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The FEBS Journal Sep 2016Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as...
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer-like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D. virilis dut gene are not identical due to several point mutations. We investigated the potential evolutionary pathway that led to the emergence of this extant fused trimeric dUTPase in D. virilis. The herein proposed scenario involves two sequential gene duplications followed by sequence divergence amongst the dut repeats. This pathway thus requires the existence of a transient two-repeat-containing fused dimeric dUTPase intermediate. We identified the corresponding ancestral dUTPase single repeat enzyme together with its tandem repeat evolutionary intermediate and characterized their enzymatic function and structural stability. We additionally engineered and characterized artificial single or tandem repeat constructs from the extant enzyme form to investigate the influence of the emergent residue alterations on the formation of a functional assembly. The observed severely impaired stability and catalytic activity of these latter constructs provide a plausible explanation for evolutionary persistence of the extant fused trimeric D. virilis dUTPase form. For the ancestral homotrimeric and the fused dimeric intermediate forms, we observed strong catalytic and structural competence, verifying viability of the proposed evolutionary pathway. We conclude that the progression along the herein described evolutionary trajectory is determined by the retained potential of the enzyme for its conserved three-fold structural symmetry.
Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Drosophila; Drosophila Proteins; Enzyme Stability; Evolution, Molecular; Gene Duplication; Genes, Insect; Models, Molecular; Phylogeny; Point Mutation; Protein Folding; Protein Structure, Quaternary; Pyrophosphatases; Sequence Homology, Amino Acid; Tandem Repeat Sequences
PubMed: 27380921
DOI: 10.1111/febs.13800 -
Cellular and Molecular Gastroenterology... 2021
Review
Topics: Animals; Female; Genotype; Immunosuppressive Agents; Mice; Pregnancy; Purines; Pyrophosphatases
PubMed: 33766784
DOI: 10.1016/j.jcmgh.2021.03.006 -
Trends in Biochemical Sciences Jul 1992Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi). Although... (Review)
Review
Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi). Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism. PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions. Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism.
Topics: Amino Acid Sequence; Animals; Bacteria; Binding Sites; Biological Evolution; Inorganic Pyrophosphatase; Kinetics; Mitochondria; Molecular Sequence Data; Mutagenesis, Site-Directed; Pyrophosphatases; Retina; Saccharomyces cerevisiae; Sequence Homology, Nucleic Acid; Structure-Activity Relationship
PubMed: 1323891
DOI: 10.1016/0968-0004(92)90406-y -
Endocrine Reviews Feb 2008Insulin resistance is a major feature of most patients with type 2 diabetes mellitus (T2D). A number of laboratories have observed that PC-1 (membrane [corrected]... (Review)
Review
The role of membrane glycoprotein plasma cell antigen 1/ectonucleotide pyrophosphatase phosphodiesterase 1 in the pathogenesis of insulin resistance and related abnormalities.
Insulin resistance is a major feature of most patients with type 2 diabetes mellitus (T2D). A number of laboratories have observed that PC-1 (membrane [corrected] glycoprotein plasma cell antigen 1; also termed [corrected] ectonucleotide pyrophosphatase phosphodiesterase 1 or ENPP1) [corrected] is either overexpressed or overactive in muscle, adipose tissue, fibroblasts, and other tissues of insulin-resistant individuals, both nondiabetic and diabetic. Moreover, PC-1 (ENPP1) overexpression [corrected] in cultured cells in vitro and in transgenic mice in vivo, [corrected] impairs insulin stimulation of insulin receptor (IR) activation and downstream signaling. PC-1 binds to the connecting domain of the IR alpha-subunit that is located in residues 485-599. The connecting domain transmits insulin binding in the alpha-subunit to activation of tyrosine kinase activation in the beta-subunit. When PC-1 is overexpressed, it inhibits insulin [corrected]induced IR beta-subunit tyrosine kinase activity. In addition, a polymorphism of PC-1 (K121Q) in various ethnic populations is closely associated with insulin resistance, T2D, and cardio [corrected] and nephrovascular diseases. The product of this polymorphism has a 2- to 3-fold increased binding affinity for the IR and is more potent than the wild-type PC-1 protein (K121K) in inhibiting the IR. These data suggest therefore that PC-1 is a candidate protein that may play a role in human insulin resistance and T2D by its overexpression, its overactivity, or both.
Topics: Animals; Diabetes Complications; Diabetes Mellitus, Type 2; Disease Models, Animal; Female; Gene Expression; Genetic Variation; Humans; Insulin Resistance; Obesity; Phosphoric Diester Hydrolases; Polycystic Ovary Syndrome; Polymorphism, Genetic; Protein Structure, Quaternary; Pyrophosphatases; Receptor, Insulin
PubMed: 18199690
DOI: 10.1210/er.2007-0004 -
The FEBS Journal Dec 2021Nudix hydrolase 9 (NUDT9) is a member of the nucleoside linked to another moiety X (NUDIX) protein superfamily, which hydrolyses a broad spectrum of organic...
Nudix hydrolase 9 (NUDT9) is a member of the nucleoside linked to another moiety X (NUDIX) protein superfamily, which hydrolyses a broad spectrum of organic pyrophosphates from metabolic processes. ADP-ribose (ADPR) has been the only known endogenous substrate accepted by NUDT9 so far. The Ca -permeable transient receptor potential melastatin subfamily 2 (TRPM2) channel contains a homologous NUDT9-homology (NUDT9H) domain and is activated by ADPR. Sustained Ca influx via ADPR-activated TRPM2 triggers apoptotic mechanisms. Thus, a precise regulation of cellular ADPR levels by NUDT9 is essential. A detailed characterization of the enzyme-substrate interaction would help to understand the high substrate specificity of NUDT9. Here, we analysed ligand binding to NUDT9 using a variety of biophysical techniques. We identified 2'-deoxy-ADPR as an additional substrate for NUDT9. Similar enzyme kinetics and binding affinities were determined for the two ligands. The high-affinity binding was preserved in NUDT9 containing the mutated NUDIX box derived from the human NUDT9H domain. NMR spectroscopy indicated that ADPR and 2'-deoxy-ADPR bind to the same binding site of NUDT9. Backbone resonance assignment and subsequent molecular docking allowed further characterization of the binding pocket. Substantial conformational changes of NUDT9 upon ligand binding were observed which might allow for the development of NUDT9-based ADPR fluorescence resonance energy transfer sensors that may help with the analysis of ADPR signalling processes in cells in the future.
Topics: Adenosine Diphosphate Ribose; Binding Sites; Binding, Competitive; Humans; Kinetics; Ligands; Magnetic Resonance Spectroscopy; Molecular Docking Simulation; Mutation; Protein Binding; Protein Conformation; Pyrophosphatases; Scattering, Small Angle; Substrate Specificity; X-Ray Diffraction
PubMed: 34189846
DOI: 10.1111/febs.16097 -
The Journal of Biological Chemistry Jul 1948
Topics: NADP; Pyrophosphatases
PubMed: 18871268
DOI: No ID Found -
Microbiological Reviews Jun 1983
Review
Topics: Adenine Nucleotides; Allosteric Regulation; Bacteria; Glutathione; Inorganic Pyrophosphatase; Kinetics; Mutation; Pyrophosphatases; Species Specificity; Subcellular Fractions
PubMed: 6135978
DOI: 10.1128/mr.47.2.169-178.1983 -
Mutation Research Nov 2010Inosine triphosphate pyrophosphatase (ITPA protein) (EC 3.6.1.19) hydrolyzes deaminated purine nucleoside triphosphates, such as ITP and dITP, to their corresponding... (Review)
Review
Inosine triphosphate pyrophosphatase (ITPA protein) (EC 3.6.1.19) hydrolyzes deaminated purine nucleoside triphosphates, such as ITP and dITP, to their corresponding purine nucleoside monophosphate and pyrophosphate. In mammals, this enzyme is encoded by the Itpa gene. Using the Itpa gene-disrupted mouse as a model, we have elucidated the biological significance of the ITPA protein and its substrates, ITP and dITP. Itpa(-/-) mice exhibited peri- or post-natal lethality dependent on the genetic background. The heart of the Itpa(-/-) mouse was found to be structurally and functionally abnormal. Significantly higher levels of deoxyinosine and inosine were detected in nuclear DNA and RNA prepared from Itpa(-/-) embryos compared to wild type embryos. In addition, an accumulation of ITP was observed in the erythrocytes of Itpa(-/-) mice. We found that Itpa(-/-) primary mouse embryonic fibroblasts (MEFs), which have no detectable ability to generate IMP from ITP in whole cell extracts, exhibited a prolonged population-doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in their nuclear DNA, in comparison to primary MEFs prepared from wild type embryos. These results revealed that (1) ITP and dITP are spontaneously produced in vivo and (2) accumulation of ITP and dITP is responsible for the harmful effects observed in the Itpa(-/-) mouse. In addition to its effect as the precursor nucleotide for RNA transcription, ITP has the potential to influence the activity of ATP/GTP-binding proteins. The biological significance of ITP and dITP in the nucleotide pool remains to be elucidated.
Topics: Animals; Chromosome Aberrations; DNA; Deamination; Heart Defects, Congenital; Humans; Mice; Mice, Transgenic; Purine Nucleotides; Pyrophosphatases; RNA; Signal Transduction
PubMed: 20601097
DOI: 10.1016/j.mrgentox.2010.06.009 -
Journal of Molecular Biology Apr 2019Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H and/or Na) translocation. We determined crystal structures and functions of Vigna...
Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H and/or Na) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH-PPase), the VrH-PPase-2P complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three VrH-PPases complexes (P-, 2P- and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2P-bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among VrH-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225-R562 is at the way out of the exit channel of VrH-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225-R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer.
Topics: Amino Acid Sequence; Crystallography, X-Ray; Hydrophobic and Hydrophilic Interactions; Ion Transport; Models, Molecular; Plant Proteins; Protein Conformation; Proton Pumps; Protons; Pyrophosphatases; Vigna
PubMed: 30878480
DOI: 10.1016/j.jmb.2019.03.009 -
International Journal of Molecular... Oct 2022Pyrophosphate (PP) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP can inhibit industrial synthesis. Pyrophosphatases (PPases) can...
Pyrophosphate (PP) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32-41%, while that of short-chain DNA was only improved by 9.5-15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.
Topics: Thermococcus; Inorganic Pyrophosphatase; Diphosphates; Archaeal Proteins; Pyrophosphatases; Uridine Diphosphate
PubMed: 36361526
DOI: 10.3390/ijms232112735