoV-2 & R. oryzae RdRps and the human IMPDH, while experimental validation is yet to be performed.

Screening for Anti-Dengue Leads from Euphorbia hirta L. through In Silico Methods

Dengue is a vector-borne viral disease caused by Flavivirus. Current treatment and medicine are inadequate to eradicate the vector mosquitoes or to prevent the disease. Traditional healers have remedies for almost all ailments using natural products and plants. The well-known medicinal plant Euphorbia hirta L. is one of the herbs conventionally used for anti-dengue treatment even if the active compound responsible for the exact activity of the plant against the disease is not known. The plant is a rich source of various secondary metabolites. In silico screening of 76 small molecules of drug-value belongs to alkaloids, polyphenols and terpenes derived from the plant against dengue virus non-structural protein-5 protease and human inosine 5'-monophosphate dehydrogenase-II determined 16 biologically active compounds with possible activity to fight against dengue virus. Based on the therapeutic importance and drug-likeness parameters, the two best leads, 2-beta, 16-alpha, 19-trihydroxy-ent-kaurane and kaempferol were selected as promising candidates for developing anti-dengue drugs.

Optimization Strategies for Purification of Mycophenolic Acid Produced by Penicillium brevicompactum.

The microbial fermentation of Penicillium brevicompactum produces secondary metabolite mycophenolic acid (MPA), which exhibits antifungal, antiviral, antibacterial, and antitumor activity. It is also a potent, selective, non-competitive, and reversible inhibitor of the human inosine monophosphate dehydrogenase (IMPDH). This study is an attempt to optimize the MPA production through a fermentation process using Penicillium brevicompactum and its further purification process optimization. In the batch fermentation process, the maximum concentration of MPA (1.84g/L) was attained in a 3.7L stirred tank reactor. Response surface methodology (RSM) using central composite design (CCD) was employed as a statistical tool to investigate the effect of pH, the volume of eluent and flow rate of the mobile phase on MPA purification process. Under optimum conditions, the experimental yield was observed to be 84.12%, which matched well with the predictive yield of 84.42%. High-performance liquid chromatography (HPLC) and Fourier-transform infrared spectroscopy (FTIR) analysis of the fermented product was carried out to confirm the presence of mycophenolic acid. The MPA purification was done by using column chromatography technique. The purification of broth involved mycophenolic acid extraction by selecting different solvents on the basis of polarity and the extraction efficiency of solvent. Various solid support materials were used for MPA purification in column chromatography. The MPA recovery through alumina column was observed to be 84.12% under the optimum conditions, which was maximum elution as compared with other support materials. The optimized purification process yielded pure MPA crystals.

Discovery of novel human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents

The enzyme inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, and thus regulates the guanine nucleotide pool required for cell proliferation. Of the two isoforms, human IMPDH type 2 (hIMPDH2) is a validated molecular target for potential immunosuppressive, antiviral and anticancer chemotherapy. In search of newer hIMPDH2 inhibitors as potential anticancer agents, three novel series (A: 5-aminoisobenzofuran-1(3H)-one, B: 3,4-dimethoxyaniline and C: benzo[d]-[1,3]dioxol-5-ylmethanamine) were synthesized and evaluated for in vitro and cell-based activities. A total of 37 molecules (29–65) were screened for their in vitro hIMPDH2 inhibition, with particular emphasis on establishing their structure–activity relationship (SAR) trends. Eight compounds (hits, 30, 31, 33–35, 37, 41 and 43) demonstrated significant enzyme inhibition (>70% @ 10 μM); especially the A series molecules were more potent than B series (<70% inhibition @ 10 μM), while C series members were found to be inactive. The hIMPDH2 IC50 values for the hits ranged from 0.36 to 7.38 μM. The hits displaying >80% hIMPDH2 inhibition (30, 33, 35, 41 and 43) were further assessed for their cytotoxic activity against cancer cell lines such as MDA-MB-231 (breast adenocarcinoma), DU145 (prostate carcinoma), U87 MG (glioblastoma astrocytoma) and a normal cell line, NIH-3T3 (mouse embryonic fibroblast) using MTT assay. Most of the compounds exhibited higher cellular potency against cancer cell lines and notably lower toxicity towards NIH-3T3 cells compared to mycophenolic acid (MPA), a prototypical hIMPDH2 inhibitor. Two of the series A hits (30 and 35) were evaluated in human peripheral blood mononuclear cells (hPBMC) assay and found to be better tolerated than MPA. The calculated/predicted molecular and physicochemical properties were satisfactory with reference to drug-likeness. The molecular docking studies clearly demonstrated crucial interactions of the hits with the cofactor-binding site of hIMPDH2, further providing critical information for refining the design strategy. The present study reports the design and discovery of structurally novel hIMPDH2 inhibitors as potential anticancer agents and provides a guide for further research on the development of safe and effective anticancer agents, especially against glioblastoma.

Human Inosine Monophosphate Dehydrogenase 2: Cryo-EM of Highly Flexible Filaments to Near Atomic Resolution

Human Inosine Monophosphate Dehydrogenase 2: Cryo-EM of Highly Flexible Filaments to Near Atomic Resolution

Newer human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents

Human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2), being an age-old target, has attracted attention recently for anticancer drug development. Mycophenolic acid (MPA), a well-known immunosuppressant drug, was used a lead structure to design and develop modestly potent and selective analogues. The steep structure–activity relationship (SAR) requirements of the lead molecule left little scope to synthesise newer analogues. Here, newer MPA amides were designed, synthesised and evaluated for hIMPDH2 inhibition and cellular efficacy in breast, prostate and glioblastoma cell lines. Few title compounds exhibited cellular activity profile better than MPA itself. The observed differences in the overall biological profile could be attributed to improved structural and physicochemical properties of the analogues over MPA. This is the first report of the activity of MPA derivatives in glioblastoma, the most aggressive brain cancer.

RNAi mediated gene silencing of ITPA using a targeted nanocarrier: Apoptosis induction in SKBR3 cancer cells.

A pure nucleotide pool is required for high-fidelity DNA replication and prevention of carcinogenesis in living cells. Human inosine triphosphatase (ITPase), encoded by the ITPA gene, plays a critical role in maintaining the purity of the cellular nucleotide pool by excluding nucleotides that enhance mutagenesis. ITPase is a nucleoside triphosphate pyrophosphatase that hydrolyzes the non-canonical nucleotides inosine triphosphate (ITP) and xanthine triphosphate (XTP). The monophosphate products of ITPase reactions are subsequently excluded from the nucleotide pool and the improper substitution of ITP and XTP into DNA and RNA is prevented. Previous studies show that deficiency in ITPA can suppress cellular growth and enhance DNA instability. In this study, we evaluated the influence of effective ITPA down-regulation on the induction of apoptosis in a human cancer cell line using folate-single wall nanotubes (SWNT) as a targeted nanocarrier. We assessed whether SWNT enhances IPTA-siRNA transfection efficiency in cancer cells using folate as a homing device. Since folate receptor is considerably overexpressed in cancer cells, conjugation of SWNTs to folate could enhance their cancer-specific penetrance. We found that nanocarrier mediated ITPA-siRNA transfection into SKBR3 cells caused significant reduction of ITPA mRNA expression level and complete down-regulation of the ITPase protein product. The silencing of ITPA led to promotion of apoptosis in SWNT-treated SKBR3 cancer cells.

Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC50 values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity.

Simulation of mutant P32T homo- and heterodimers of human inosine triphosphate pyrophosphatase hITPA

The structure of three forms of a dimeric enzyme, human inosine triphosphate pyrophosphatase, is considered to identify the enzyme conformation changes causing the inactivation effect of a P32T mutation. Analysis of a nanosecond molecular dynamics is performed; the mean square deviations of the atoms between the wild-type and mutant homodimers, and also the heterodimer are calculated. A 3 ns modeling shows a greater displacement of atoms in mutant protomers. During molecular dynamics simulation, the strongest changes are observed in the loop between α2 and β2 (amino acid residues 28-33, an area of the P32T mutation), the loop between β5 and β6, and the C-terminal amino acid residues. The loop between (α2 and β2 has two conformations characterized by different positions of the Phe31 aromatic group. The distance between Cys33 (Cα) and Phe31 (C(z)) for wild-type and mutant protomers was -9 and 5.5 Å, respectively. These conformations were kept constant.

A real‐time and hands‐on research course in protein purification and characterization: Purification and crystal growth of human inosine triphosphate pyrophosphatase

A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at the University of Nebraska at Omaha. This unique course offers students an opportunity to work hands-on with modern instrumentation not normally found in a predominately undergraduate institution, and to complete an entire research project in a realistic timeframe via a time-intensive curriculum as a special summer session. The course content gives a strong background in protein structure/chemistry, purification principles, protocol development, optimization strategies, use and programming of an automated chromatography instrument, and characterization strategies with an emphasis on X-ray crystallography. The laboratory portion offers students the chance to purify a protein (human inosine triphosphate pyrophosphatase) from start to finish, program and use an ÄKTA fast protein liquid chromatography instrument, and to grow and analyze their own protein crystals using their purified protein. This innovative laboratory experience gives the participating students the opportunity to complete a miniresearch project in real time and enhances their overall understanding of important biochemical research techniques and the instrumentation involved, fostering a better understanding of the research process all within a classroom setting. Evaluations and feedback concerning this course indicated a positive learning environment, a retention of knowledge and skills, a belief that the skill set learned continues to be useful in current endeavors, and a sense of accomplishment in the completion of an actual research project within the confines of a class setting.

Commentary: Innovation in the biochemistry/molecular biology lab

In 2009, we initiated this special section on innovative labs with the publication of four outstanding experiments for use in BMB courses [1], and it was continued in 2010 with another four experiments [2]. A primary goal for this series is to provide examples of projects describing lab activities based on modern themes presented in an innovative, engaging manner, preferably using student-centered learning styles such as problem-based learning (PBL), research-based learning, process oriented guided inquiry learning (POGIL), and others [3]. We seek, especially, manuscripts describing laboratory activities that use high impact educational practices. Among the activities that have been shown to enhance deep learning, several are regularly employed in student-centered laboratories including undergraduate research, writing-intensive courses, collaborative assignments and projects, and internships [4]. In actual practice, this means that students are actively involved in the following: the design and performance of an experiment, the collection and analysis of data, and final write-up and/or oral presentation of the experimental results. In this issue, we introduce the special lab series for 2011 with the four excellent articles listed below. In addition to the wide topical range of the articles, they are characterized by other diverse aspects. Diversity is seen in the level of students for which the experimental projects were written. One article is written for undergraduate medical students (the typical path for medical study outside the United States); another is for graduate students in BMB programs; a third is for advanced undergraduates and starting graduate students in BMB; finally, another article is for undergraduate majors that, with modification, may be used in the range of high school biology labs to advanced undergraduate labs. All eight of the articles in the first two years of the series were designed for undergraduate labs. Another example of diversity is the global regions from which the 2011 articles originated. Two are of international origin (Turkey and Brazil) and two are from the United States. All articles from previous years were from the United States. The global representation is especially encouraging, as it will truly serve our international audience of readers. We hope that instructors find the articles useful for their classes and pedagogically engaging for their students. Manuscripts and suggestions for future articles in the series are welcomed. Special study modules in a problem-based-learning medical curriculum: An innovative laboratory research practice supporting introduction to research methodology in the undergraduate curriculum. Gül Güner-Akdogan, Zahide Çavdar, Nilgün Yener, Tuncay Küme, Mehtap-Yüksel Egrilmez, and Halil Resmi. Department of Medical Biochemistry and Central Research Laboratory, School of Medicine, Dokuz Eylül University, Izmir, Turkey. A practical teaching course in directed protein evolution using the green fluorescent protein as a model. Roberto Ruller, Rafael Silva-Rocha, Artur Silva, Maria Paula Cruz Schneider, and Richard John Ward. Departamento de Química, Faculdade de Filosofia Cieˇncias e Letras de Ribeirão Preto, Universidade São Paulo, São Paulo, Brazil and Departamento de Genética, Laboratório de Polimorfismo de DNA, Universidade Federal do Pará, Campus Universitá rio do Guamá, Rua Augusto Correa 01, 66075-970 Belé m-PA, Brazil. From trace evidence to bioinformatics: Putting bryophytes into molecular biology education. Linda Fuselier, Azhar Bougary, and Michelle Malott. Department of Biosciences, Minnesota State University-Moorhead, Moorhead, MN 56563. A real-time and hands-on research course in protein purification and characterization: Purification and crystal growth of human inosine triphosphate pyrophosphatase. Jodi L. Kreiling, Kerry Brader, Carol Kolar, and Gloria E. O. Borgstahl. Department of Chemistry, University of Nebraska at Omaha, Omaha, NE 68182 and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198.

Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

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.

Interpopulation Variation Frequency of Human Inosine 5′-Monophosphate Dehydrogenase Type II (IMPDH2) Genetic Polymorphisms

Inosine monophosphate dehydrogenase type II (IMPDH2) is the target for immunosuppression by mycophenolic acid and has been linked to resistance of tumor cells to chemotherapy. Determining the frequency of IMPDH2 genetic polymorphisms can inform the design of clinical studies investigating the impact of IMPDH2 genetic variability on both cancer therapy and immunosuppression. Frequencies of three IMPDH2 polymorphisms (rs4974081, rs5848860, and rs11557540) in >400 DNA samples from four different racial/ethnic groups (Caucasian, African American, Hispanic, and Asian populations) were characterized by the pyrosequencing genotyping method. For rs5848860 1591 CTT/- (500 G/EG) and rs11557540 1345 A/G (418 D/G), we did not observe any variant alleles in all DNA samples from these populations, which suggests that these variants could simply be sequencing errors rather than real polymorphisms. The observed frequency of the 5'-upstream single-nucleotide polymorphism (SNP) rs4974081 A/G was similar to that previously reported in the NCBI databank (dbSNP). An in silico functional analysis using FASTSNP predicts that this promoter SNP rs4974081 (-3624 A/G) could be a potential transcription factor binding site. This finding suggests that rs4974081 could be a good candidate SNP for association studies with immunosuppressive and chemotherapeutic therapy outcomes.

A novel variant L263F in human inosine 5′-monophosphate dehydrogenase 2 is associated with diminished enzyme activity

Inosine 5'-monophosphate dehydrogenase 2 is required for purine synthesis in activated lymphocytes. Variants in the IMPDH2 gene may account for the large inter-individual variability in baseline enzyme activity, immunosuppressive efficacy and side effects in transplant recipients receiving mycophenolic acid. Therefore, the objective of this study was to identify and functionally characterize IMPDH2 variants. DNA samples from 152 solid organ transplant patients were screened at exons and exon/intron junctions of the IMPDH2 genes by PCR amplification followed by bidirectional direct DNA sequencing. Genetic variant was constructed by site-directed mutagenesis and transformed to an inosine 5'-monophosphate dehydrogenase-deficient strain of Escherichia coli h712. Proteins were purified to homogeneity and the enzymatic activity was measured by reduced nicotinamide adenine dinucleotide production. Nine genetic variants were identified in the IMPDH2 gene, with frequencies of the rarer alleles ranging from 0.5 to 10.2%. A novel nonsynonymous variant L263F was identified, and the kinetic assay demonstrated that the inosine 5'-monophosphate dehydrogenase activity of L263F variant was decreased to 10% of the wild-type. The Ki for mycophenolic acid inhibition of the L263F variant was comparable with the wild-type, and the variant Km for inosine 5'-monophosphate and nicotinamide adenine dinucleotide did not change significantly. IMPDH2 has low genetic diversity, but the nonsynonymous variant L263F has a significant impact on inosine 5'-monophosphate dehydrogenase activity. This novel functional variant may be one of the factors contributing to the inter-individual difference of baseline inosine 5'-monophosphate dehydrogenase activity as well as drug efficacy and adverse events in transplant patients.

HOMOLOGY MODELING AND MOLECULAR DYNAMICS STUDY OF HUMAN INOSINE TRIPHOSPHATE PYROPHOSPHATASE

With homology-modeling techniques, molecular mechanics and molecular dynamics methods, a 3D structure model of the human inosine triphosphatase (ITPase; EC 3.6.1.19) is created and refined. This model is further assessed by Profile-3D and ProStat, which confirm that the refined model is reliable. With this model, a flexible docking study is performed, and the results indicate that Arg178, Lys19 and Glu44 are three important determinant residues in substrate binding because they have prominent interaction energies with ITP and form strong hydrogen bonds with ITP. In addition, we further find that the P32T substitution alters the α-helices of ITPase but the β-sheets are almost not changed, and the mutation induces the interaction energy between ITPase and ITP to increase, which are consistent with the conclusion predicted by Sumi et al.8 The results from the mutagenesis imply that Pro32 is vital for the catalytic activity.

Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase.

The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 A resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix alpha1, the loop before alpha6 and helix alpha7 to cap off the active site when substrate is bound.