Structures of human cytosolic and mitochondrial nucleotidases: implications for structure-based design of selective inhibitors

2014 ◽  
Vol 70 (2) ◽  
pp. 461-470 ◽  
Author(s):  
Petr Pachl ◽  
Milan Fábry ◽  
Ivan Rosenberg ◽  
Ondřej Šimák ◽  
Pavlína Řezáčová ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Specific Inhibitor ◽  
Detailed Comparison ◽  
Atomic Resolution ◽  
Phosphate Anion ◽  
Protonation State ◽  
Interatomic Distances ◽  
Enzyme Active Site ◽  
Resolution Structure

The human 5′(3′)-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.

scholarly journals Atomic resolution structure of serine protease proteinase K at ambient temperature

2017 ◽  
Author(s):  
Tetsuya Masuda ◽  
Mamoru Suzuki ◽  
Shigeyuki Inoue ◽  
Changyong Song ◽  
Takanori Nakane ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
Active Sites ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Water Molecules ◽  
Specific Substrate ◽  
Hydrogen Atoms ◽  
X Ray Crystallography ◽  
Chain Conformations ◽  
Resolution Structure

AbstractAtomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

scholarly journals Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site

2015 ◽  
Vol 6 (1) ◽  
pp. 505-516 ◽  
Author(s):  
Katrin Adamczyk ◽  
Niall Simpson ◽  
Gregory M. Greetham ◽  
Andrea Gumiero ◽  
Martin A. Walsh ◽  
...  
Keyword(s):  
Infrared Spectroscopy ◽  
Active Site ◽  
Active Sites ◽  
Dynamic Nature ◽  
Enzyme Active Site ◽  
Coherent Dynamics ◽  
Peroxidase Enzymes ◽  
Ultrafast Infrared

Ultrafast infrared spectroscopy provides insights into the dynamic nature of water in the active sites of catalase and peroxidase enzymes.

scholarly journals Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles

2020 ◽  
Vol 117 (52) ◽  
pp. 33204-33215
Author(s):  
Filip Yabukarski ◽  
Justin T. Biel ◽  
Margaux M. Pinney ◽  
Tzanko Doukov ◽  
Alexander S. Powers ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Enzyme Design ◽  
Reaction Cycle ◽  
New Era ◽  
X Ray ◽  
Enzyme Active Site ◽  
Conformational Ensembles ◽  
X Ray Crystallography ◽  
General Base

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.

scholarly journals Structure ofStaphylococcus aureusadenylosuccinate lyase (PurB) and assessment of its potential as a target for structure-based inhibitor discovery

2010 ◽  
Vol 66 (8) ◽  
pp. 881-888 ◽  
Author(s):  
Paul K. Fyfe ◽  
Alice Dawson ◽  
Marie-Theres Hutchison ◽  
Scott Cameron ◽  
William N. Hunter
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Bacterial Pathogen ◽  
Enzyme Mechanism ◽  
Enzyme Active Site ◽  
Adenylosuccinate Lyase ◽  
Medium Resolution ◽  
Inhibitor Discovery ◽  
Species Specific ◽  
Distinct Subunit

The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogenStaphylococcus aureusin complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival ofS. aureusand to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme fromEscherichia coliare presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit–subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization.

Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease fromCandida parapsilosis

2015 ◽  
Vol 71 (12) ◽  
pp. 2494-2504 ◽  
Author(s):  
Jiří Dostál ◽  
Adam Pecina ◽  
Olga Hrušková-Heidingsfeldová ◽  
Lucie Marečková ◽  
Iva Pichová ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Candida Parapsilosis ◽  
Chemical Interaction ◽  
Detailed Comparison ◽  
Aspartic Protease ◽  
Atomic Resolution ◽  
Active Site Residues ◽  
Aspartic Proteases ◽  
Pepstatin A

The virulence of theCandidapathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p fromCandida parapsilosiswas determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundantC. parapsilosissecreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

scholarly journals High-resolution structure of an atypical α-phosphoglucomutase related to eukaryotic phosphomannomutases

2013 ◽  
Vol 69 (10) ◽  
pp. 2008-2016 ◽  
Author(s):  
Przemyslaw Nogly ◽  
Pedro M. Matias ◽  
Matteo de Rosa ◽  
Rute Castro ◽  
Helena Santos ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Haloacid Dehalogenase ◽  
Enzyme Active Site ◽  
High Resolution Structure ◽  
Dimeric Form ◽  
Strict Specificity ◽  
Resolution Structure

The first structure of a bacterial α-phosphoglucomutase with an overall fold similar to eukaryotic phosphomannomutases is reported. Unlike most α-phosphoglucomutases within the α-D-phosphohexomutase superfamily, it belongs to subclass IIb of the haloacid dehalogenase superfamily (HADSF). It catalyzes the reversible conversion of α-glucose 1-phosphate to glucose 6-phosphate. The crystal structure of α-phosphoglucomutase fromLactococcus lactis(APGM) was determined at 1.5 Å resolution and contains a sulfate and a glycerol bound at the enzyme active site that partially mimic the substrate. A dimeric form of APGM is present in the crystal and in solution, an arrangement that may be functionally relevant. The catalytic mechanism of APGM and its strict specificity towards α-glucose 1-phosphate are discussed.

scholarly journals Traditional Phytochemistry: Identification of Drug by ‘Taste’

2007 ◽  
Vol 4 (2) ◽  
pp. 145-148 ◽  
Author(s):  
Kalpana Joshi ◽  
Alex Hankey ◽  
Bhushan Patwardhan
Keyword(s):  
Traditional Medicine ◽  
Active Site ◽  
Active Sites ◽  
The Body ◽  
Pharmacological Properties ◽  
Specific Enzyme ◽  
Materia Medica ◽  
Enzyme Active Site ◽  
Scientific Approach ◽  
Hypo Thesis

Ayurveda, the system of traditional medicine from India, holds that ‘Rasa’, a concept roughly corresponding to taste, is a basis for identifying pharmacological properties of plants and other materia medica used in Dravyaguna—its system of phytomedicine. This idea has recently found support in studies of ibuprofen, the pharmacological properties of which are similar to those of oleocanthal, because the two substances have very similar tastes. This paper discusses a possible scientific approach to understanding the Ayurvedic (hypo)thesis in terms of the stereochemical basis of both pharamaco-activity and taste, and the numbers of possible pharmaco-active compounds that ‘Rasa’ may be able to distinguish. We conclude that molecules binding to a specific enzyme active site should have their own ‘Rasa’, and that the number of different subjectively experienced ‘tastes’ is more than enough to distinguish between molecular shapes binding to all enzyme active sites in the body.

Near-atomic resolution structures of urate oxidase complexed with its substrate and analogues: the protonation state of the ligand

2010 ◽  
Vol 66 (6) ◽  
pp. 714-724 ◽  
Author(s):  
Laure Gabison ◽  
Mohamed Chiadmi ◽  
Mohamed El Hajji ◽  
Bertrand Castro ◽  
Nathalie Colloc'h ◽  
...  
Keyword(s):  
Uric Acid ◽  
Metal Ion ◽  
Active Sites ◽  
Reaction Pathway ◽  
Catalytic Triad ◽  
Urate Oxidase ◽  
Atomic Resolution ◽  
Base System ◽  
Protonation State ◽  
Large Water

Urate oxidase (uricase; EC 1.7.3.3; UOX) fromAspergillus flavuscatalyzes the oxidation of uric acid in the presence of molecular oxygen to 5-hydroxyisourate in the degradation cascade of purines; intriguingly, catalysis proceeds using neither a metal ion (Fe, Cuetc.) nor a redox cofactor. UOX is a tetrameric enzyme with four active sites located at the interface of two subunits; its structure was refined at atomic resolution (1 Å) using new crystal data in the presence of xanthine and at near-atomic resolution (1.3–1.7 Å) in complexes with the natural substrate (urate) and two inhibitors: 8-nitroxanthine and 8-thiouric acid. Three new features of the structural and mechanistic behaviour of the enzyme were addressed. Firstly, the high resolution of the UOX–xanthine structure allowed the solution of an old structural problem at a contact zone within the tetramer; secondly, the protonation state of the substrate was determined from both a halochromic inhibitor complex (UOX–8-nitroxanthine) and from the H-atom distribution in the active site, using the structures of the UOX–xanthine and the UOX–uric acid complexes; and thirdly, it was possible to extend the general base system, characterized by the conserved catalytic triad Thr–Lys–His, to a large water network that is able to buffer and shuttle protons back and forth between the substrate and the peroxo hole along the reaction pathway.

scholarly journals Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles

2019 ◽  
Author(s):  
Filip Yabukarski ◽  
Justin T Biel ◽  
Margaux M Pinney ◽  
Tzanko Doukov ◽  
Alexander S Powers ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Enzyme Design ◽  
Reaction Cycle ◽  
New Era ◽  
X Ray ◽  
Enzyme Active Site ◽  
Conformational Ensembles ◽  
X Ray Crystallography ◽  
General Base

AbstractHow enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for P. putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1-1.5 Å scale, and was not exceptional compared to non-catalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.

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