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Acta Crystallographica Section A: Foundations of Crystallography (1)
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry (1)
The Journal of Biological Chemistry (1)
Vorontsov, Ivan I. (2)
Anderson, Wayne F. (1)
Benedict, Jason (1)
Bennett, Brian (1)
Breece, Robert M. (1)
Brunzelle, Joseph S. (1)
Chen, Yu-Sheng (1)
Coppens, Philip (1)
Crowder, Michael W. (1)
Easton, J. Allen (1)
Graber, Tim (1)
Kim, Lydia R. (1)
Kiryukhina, Olga (1)
Messerschmidt, Marc (1)
Minasov, George (1)
Novozhilova, Irina (1)
Rosenzweig, Amy C. (1)
Scheins, Stephan (1)
Shuvalova, Ludmilla (1)
Sugarbaker, Stacy A. (1)
Tierney, David L. (1)
Vorontsov, Ivan (1)
Yatsunyk, Liliya A. (1)
Zheng, Shao-Liang (1)
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Characterization of the Deoxynucleotide Triphosphate Triphosphohydrolase (dNTPase) Activity of the EF1143 Protein from Enterococcus faecalis and Crystal Structure of the Activator-Substrate Complex*
Brunzelle, Joseph S.
Anderson, Wayne F.
The Journal of Biological Chemistry
The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn2+. In contrast, with Mg2+ hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the α-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the α3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.
Allosteric Regulation; Crystal Structure; Enzyme Catalysis; Enzyme Structure; Metalloenzymes; Nucleoside Nucleotide Metabolism; Phosphodiesterases; Deoxynucleotide Triphosphate Triphosphohydrolase
Time-resolved synchrotron diffraction and theoretical studies of very short-lived photo-induced molecular species
Acta Crystallographica Section A: Foundations of Crystallography
Excited-state geometries determined by time-resolved synchrotron diffraction are summarized with emphasis on their comparison with a series of theoretical results. The relative merits of monochromatic and polychromatic (Laue) techniques are discussed.
Definitive experimental results on the geometry of fleeting species are at the time of writing still limited to monochromatic data collection, but methods for modifications of the polychromatic Laue data to increase their accuracy and their suitability for pump–probe experiments have been implemented and are reviewed. In the monochromatic experiments summarized, excited-state conversion percentages are small when neat crystals are used, but are higher when photoactive species are embedded in an inert framework in supramolecular crystals. With polychromatic techniques and increasing source brightness, smaller samples down to tenths of a micrometre or less can be used, increasing homogeneity of exposure and the fractional population of the excited species. Experiments described include a series of transition metal complexes and a fully organic example involving excimer formation. In the final section, experimental findings are compared with those from theoretical calculations on the isolated species. Qualitative agreement is generally obtained, but the theoretical results are strongly dependent on the details of the calculation, indicating the need for further systematic analysis.
pump–probe experiments; time-resolved diffraction; excited-state molecular geometries; excimers
Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli
Yatsunyk, Liliya A.
Easton, J. Allen
Kim, Lydia R.
Sugarbaker, Stacy A.
Breece, Robert M.
Tierney, David L.
Crowder, Michael W.
Rosenzweig, Amy C.
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
ZnuA is the periplasmic Zn2+-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn2+-bound, and Co2+-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn2+ with Co2+ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn2+ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn2+ (estimated Kd < 20 nM), Co2+, Ni2+, Cu2+, Cu+, and Cd2+, but not Mn2+. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn2+ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer.
ZnuA; Zinc-specific uptake system; Zinc binding; ATP binding cassette transporter
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