scholarly journals Binding of Phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography

2001 ◽  
Vol 10 (8) ◽  
pp. 1669-1676 ◽  
Author(s):  
Demetres D. Leonidas ◽  
Gayatri B. Chavali ◽  
Anwar M. Jardine ◽  
Songlin Li ◽  
Robert Shapiro ◽  
...  
Keyword(s):  
Active Site ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1211-C1211
Author(s):  
Joseph Ng ◽  
Ronny Hughes ◽  
Michelle Morris ◽  
Leighton Coates ◽  
Matthew Blakeley ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Crystallographic Structures ◽  
Hydrolysis Of ◽  
Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

scholarly journals Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography

2010 ◽  
Vol 19 (12) ◽  
pp. 2430-2439 ◽  
Author(s):  
Louise J. Gourlay ◽  
Silvia Sommaruga ◽  
Marco Nardini ◽  
Paola Sperandeo ◽  
Gianni Dehò ◽  
...  
Keyword(s):  
Active Site ◽  
X Ray ◽  
E Coli ◽  
X Ray Crystallography

scholarly journals Mechanism-based inactivator of isocitrate lyases 1 and 2 fromMycobacterium tuberculosis

2017 ◽  
Vol 114 (29) ◽  
pp. 7617-7622 ◽  
Author(s):  
Truc V. Pham ◽  
Andrew S. Murkin ◽  
Margaret M. Moynihan ◽  
Lawrence Harris ◽  
Peter C. Tyler ◽  
...  
Keyword(s):  
Active Site ◽  
High Efficiency ◽  
Isocitrate Lyase ◽  
Partition Ratio ◽  
Kinetic Properties ◽  
Ionization Mass ◽  
X Ray ◽  
X Ray Crystallography ◽  
Covalent Adduct ◽  
Reversible Covalent

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway forMycobacterium tuberculosis(Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator ofMtbICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191. 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalentS-homopyruvoyl adduct of the active-site Cys191.

scholarly journals Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aron Broom ◽  
Rojo V. Rakotoharisoa ◽  
Michael C. Thompson ◽  
Niayesh Zarifi ◽  
Erin Nguyen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Protein Design ◽  
Active Site ◽  
Room Temperature ◽  
De Novo ◽  
Conformational Ensemble ◽  
Enzyme Design ◽  
X Ray ◽  
Conformational Ensembles ◽  
X Ray Crystallography

Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

scholarly journals Extending electron paramagnetic resonance to nanoliter volume protein single crystals using a self-resonant microhelix

2019 ◽  
Vol 5 (10) ◽  
pp. eaay1394 ◽  
Author(s):  
Jason W. Sidabras ◽  
Jifu Duan ◽  
Martin Winkler ◽  
Thomas Happe ◽  
Rana Hussein ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Single Crystals ◽  
Active Site ◽  
Measured Signal ◽  
Paramagnetic Resonance ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Band ◽  
Electron Paramagnetic ◽  
Epr Experiments

Electron paramagnetic resonance (EPR) spectroscopy on protein single crystals is the ultimate method for determining the electronic structure of paramagnetic intermediates at the active site of an enzyme and relating the magnetic tensor to a molecular structure. However, crystals of dimensions typical for protein crystallography (0.05 to 0.3mm) provide insufficient signal intensity. In this work, we present a microwave self-resonant microhelix for nanoliter samples that can be implemented in a commercial X-band (9.5 GHz) EPR spectrometer. The self-resonant microhelix provides a measured signal-to-noise improvement up to a factor of 28 with respect to commercial EPR resonators. This work opens up the possibility to use advanced EPR techniques for studying protein single crystals of dimensions typical for x-ray crystallography. The technique is demonstrated by EPR experiments on single crystal [FeFe]-hydrogenase (Clostridium pasteurianum; CpI) with dimensions of 0.3 mm by 0.1 mm by 0.1 mm, yielding a proposed g-tensor orientation of the Hox state.

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 Biochemical and structural explorations of α-hydroxyacid oxidases reveal a four-electron oxidative decarboxylation reaction

2019 ◽  
Vol 75 (8) ◽  
pp. 733-742 ◽  
Author(s):  
Hsien-Wei Yeh ◽  
Kuan-Hung Lin ◽  
Syue-Yi Lyu ◽  
Yi-Shan Li ◽  
Chun-Man Huang ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Active Site ◽  
Oxidation Reaction ◽  
Isotope Labeling ◽  
Molecular Level ◽  
Flavin Mononucleotide ◽  
Oxidative Decarboxylation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Decarboxylation Reaction

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.

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