scholarly journals Tyrosine 121 moves revealing a druggable pocket that couples catalysis to ATP-binding in serine racemase

2021 ◽  
Author(s):  
Chloe R. Koulouris ◽  
Sian E. Gardiner ◽  
Tessa K. Harris ◽  
Karen T. Elvers ◽  
S. Mark Roe ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Active Site ◽  
Serine Racemase ◽  
Hydroxyl Group ◽  
Catalytic Residue ◽  
Dimer Interface ◽  
X Ray ◽  
The Core ◽  
Small Domain ◽  
Allosteric Activator

ABSTRACTHuman serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a ‘closed’ hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the ATP a-phosphate. In contrast, in ‘open’ hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzymefragment structures show that Tyr121 is flipped out of its pocket, suggesting that this pocket is druggable.

scholarly journals Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alice Douangamath ◽  
Daren Fearon ◽  
Paul Gehrtz ◽  
Tobias Krojer ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Design ◽  
Active Site ◽  
Large Scale ◽  
Structure Based Drug Design ◽  
Dimer Interface ◽  
X Ray ◽  
Fragment Screening ◽  
Viral Proteases ◽  
Main Protease

Abstract COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

scholarly journals X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from Burkholderia sp. Strain FA1

2009 ◽  
Vol 191 (8) ◽  
pp. 2630-2637 ◽  
Author(s):  
Keiji Jitsumori ◽  
Rie Omi ◽  
Tatsuo Kurihara ◽  
Atsushi Kurata ◽  
Hisaaki Mihara ◽  
...  
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Chloride Ion ◽  
Catalytic Triad ◽  
Dimensional Structure ◽  
Active Site Residues ◽  
Density Peak ◽  
Core Domain ◽  
X Ray ◽  
The Core

ABSTRACT Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the α/β hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the Nε1 atom of Trp150 and the Nε2 atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA

2012 ◽  
Vol 40 (3) ◽  
pp. 567-572 ◽  
Author(s):  
Abdussalam Adina-Zada ◽  
Tonya N. Zeczycki ◽  
Martin St. Maurice ◽  
Sarawut Jitrapakdee ◽  
W. Wallace Cleland ◽  
...  
Keyword(s):  
Active Site ◽  
Pyruvate Carboxylase ◽  
Structural Effects ◽  
Reaction Conditions ◽  
Acetyl Coa ◽  
X Ray ◽  
Allosteric Interactions ◽  
Crystallographic Structures ◽  
And Staphylococcus Aureus ◽  
Allosteric Activator

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.

scholarly journals Identification of Mutation Resistance Coldspots for Targeting the SARS-CoV2 Main Protease

2020 ◽  
Author(s):  
Navaneethakrishnan Krishnamoorthy ◽  
Khalid Fakhro
Keyword(s):  
Active Site ◽  
The Novel ◽  
Dimer Interface ◽  
X Ray ◽  
Main Protease ◽  
New Perspective ◽  
Function Relationship ◽  
Novel Coronavirus ◽  
Relationship Of ◽  
Structure And Activity

Abstract Most attempts to target the novel coronavirus SARS-CoV2 are focusing on the main protease (Mpro) 1-9. However, >19,000 mutations in the Mpro have already been reported 10. The mutations encompassing 282 amino acid positions and these “hotspots” might change the Mpro structure and activity, potentially rendering novel antivirals and vaccines ineffective. Here we identified 24 mutational “coldspots” that have resisted mutation since the virus was first detected. We compared the structure-function relationship of these coldspots with several SARS-CoV2 Mpro X-ray crystal structures. We found that three coldspot residues (Leu141, Phe185 and Gln192) help to form the active site, while six (Gly2, Arg4, Tyr126, Lys137, Leu141 and Leu286) contribute to dimer formation that is required for Mpro activity. The surface of the dimer interface is more resistant to mutations compared to the active site. Interestingly, 16 coldspots are found in conserved patterns when compared with other coronaviruses. Importantly, several conserved coldpots are available on the surface of the active site and at the dimer interface for targeting. The identification and short list of these coldspots offers a new perspective to target the SARS-CoV2 Mpro while avoiding mutation-based drug resistance.

scholarly journals Allosteric modulation of caspase 3 through mutagenesis

2012 ◽  
Vol 32 (4) ◽  
pp. 401-411 ◽  
Author(s):  
Jad Walters ◽  
Joshua L. Schipper ◽  
Paul Swartz ◽  
Carla Mattos ◽  
A. Clay Clark
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Caspase 3 ◽  
Structural Defects ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Dimer Interface ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dynamics Simulations

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.

Neutron structure of the cyclic glucose-bound xylose isomerase E186Q mutant

2014 ◽  
Vol 70 (2) ◽  
pp. 414-420 ◽  
Author(s):  
Parthapratim Munshi ◽  
Edward H. Snell ◽  
Mark J. van der Woerd ◽  
Russell A. Judge ◽  
Dean A. A. Myles ◽  
...  
Keyword(s):  
Active Site ◽  
Ring Opening ◽  
Xylose Isomerase ◽  
Hydroxyl Group ◽  
Water Molecules ◽  
Conserved Residues ◽  
X Ray ◽  
Hydrogen Bond Networks ◽  
Acid Acid ◽  
Conserved Water Molecules

Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl groupviaan activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation.

scholarly journals Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

2020 ◽  
Author(s):  
Alice Douangamath ◽  
Daren Fearon ◽  
Paul Gehrtz ◽  
Tobias Krojer ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Design ◽  
Active Site ◽  
Large Scale ◽  
Structure Based Drug Design ◽  
Dimer Interface ◽  
X Ray ◽  
Fragment Screening ◽  
Viral Proteases ◽  
Main Protease

SummaryCOVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Tele-Substitution Reactions in the Synthesis of a Promising Class of Antimalarials

2020 ◽  
Author(s):  
Marat Korsik ◽  
Edwin Tse ◽  
David Smith ◽  
William Lewis ◽  
Peter J. Rutledge ◽  
...  
Keyword(s):  
Heterocyclic Ring ◽  
Ring System ◽  
Substitution Reactions ◽  
Laboratory Notebook ◽  
Polar Solvents ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Core ◽  
Nmr Data

<p></p><p>We have discovered and studied a <i>tele</i>substitution reaction in a biologically important heterocyclic ring system. Conditions that favour the <i>tele</i>-substitution pathway were identified: the use of increased equivalents of the nucleophile or decreased equivalents of base, or the use of softer nucleophiles, less polar solvents and larger halogens on the electrophile. Using results from X-ray crystallography and isotope labelling experiments a mechanism for this unusual transformation is proposed. We focused on this triazolopyrazine as it is the core structure of the <i>in vivo </i>active anti-plasmodium compounds of Series 4 of the Open Source Malaria consortium.</p> <p> </p> <p>Archive of the electronic laboratory notebook with the description of all conducted experiments and raw NMR data could be accessed via following link <a href="https://ses.library.usyd.edu.au/handle/2123/21890">https://ses.library.usyd.edu.au/handle/2123/21890</a> . For navigation between entries of laboratory notebook please use file "Strings for compounds in the article.pdf" that works as a reference between article codes and notebook codes, also this file contain SMILES for these compounds. </p><br><p></p>

scholarly journals The role of AGN activity in the building up of the BCG at z ∼ 1.6

2019 ◽  
Vol 15 (S356) ◽  
pp. 280-284
Author(s):  
Angela Bongiorno ◽  
Andrea Travascio
Keyword(s):  
Galaxy Cluster ◽  
Cluster Galaxy ◽  
X Ray ◽  
Groups Of Galaxies ◽  
Star Forming ◽  
The Core ◽  
The Galaxy ◽  
Multi Wavelength

AbstractXDCPJ0044.0-2033 is one of the most massive galaxy cluster at z ∼1.6, for which a wealth of multi-wavelength photometric and spectroscopic data have been collected during the last years. I have reported on the properties of the galaxy members in the very central region (∼ 70kpc × 70kpc) of the cluster, derived through deep HST photometry, SINFONI and KMOS IFU spectroscopy, together with Chandra X-ray, ALMA and JVLA radio data.In the core of the cluster, we have identified two groups of galaxies (Complex A and Complex B), seven of them confirmed to be cluster members, with signatures of ongoing merging. These galaxies show perturbed morphologies and, three of them show signs of AGN activity. In particular, two of them, located at the center of each complex, have been found to host luminous, obscured and highly accreting AGN (λ = 0.4−0.6) exhibiting broad Hα line. Moreover, a third optically obscured type-2 AGN, has been discovered through BPT diagram in Complex A. The AGN at the center of Complex B is detected in X-ray while the other two, and their companions, are spatially related to radio emission. The three AGN provide one of the closest AGN triple at z > 1 revealed so far with a minimum (maximum) projected distance of 10 kpc (40 kpc). The discovery of multiple AGN activity in a highly star-forming region associated to the crowded core of a galaxy cluster at z ∼ 1.6, suggests that these processes have a key role in shaping the nascent Brightest Cluster Galaxy, observed at the center of local clusters. According to our data, all galaxies in the core of XDCPJ0044.0-2033 could form a BCG of M* ∼ 1012Mȯ hosting a BH of 2 × 108−109Mȯ, in a time scale of the order of 2.5 Gyrs.

Crystal structure of oxybutynin hydrochloride hemihydrate, C22H32NO3Cl(H2O)0.5

2019 ◽  
Vol 34 (1) ◽  
pp. 50-58
Author(s):  
James A. Kaduk ◽  
Nicholas C. Boaz ◽  
Amy M. Gindhart ◽  
Thomas N. Blanton
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Powder Diffraction ◽  
Density Functional ◽  
Hydroxyl Group ◽  
Layered Crystal ◽  
Powder Diffraction File ◽  
X Ray ◽  
Layered Crystal Structure ◽  
Oxybutynin Hydrochloride

The crystal structure of oxybutynin hydrochloride hemihydrate has been solved and refined using synchrotron X-ray powder diffraction data, and optimized using density functional techniques. Oxybutynin hydrochloride hemihydrate crystallizes in space group I2/a (#15) with a = 14.57266(8), b = 8.18550(6), c = 37.16842(26) Å, β = 91.8708(4)°, V = 4421.25(7) Å3, and Z = 8. The compound exhibits X-ray-induced photoreduction of the triple bond. Prominent in the layered crystal structure is the N–H⋅⋅⋅Cl hydrogen bond between the cation and anion, as well as O–H⋅⋅⋅Cl hydrogen bonds from the water molecule and hydroxyl group of the oxybutynin cation. C–H⋅⋅⋅Cl hydrogen bonds also contribute to the crystal energy, and help determine the conformation of the cation. The powder pattern is included in the Powder Diffraction File™ as entry 00-068-1305.

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