scholarly journals The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function

2015 ◽  
Vol 43 (14) ◽  
pp. 6934-6944 ◽  
Author(s):  
Claire Breslin ◽  
Peter Hornyak ◽  
Andrew Ridley ◽  
Stuart L. Rulten ◽  
Hana Hanzlikova ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Phosphate Binding

scholarly journals Insights into the Importance of Hydrogen Bonding in the γ-Phosphate Binding Pocket of Myosin: Structural and Functional Studies of Serine 236,

Biochemistry ◽  
2010 ◽  
Vol 49 (23) ◽  
pp. 4897-4907 ◽  
Author(s):  
Jeremiah J. Frye ◽  
Vadim A. Klenchin ◽  
Clive R. Bagshaw ◽  
Ivan Rayment
Keyword(s):  
Hydrogen Bonding ◽  
Binding Pocket ◽  
Phosphate Binding ◽  
Functional Studies

3′-Azidothymidine in the active site ofEscherichia colithymidine phosphorylase: the peculiarity of the binding on the basis of X-ray study

2014 ◽  
Vol 70 (4) ◽  
pp. 1155-1165 ◽  
Author(s):  
Vladimir Timofeev ◽  
Yulia Abramchik ◽  
Nadezda Zhukhlistova ◽  
Tatiana Muravieva ◽  
Ilya Fateev ◽  
...  
Keyword(s):  
Active Site ◽  
Thymidine Phosphorylase ◽  
Binding Pocket ◽  
Chemotherapeutic Agents ◽  
Nucleoside Analogues ◽  
Dimensional Structure ◽  
Phosphate Binding ◽  
X Ray ◽  
E Coli ◽  
Replacement Method

The structural study of complexes of thymidine phosphorylase (TP) with nucleoside analogues which inhibit its activity is of special interest because many of these compounds are used as chemotherapeutic agents. Determination of kinetic parameters showed that 3′-azido-3′-deoxythymidine (3′-azidothymidine; AZT), which is widely used for the treatment of human immunodeficiency virus, is a reversible noncompetitive inhibitor ofEscherichia colithymidine phosphorylase (TP). The three-dimensional structure ofE. coliTP complexed with AZT was solved by the molecular-replacement method and was refined at 1.52 Å resolution. Crystals for X-ray study were grown in microgravity by the counter-diffusion technique from a solution of the protein in phosphate buffer with ammonium sulfate as a precipitant. The AZT molecule was located with full occupancy in the electron-density maps in the nucleoside-binding pocket of TP, whereas the phosphate-binding pocket of the enzyme was occupied by phosphate (or sulfate) ion. The structure of the active-site cavity and conformational changes of the enzyme upon AZT binding are described in detail. It is found that the position of AZT differs remarkably from the positions of the pyrimidine bases and nucleoside analogues in other known complexes of pyrimidine phosphorylases, but coincides well with the position of 2′-fluoro-3′-azido-2′,3′-dideoxyuridine (N3FddU) in the recently investigated complex ofE. coliTP with this ligand (Timofeevet al., 2013). The peculiarities of the arrangement of N3FddU and 3′-azidothymidine in the nucleoside binding pocket of TP and correlations between the arrangement and inhibitory properties of these compounds are discussed.

scholarly journals The C113D Mutation in Human Pin1 Causes Allosteric Structural Changes in the Phosphate Binding Pocket of the PPIase Domain through the Tug of War in the Dual-Histidine Motif

Biochemistry ◽  
2014 ◽  
Vol 53 (34) ◽  
pp. 5568-5578 ◽  
Author(s):  
Ning Xu ◽  
Naoya Tochio ◽  
Jing Wang ◽  
Yu Tamari ◽  
Jun-ichi Uewaki ◽  
...  
Keyword(s):  
Structural Changes ◽  
Binding Pocket ◽  
Phosphate Binding ◽  
Ppiase Domain

scholarly journals Engineering Candida tenuis Xylose Reductase for Improved Utilization of NADH: Antagonistic Effects of Multiple Side Chain Replacements and Performance of Site-Directed Mutants under Simulated In Vivo Conditions

2005 ◽  
Vol 71 (10) ◽  
pp. 6390-6393 ◽  
Author(s):  
Barbara Petschacher ◽  
Bernd Nidetzky
Keyword(s):  
Xylose Reductase ◽  
Binding Pocket ◽  
Kinetic Mechanism ◽  
Side Chain ◽  
Multiple Site ◽  
Wild Type ◽  
Structural Effects ◽  
Phosphate Binding ◽  
And Performance

ABSTRACT Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2′-phosphate binding pocket were tested for NADPH versus NADH selectivity (R sel) in the presence of physiological reactant concentrations. The experimental R sel values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274→Arg and Arg-280→His substitutions, which individually improved wild-type R sel 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.

Structural and kinetic differences between human and Aspergillus fumigatusD-glucosamine-6-phosphate N-acetyltransferase

2008 ◽  
Vol 415 (2) ◽  
pp. 217-223 ◽  
Author(s):  
Ramon Hurtado-Guerrero ◽  
Olawale G. Raimi ◽  
Jinrong Min ◽  
Hong Zeng ◽  
Laura Vallius ◽  
...  
Keyword(s):  
Active Site ◽  
Gene Disruption ◽  
Biosynthetic Pathway ◽  
Binding Pocket ◽  
Immunocompromised Patients ◽  
Kinetic Characterization ◽  
Phosphate Binding ◽  
Fungal Enzyme ◽  
Pathway Gene ◽  
Steady State Kinetics

Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.

scholarly journals A Phosphate-Binding Pocket within the Platform-PAZ-Connector Helix Cassette of Human Dicer

2014 ◽  
Vol 53 (4) ◽  
pp. 606-616 ◽  
Author(s):  
Yuan Tian ◽  
Dhirendra K. Simanshu ◽  
Jin-Biao Ma ◽  
Jong-Eun Park ◽  
Inha Heo ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Phosphate Binding

scholarly journals Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

2016 ◽  
Vol 113 (49) ◽  
pp. 14031-14036 ◽  
Author(s):  
Suresh K. Kandasamy ◽  
Ryuya Fukunaga
Keyword(s):  
Rna Silencing ◽  
Point Mutations ◽  
Binding Pocket ◽  
Rna Cleavage ◽  
High Fidelity ◽  
Small Interfering Rnas ◽  
Phosphate Binding ◽  
Micro Rnas ◽  
Paz Domain

The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

scholarly journals Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

2007 ◽  
Vol 16 (11) ◽  
pp. 2368-2377 ◽  
Author(s):  
Manoj Cheriyan ◽  
Eric J. Toone ◽  
Carol A. Fierke
Keyword(s):  
Binding Pocket ◽  
Phosphate Binding ◽  
Kdpg Aldolase

scholarly journals The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4- benzoquinone and to FMN.

2021 ◽  
Author(s):  
Martin Alan Day ◽  
David Jarrom ◽  
Andrew J Christofferson ◽  
Antonio E Graziano ◽  
Ross Anderson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Furan Ring ◽  
Hydride Transfer ◽  
Binding Pocket ◽  
Phosphate Binding ◽  
Free Protein ◽  
E Coli ◽  
Nitrofuran Antibiotics ◽  
Dynamics Simulations

NfsA is a dimeric flavoprotein that catalyses the reduction of nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free E. coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.

scholarly journals Structural basis for DNA 5´-end resection by RecJ

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Kaiying Cheng ◽  
Hong Xu ◽  
Xuanyi Chen ◽  
Liangyan Wang ◽  
Bing Tian ◽  
...  
Keyword(s):  
Active Site ◽  
Deinococcus Radiodurans ◽  
Divalent Cations ◽  
Binding Pocket ◽  
Structural Basis ◽  
Phosphate Binding ◽  
Terminal Domain ◽  
Dna Strand ◽  
Recombination Pathway ◽  
End Resection

The resection of DNA strand with a 5´ end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5´ nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5´-phosphate-binding pocket above the active site determines the 5´-3´ polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination.

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