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International Journal of Molecular... May 2024Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of...
Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.
Topics: Cystathionine beta-Synthase; Mutation; Protein Binding; Mutagenesis, Site-Directed; Adenine Nucleotides; Protein Domains; Pyrophosphatases; Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Inorganic Pyrophosphatase; Models, Molecular; Binding Sites
PubMed: 38891956
DOI: 10.3390/ijms25115768 -
Acta Crystallographica. Section D,... Sep 2020Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to...
Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design campaigns against this attractive cancer therapeutic target.
Topics: Enzyme Inhibitors; Humans; Membrane Proteins; Neoplasms; Phosphoric Diester Hydrolases; Protein Binding; Protein Conformation; Pyrophosphatases
PubMed: 32876064
DOI: 10.1107/S2059798320010505 -
The Journal of Biological Chemistry Nov 1948
Topics: NAD; Pyrophosphatases
PubMed: 18889922
DOI: No ID Found -
Nature Chemical Biology Sep 2016RNA capping and decapping are thought to be distinctive features of eukaryotes. The redox cofactor NAD was recently discovered to be attached to small regulatory RNAs in...
RNA capping and decapping are thought to be distinctive features of eukaryotes. The redox cofactor NAD was recently discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner, and Nudix hydrolase NudC was found to act as a NAD-decapping enzyme in vitro and in vivo. Here, crystal structures of Escherichia coli NudC in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product. Biochemical mutation analysis identifies the conserved Nudix motif as the catalytic center of the enzyme, which needs to be homodimeric, as the catalytic pocket is composed of amino acids from both monomers. NudC is single-strand specific and has a purine preference for the 5'-terminal nucleotide. The enzyme strongly prefers NAD-linked RNA (NAD-RNA) over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.
Topics: Biocatalysis; Crystallography, X-Ray; Escherichia coli; Models, Molecular; Protein Conformation; Pyrophosphatases; Nudix Hydrolases
PubMed: 27428510
DOI: 10.1038/nchembio.2132 -
Biochemistry and Molecular Biology... 2011A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at...
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.
Topics: Chemistry; Crystallization; Crystallography, X-Ray; Educational Technology; Electrophoresis, Polyacrylamide Gel; Humans; Nebraska; Problem-Based Learning; Pyrophosphatases; Reproducibility of Results; Research; Research Design; Time Factors; Universities
PubMed: 21433250
DOI: 10.1002/bmb.20432 -
PloS One Sep 2007It is recognized that the ability of adipose tissue to expand in response to energy excess, i.e. adipocyte maturation, is important in determining systemic abnormalities...
BACKGROUND
It is recognized that the ability of adipose tissue to expand in response to energy excess, i.e. adipocyte maturation, is important in determining systemic abnormalities in glucose and lipid metabolism. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1, also known as PC-1) has been recently reported to be involved in the pathogenesis of insulin resistance and related diseases. However, its role on adipose tissue physiology as a mechanism of systemic insulin resistance is not understood. This study was performed to evaluate whether ENPP1 is regulated during adipogenesis and whether over-expression in adipocytes can affect adipocyte maturation, a potential novel mechanism of ENPP1-related insulin resistance.
METHODOLOGY/PRINCIPAL FINDINGS
ENPP1 expression was found down-regulated during 3T3-L1 maturation, and over-expression of human ENPP1 in 3T3-L1 (pQCXIP-ENPP1 vector) resulted in adipocyte insulin resistance and in defective adipocyte maturation. Adipocyte maturation was more efficient in mesenchymal embryonal cells from ENPP1 knockout mice than from wild-type.
CONCLUSIONS
We identify ENPP1 as a novel mechanism of defective adipocyte maturation. This mechanism could contribute to the pathogenesis of insulin resistance in absence of obesity.
Topics: 3T3-L1 Cells; Adipocytes; Animals; Blotting, Western; Humans; Mice; Phosphoric Diester Hydrolases; Pyrophosphatases; RNA, Messenger
PubMed: 17849011
DOI: 10.1371/journal.pone.0000882 -
Molecules and Cells Apr 2002The gene encoding Aquifex pyrophilus (Apy) pyrophosphatase was cloned and sequenced. The deduced amino acid sequence of Apy pyrophosphatase showed a 94.2% homology to...
The gene encoding Aquifex pyrophilus (Apy) pyrophosphatase was cloned and sequenced. The deduced amino acid sequence of Apy pyrophosphatase showed a 94.2% homology to Aquifex aeolicus (Aae) pyrophosphatase. The gene exhibits a difference in the codon usage at the third position from Aae pyrophosphatase. The gene was expressed under the control of a tac promoter in E. coli. The recombinant Apy pyrophosphatase was purified 18.7-fold with a 52.8% yield and a specific activity of 26.2 U mg(-1) protein. The native enzyme has a homotetramer of 177 amino acids. The enzyme shows optimal activity in pH 7.5. The optimum temperature was approximately 70 degrees C. A divalent cation was absolutely required for the enzyme activity; Mg2+ was the most effective.
Topics: Amino Acid Sequence; Bacteria; Base Sequence; Cloning, Molecular; Genes, Bacterial; Molecular Sequence Data; Molecular Weight; Pyrophosphatases; Sequence Alignment; Sequence Analysis, DNA; Temperature
PubMed: 12018853
DOI: No ID Found -
Scientific Reports May 2021The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct...
The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry.
Topics: Adenosine Monophosphate; Aminohydrolases; Catalysis; Catalytic Domain; Histidine; Medicago truncatula; Metabolic Networks and Pathways; Phylogeny; Protein Structure, Tertiary; Pyrophosphatases; Sequence Alignment
PubMed: 33958623
DOI: 10.1038/s41598-021-88920-2 -
The FEBS Journal Nov 2017The ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins mediates purinergic signaling by degrading extracellular nucleotides and also participates...
UNLABELLED
The ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins mediates purinergic signaling by degrading extracellular nucleotides and also participates in phospholipid metabolism. NPP5 (ENPP5) is the least characterized member of this group and its specific role is unknown. This enzyme does not display activity on certain nucleotides and on other typical NPP substrates. In order to gain insights into its function, we determined the crystal structure of human and murine NPP5. Structural comparison with close homologs revealed a key phenylalanine to tyrosine substitution that prevents efficient hydrolysis of nucleotide diphosphates and triphosphates; reversal of this mutation enabled degradation of these molecules. Interestingly, NPP5 is able to cleave nicotinamide adenine dinucleotide (NAD), suggesting a potential role of this enzyme in NAD-based neurotransmission. An NPP5-specific metal binding motif is found adjacent to the active site, although its significance is unclear. These findings expand our understanding of substrate specificity within the NPP family.
DATABASE
Structural data are available in the Protein Data Bank under the accession numbers 5VEM, 5VEN, and 5VEO.
Topics: Animals; Crystallography, X-Ray; Humans; Hydrolysis; Mice; Models, Molecular; NAD; Phosphoric Diester Hydrolases; Pyrophosphatases; Recombinant Proteins; Tyrosine
PubMed: 28898552
DOI: 10.1111/febs.14266 -
Journal of Medicinal Chemistry Feb 2016The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates...
The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.
Topics: Dose-Response Relationship, Drug; Drug Discovery; Enzyme Inhibitors; HL-60 Cells; Humans; Ligands; Molecular Structure; Pyrophosphatases; Structure-Activity Relationship
PubMed: 26771665
DOI: 10.1021/acs.jmedchem.5b01741