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

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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 Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

2014 ◽  
Vol 70 (9) ◽  
pp. 1143-1149 ◽  
Author(s):  
Mika Aoyagi-Scharber ◽  
Anna S. Gardberg ◽  
Bryan K. Yip ◽  
Bing Wang ◽  
Yuqiao Shen ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Clinical Development ◽  
Structural Basis ◽  
Water Molecules ◽  
The Novel ◽  
Breast Cancers ◽  
High Potency ◽  
Damage Response ◽  
Site Water ◽  
Inhibitor Selectivity

Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocketviaan extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.

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 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.

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
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