scholarly journals Helices on interdomain interface couple catalysis in the ATPPase domain with allostery in Plasmodium falciparum GMP synthetase

2019 ◽  
Author(s):  
Santosh Shivakumaraswamy ◽  
Nivedita Pandey ◽  
Lionel Ballut ◽  
Sébastien Violot ◽  
Nushin Aghajari ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Basis ◽  
Drug Targets ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Allosteric Activation ◽  
Purine Base ◽  
Interdomain Interactions ◽  
Hydrolysis Of ◽  
Gmp Synthetase

AbstractGMP synthetase catalyzes the substitution of the C2 oxo-group of the purine base in XMP with an amino-group generating GMP, the last step in the biosynthesis of GMP. This reaction involves a series of catalytic events that include hydrolysis of Gln generating ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. Inherent to the functioning of GMP synthetases is bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain by substrates binding to the ATPPase domain, synchronization of the two catalytic events and tunnelling of ammonia from the GATase to the ATPPase domain. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and, steady-state and transient kinetic assays on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. The results map the residues critical for catalysis in the ATPPase domain to the helices α11 and α12 that are located at the interdomain interface, and the lid-loop that follows α11. This apart, perturbing interdomain interactions involving residues on α11 and α12 impairs GATase activation. These results imply that this arrangement of helices at the domain interface with residues that play roles in ATPPase catalysis as well as domain crosstalk enables coupling ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

scholarly journals Molecular determinants for agonist recognition and discrimination in P2X2 receptors

2019 ◽  
Vol 151 (7) ◽  
pp. 898-911 ◽  
Author(s):  
Federica Gasparri ◽  
Jesper Wengel ◽  
Thomas Grutter ◽  
Stephan A. Pless
Keyword(s):  
Drug Targets ◽  
P2x Receptors ◽  
Carbonyl Oxygen ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Ligand Recognition ◽  
Directed Mutagenesis ◽  
Backbone Carbonyl ◽  
Molecular Determinants ◽  
Atp Analogues

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.

Site-directed mutagenesis provides insights into the selective binding of trityl derivatives to Plasmodium falciparum dUTPase

2011 ◽  
Vol 46 (8) ◽  
pp. 3309-3314 ◽  
Author(s):  
Eliseo Recio ◽  
Alexander Musso-Buendía ◽  
Antonio E. Vidal ◽  
Gian Filippo Ruda ◽  
Ganasan Kasinathan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Selective Binding

scholarly journals Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

2005 ◽  
Vol 49 (8) ◽  
pp. 3421-3427 ◽  
Author(s):  
Fahd K. Majiduddin ◽  
Timothy Palzkill
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Class A ◽  
Study Support ◽  
Functional Mutants ◽  
Hydrolysis Of ◽  
Traditional Class

ABSTRACT Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

scholarly journals Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity

2015 ◽  
Vol 466 (1) ◽  
pp. 177-188 ◽  
Author(s):  
Bethany Nancolas ◽  
Richard B. Sessions ◽  
Andrew P. Halestrap
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Binding Site ◽  
Molecular Basis ◽  
Specific Inhibitor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Isoform Specificity ◽  
Isoform Selectivity ◽  
Dynamics Simulations

A combination of molecular modelling, site-directed mutagenesis and molecular dynamics simulations define the binding site of MCT1 for AR-C155858, a potent and specific inhibitor. Key amino acids within the binding site differ between MCT1 and MCT4 accounting for isoform specificity.

scholarly journals Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I

1992 ◽  
Vol 288 (3) ◽  
pp. 1045-1051 ◽  
Author(s):  
S J Thornewell ◽  
S G Waley
Keyword(s):  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Extracellular Medium ◽  
The Stability ◽  
Hydrolysis Of ◽  
Reduced Activity

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

scholarly journals Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif

2015 ◽  
Vol 59 (9) ◽  
pp. 5714-5720 ◽  
Author(s):  
Daria Soroka ◽  
Inès Li de la Sierra-Gallay ◽  
Vincent Dubée ◽  
Sébastien Triboulet ◽  
Herman van Tilbeurgh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fold Increase ◽  
Mycobacterium Abscessus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Emergence Of Resistance ◽  
Hydrolysis Of ◽  
Sequential Formation

ABSTRACTCombinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. SinceMycobacterium tuberculosisproduces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase BlaMAbofMycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAband BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G132N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 104-fold increase inkcat(0.41 s−1), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G132N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G132N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.

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