scholarly journals Structural evidence for the covalent modification of FabH by 4,5-dichloro-1,2-dithiol-3-one (HR45)

2017 ◽  
Vol 15 (30) ◽  
pp. 6310-6313 ◽  
Author(s):  
Alexander G. Ekström ◽  
Van Kelly ◽  
Jon Marles-Wright ◽  
Scott L. Cockroft ◽  
Dominic J. Campopiano
Keyword(s):  
Mass Spectrometry ◽  
Reaction Mechanism ◽  
Active Site ◽  
Covalent Modification ◽  
Elimination Reaction ◽  
Covalent Adduct ◽  
Michael Type Addition

Mass spectrometry and modelling shows the antimicrobial inhibitor 4,5-dichloro-1,2-dithiol-3-one (HR45) acts by forming a covalent adduct with the target β-ketoacyl-ACP synthase III (FabH). The 5-chloro substituent directs attack of the essential active site thiol (C112) via a Michael type addition elimination reaction mechanism.

Novel NADPH–cysteine covalent adduct found in the active site of an aldehyde dehydrogenase

2011 ◽  
Vol 439 (3) ◽  
pp. 443-455 ◽  
Author(s):  
Ángel G. Díaz-Sánchez ◽  
Lilian González-Segura ◽  
Enrique Rudiño-Piñera ◽  
Alfonso Lira-Rocha ◽  
Alfredo Torres-Larios ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
Enzyme Inactivation ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Oxidative Stresses ◽  
Covalent Adduct

PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the ‘resting’ conformation to the double bond of the α,β-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H–Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

scholarly journals Holospiniferoside: A New Antitumor Cerebroside from The Red Sea Cucumber Holothuria spinifera: In Vitro and In Silico Studies

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1555
Author(s):  
Enas E. Eltamany ◽  
Usama Ramadan Abdelmohsen ◽  
Dina M. Hal ◽  
Amany K. Ibrahim ◽  
Hashim A. Hassanean ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Red Sea ◽  
Active Site ◽  
Sea Cucumber ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometric Analysis ◽  
Chemical Structures ◽  
In Silico Studies ◽  
Red Sea Cucumber

Chemical investigation of the methanolic extract of the Red Sea cucumber Holothuria spinifera led to the isolation of a new cerebroside, holospiniferoside (1), together with thymidine (2), methyl-α-d-glucopyranoside (3), a new triacylglycerol (4), and cholesterol (5). Their chemical structures were established by NMR and mass spectrometric analysis, including gas chromatography–mass spectrometry (GC–MS) and high-resolution mass spectrometry (HRMS). All the isolated compounds are reported in this species for the first time. Moreover, compound 1 exhibited promising in vitro antiproliferative effect on the human breast cancer cell line (MCF-7) with IC50 of 20.6 µM compared to the IC50 of 15.3 µM for the drug cisplatin. To predict the possible mechanism underlying the cytotoxicity of compound 1, a docking study was performed to elucidate its binding interactions with the active site of the protein Mdm2–p53. Compound 1 displayed an apoptotic activity via strong interaction with the active site of the target protein. This study highlights the importance of marine natural products in the design of new anticancer agents.

scholarly journals Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry

Amino Acids ◽  
2021 ◽  
Author(s):  
Magdalena Widgren Sandberg ◽  
Jakob Bunkenborg ◽  
Stine Thyssen ◽  
Martin Villadsen ◽  
Thomas Kofoed
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Peptide Mapping ◽  
Covalent Modification ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
E Coli ◽  
Gm Csf ◽  
Fragmentation Behavior ◽  
Macrophage Colony

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

Alkylation of Escherichia coli Thioredoxin by S-(2-Chloroethyl)glutathione and Identification of the Adduct on the Active Site Cysteine-32 by Mass Spectrometry

1995 ◽  
Vol 8 (7) ◽  
pp. 934-941 ◽  
Author(s):  
John C. L. Erve ◽  
Elisabeth Barofsky ◽  
Douglas F. Barofsky ◽  
Max L. Deinzer ◽  
Donald J. Reed
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Active Site ◽  
Active Site Cysteine

Synthesis and properties of small molecules designed to covalently capture native and oxidized forms of protein tyrosine phosphatase 1B (PTP1B)

2016 ◽  
Author(s):  
◽  
Kasi Viswanatharaju Ruddraraju
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Active Site ◽  
Tyrosine Phosphatase ◽  
Covalent Modification ◽  
Affinity Labeling ◽  
Protein Tyrosine Phosphatase 1B ◽  
University Of Missouri ◽  
Protein Tyrosine ◽  
Enzyme Active Site ◽  
Carbon Nucleophiles

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Protein tyrosine phosphatase 1B (PTP1B) is a validated target for the treatment of type 2 diabetes and obesity. The discovery of selective inhibitors with drug-like properties has proven to be challenging because there are [about]80 PTP family members that share a similar and positively charged active site. To overcome these challenges, we have pursued two novel approaches for the covalent inactivation of PTP1B. Exo-affinity labeling agents exploit covalent reactions with amino acids outside the enzyme active site to gain both affinity and selectivity. We prepared several affinity labeling agents using a 12-step convergent synthesis. Enzyme assays revealed that some of these agents are capable of inactivating the enzyme by covalent modification. In another project, we prepared a low molecular weight mimic of the oxidized form of PTP1B that is generated in cells, during insulin signaling events. Seeking molecules capable of covalent capture of oxidized PTP1B, we treated this chemical model with several carbon nucleophiles, such as 1,3-diketones and sulfone-stabilized carbon anions. These carbon nucleophiles readily reacted with the model compound, under mild conditions to give stable adducts. Inactivation experiments revealed that 1,3-diketones are capable of inactivating the oxidized PTP1B at micromolar concentrations.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

2014 ◽  
Vol 70 (12) ◽  
pp. 3212-3225 ◽  
Author(s):  
Tiila-Riikka Kiema ◽  
Rajesh K. Harijan ◽  
Malgorzata Strozyk ◽  
Toshiyuki Fukao ◽  
Stefan E. H. Alexson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reaction Mechanism ◽  
Active Site ◽  
Quaternary Structure ◽  
Fatty Acyl ◽  
Physiological Importance ◽  
Loop Domain ◽  
Solution Studies ◽  
Hydrolysis Of ◽  
Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

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.

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