Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B

2013 ◽  
Vol 81 (5) ◽  
pp. 788-804 ◽  
Author(s):  
Ahmet Özcan ◽  
Elif Ozkirimli Olmez ◽  
Burak Alakent
Keyword(s):  
Active Site ◽  
Water Molecules ◽  
Protonation State ◽  
Site Water

scholarly journals Relebactam Is a Potent Inhibitor of the KPC-2 β-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Melissa D. Barnes ◽  
Jim Alsop ◽  
Magdalena A. Taracila ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rate Constant ◽  
Active Site ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Clinical Isolates ◽  
Piperidine Ring ◽  
Water Molecules ◽  
Content Type ◽  
Site Water

ABSTRACT The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

scholarly journals Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 22 ◽  
Author(s):  
Ignacio Boron ◽  
Juan Pablo Bustamante ◽  
Kelly S Davidge ◽  
Sandip Singh ◽  
Lesley AH Bowman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Water Molecules ◽  
Single Mutation ◽  
Truncated Hemoglobin ◽  
Kinetic Measurements ◽  
Migration Rates ◽  
Ligand Migration ◽  
Site Water ◽  
Dynamics Simulations

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

The Structural Basis for Pseudoreversion of the E165D Lesion by the Secondary S96P Mutation in Triosephosphate Isomerase Depends on the Positions of Active Site Water Molecules

Biochemistry ◽  
1995 ◽  
Vol 34 (41) ◽  
pp. 13612-13621 ◽  
Author(s):  
Elizabeth A. Komives ◽  
Julie C. Lougheed ◽  
Kathleen Liu ◽  
Shigetoshi Sugio ◽  
Zhidong Zhang ◽  
...  
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Structural Basis ◽  
Water Molecules ◽  
Site Water

scholarly journals SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

2010 ◽  
Vol 285 (41) ◽  
pp. 31849-31858 ◽  
Author(s):  
Paul A. Del Rizzo ◽  
Jean-François Couture ◽  
Lynnette M. A. Dirk ◽  
Bethany S. Strunk ◽  
Marijo S. Roiko ◽  
...  
Keyword(s):  
Active Site ◽  
Water Molecules ◽  
Site Water

Key Role of Active-Site Water Molecules in Bacteriorhodopsin Proton-Transfer Reactions

2008 ◽  
Vol 112 (47) ◽  
pp. 14729-14741 ◽  
Author(s):  
Ana-Nicoleta Bondar ◽  
Jerome Baudry ◽  
Sándor Suhai ◽  
Stefan Fischer ◽  
Jeremy C. Smith
Keyword(s):  
Proton Transfer ◽  
Active Site ◽  
Transfer Reactions ◽  
Water Molecules ◽  
Proton Transfer Reactions ◽  
Site Water

Active site water molecules revealed in the 2.1 Å resolution structure of a site-directed mutant of isocitrate dehydrogenase 1 1Edited by I. A. Wilson

2000 ◽  
Vol 295 (3) ◽  
pp. 377-385 ◽  
Author(s):  
Diana B Cherbavaz ◽  
Myoung E Lee ◽  
Robert M Stroud ◽  
Daniel E Koshland
Keyword(s):  
Active Site ◽  
Isocitrate Dehydrogenase ◽  
Water Molecules ◽  
Isocitrate Dehydrogenase 1 ◽  
Site Water ◽  
A Site ◽  
Resolution Structure

scholarly journals Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 22 ◽  
Author(s):  
Ignacio Boron ◽  
Juan Pablo Bustamante ◽  
Kelly S Davidge ◽  
Sandip Singh ◽  
Lesley AH Bowman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Water Molecules ◽  
Single Mutation ◽  
Truncated Hemoglobin ◽  
Kinetic Measurements ◽  
Migration Rates ◽  
Ligand Migration ◽  
Site Water ◽  
Dynamics Simulations

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels interrupted by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations affect both the tunnels accessibility as well as the affinity of distal site water molecules, thus modifying the ligand access to the iron. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

Ligand-bound structures of 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase fromMoraxella catarrhalisreveal a water channel connecting to the active site for the second step of catalysis

2015 ◽  
Vol 71 (2) ◽  
pp. 239-255 ◽  
Author(s):  
Sonali Dhindwal ◽  
Priyanka Priyadarshini ◽  
Dipak N. Patil ◽  
Satya Tapas ◽  
Pramod Kumar ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Metal Ion ◽  
Water Channel ◽  
Second Step ◽  
Phosphoryl Transfer ◽  
Water Molecules ◽  
Crystal Forms ◽  
Tetrameric Structure ◽  
Site Water

KdsC, the third enzyme of the 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic pathway, catalyzes a substrate-specific reaction to hydrolyze 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of KDO and phosphate. KdsC is a phosphatase that belongs to the C0 subfamily of the HAD superfamily. To understand the molecular basis for the substrate specificity of this tetrameric enzyme, the crystal structures of KdsC fromMoraxella catarrhalis(Mc-KdsC) with several combinations of ligands, namely metal ion, citrate and products, were determined. Various transition states of the enzyme have been captured in these crystal forms. The ligand-free and ligand-bound crystal forms reveal that the binding of ligands does not cause any specific conformational changes in the active site. However, the electron-density maps clearly showed that the conformation of KDO as a substrate is different from the conformation adopted by KDO when it binds as a cleaved product. Furthermore, structural evidence for the existence of an intersubunit tunnel has been reported for the first time in the C0 subfamily of enzymes. A role for this tunnel in transferring water molecules from the interior of the tetrameric structure to the active-site cleft has been proposed. At the active site, water molecules are required for the formation of a water bridge that participates as a proton shuttle during the second step of the two-step phosphoryl-transfer reaction. In addition, as the KDO biosynthesis pathway is a potential antibacterial target, pharmacophore-based virtual screening was employed to identify inhibitor molecules for theMc-KdsC enzyme.

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