Publications

• The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity.

  Barnes C.O., Wu Y., Song J., Lin G., Baxter E.L., Brewster A.S., Nagarajan V., Holmes A., Soltis S.M., Sauter N.K., Ahn J., Cohen A.E., Calero G.

  Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks revers ... >> More

  Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, <i>Escherichia coli</i> (<i>Ec</i>)<i>-</i>dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric <i>Ec-</i>dGTPase, including an X-ray free-electron laser structure of the free <i>Ec</i>-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo-form. Moreover, despite no sequence homology, <i>Ec</i>-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, <i>Ec</i>-dGTPase residues at the end of the substrate-binding pocket mimic Watson-Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally "opening" a tyrosine gate allowing enhanced substrate binding. << Less

  Proc Natl Acad Sci U S A 116:9333-9339(2019) [PubMed] [EuropePMC]

• Impaired dNTPase activity of SAMHD1 by phosphomimetic mutation of Thr-592.

  Tang C., Ji X., Wu L., Xiong Y.

  SAMHD1 is a cellular protein that plays key roles in HIV-1 restriction and regulation of cellular dNTP levels. Mutations in SAMHD1 are also implicated in the pathogenesis of chronic lymphocytic leukemia and Aicardi-Goutières syndrome. The anti-HIV-1 activity of SAMHD1 is negatively modulated by ph ... >> More

  SAMHD1 is a cellular protein that plays key roles in HIV-1 restriction and regulation of cellular dNTP levels. Mutations in SAMHD1 are also implicated in the pathogenesis of chronic lymphocytic leukemia and Aicardi-Goutières syndrome. The anti-HIV-1 activity of SAMHD1 is negatively modulated by phosphorylation at residue Thr-592. The mechanism underlying the effect of phosphorylation on anti-HIV-1 activity remains unclear. SAMHD1 forms tetramers that possess deoxyribonucleotide triphosphate triphosphohydrolase (dNTPase) activity, which is allosterically controlled by the combined action of GTP and all four dNTPs. Here we demonstrate that the phosphomimetic mutation T592E reduces the stability of the SAMHD1 tetramer and the dNTPase activity of the enzyme. To better understand the underlying mechanisms, we determined the crystal structures of SAMHD1 variants T592E and T592V. Although the neutral substitution T592V does not perturb the structure, the charged T592E induces large conformational changes, likely triggered by electrostatic repulsion from a distinct negatively charged environment surrounding Thr-592. The phosphomimetic mutation results in a significant decrease in the population of active SAMHD1 tetramers, and hence the dNTPase activity is substantially decreased. These results provide a mechanistic understanding of how SAMHD1 phosphorylation at residue Thr-592 may modulate its cellular and antiviral functions. << Less

  J. Biol. Chem. 290:26352-26359(2015) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The purification and properties of deoxyguanosine triphosphate triphosphohydrolase from Escherichia coli.

  Seto D., Bhatnagar S.K., Bessman M.J.

  Deoxyguanosine triphosphate (dGTP) triphosphohydrolase (EC 3.1.5.1) has been purified approximately 16,000-fold to apparent homogeneity from extracts of Escherichia coli. The enzyme has a native molecular weight of 230,000 and a sedimentation coefficient of 9.3 S. Its subunit molecular weight deri ... >> More

  Deoxyguanosine triphosphate (dGTP) triphosphohydrolase (EC 3.1.5.1) has been purified approximately 16,000-fold to apparent homogeneity from extracts of Escherichia coli. The enzyme has a native molecular weight of 230,000 and a sedimentation coefficient of 9.3 S. Its subunit molecular weight derived from electrophoresis in denaturing polyacrylamide gels is 58,900, and it has a unique N-terminal sequence for the first 25 amino acids, which indicate that the native enzyme is composed of 4 homologous subunits. It is insensitive to sulfhydryl reagents and EDTA and can be heated to 60 degrees C for 60 min without loss of activity. The enzyme requires Mg2+ for activity, is highly specific for dGTP among the canonical deoxynucleoside triphosphates, and has a unique activity among nucleoside triphosphatases in that the products of the reaction are deoxyguanosine and inorganic tripolyphosphate. Preliminary evidence suggest that this enzyme is responsible for the optA mutant phenotype first described by Saito and Richardson (Saito, H., and Richardson, C.C. (1981) J. Virol. 37, 343-351). << Less

  J. Biol. Chem. 263:1494-1499(1988) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Enzymatic cleavage of deoxyguanosine triphosphate to deoxyguanosine and tripolyphosphate.

  KORNBERG S.R., LEHMAN I.R., BESSMAN M.J., SIMMS E.S., KORNBERG A.

  J Biol Chem 233:159-162(1958) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Discovery of a novel nucleoside immune signaling molecule 2'-deoxyguanosine in microbes and plants.

  Lu C., Wang Q., Jiang Y., Zhang M., Meng X., Li Y., Liu B., Yin Z., Liu H., Peng C., Li F., Yue Y., Hao M., Sui Y., Wang L., Cheng G., Liu J., Chu Z., Zhu C., Dong H., Ding X.

  <h4>Introduction</h4>Beneficial microorganisms play essential roles in plant growth and induced systemic resistance (ISR) by releasing signaling molecules. Our previous study obtained the crude extract from beneficial endophyte Paecilomyces variotii, termed ZNC (ZhiNengCong), which significantly e ... >> More

  <h4>Introduction</h4>Beneficial microorganisms play essential roles in plant growth and induced systemic resistance (ISR) by releasing signaling molecules. Our previous study obtained the crude extract from beneficial endophyte Paecilomyces variotii, termed ZNC (ZhiNengCong), which significantly enhanced plant resistance to pathogen even at 100 ng/ml. However, the immunoreactive components of ZNC remain unclear. Here, we further identified one of the immunoreactive components of ZNC is a nucleoside 2'-deoxyguanosine (2-dG).<h4>Objectives</h4>This paper intends to reveal the molecular mechanism of microbial-derived 2'-deoxyguanosine (2-dG) in activating plant immunity, and the role of plant-derived 2-dG in plant immunity.<h4>Methods</h4>The components of ZNC were separated using a high-performance liquid chromatography (HPLC), and 2-dG is identified using a HPLC-mass spectrometry system (LC-MS). Transcriptome analysis and genetic experiments were used to reveal the immune signaling pathway dependent on 2-dG activation of plant immunity.<h4>Results</h4>This study identified 2'-deoxyguanosine (2-dG) as one of the immunoreactive components from ZNC. And 2-dG significantly enhanced plant pathogen resistance even at 10 ng/ml (37.42 nM). Furthermore, 2-dG-induced resistance depends on NPR1, pattern-recognition receptors/coreceptors, ATP receptor P2K1 (DORN1), ethylene signaling but not salicylic acid accumulation. In addition, we identified Arabidopsis VENOSA4 (VEN4) was involved in 2-dG biosynthesis and could convert dGTP to 2-dG, and vne4 mutant plants were more susceptible to pathogens.<h4>Conclusion</h4>In summary, microbial-derived 2-dG may act as a novel immune signaling molecule involved in plant-microorganism interactions, and VEN4 is 2-dG biosynthesis gene and plays a key role in plant immunity. << Less

  J Adv Res 46:1-15(2023) [PubMed] [EuropePMC]

• Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase.

  Powell R.D., Holland P.J., Hollis T., Perrino F.W.

  The SAMHD1 protein is an HIV-1 restriction factor that is targeted by the HIV-2 accessory protein Vpx in myeloid lineage cells. Mutations in the SAMHD1 gene cause Aicardi-Goutières syndrome, a genetic disease that mimics congenital viral infection. To determine the physiological function of the SA ... >> More

  The SAMHD1 protein is an HIV-1 restriction factor that is targeted by the HIV-2 accessory protein Vpx in myeloid lineage cells. Mutations in the SAMHD1 gene cause Aicardi-Goutières syndrome, a genetic disease that mimics congenital viral infection. To determine the physiological function of the SAMHD1 protein, the SAMHD1 gene was cloned, recombinant protein was produced, and the catalytic activity of the purified enzyme was identified. We show that SAMHD1 contains a dGTP-regulated deoxynucleotide triphosphohydrolase. We propose that Vpx targets SAMHD1 for degradation in a viral strategy to control cellular deoxynucleotide levels for efficient replication. << Less

  J. Biol. Chem. 286:43596-43600(2011) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.