Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct

2015 ◽  
Vol 137 (29) ◽  
pp. 9333-9343 ◽  
Author(s):  
Concepción González-Bello ◽  
Lorena Tizón ◽  
Emilio Lence ◽  
José M. Otero ◽  
Mark J. van Raaij ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Bacterial Virulence ◽  
Ammonium Derivative ◽  
Covalent Adduct

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

2021 ◽  
Author(s):  
Ryuji Yamazawa ◽  
Ritsuko Kuwana ◽  
Kenji Takeuchi ◽  
Hiromu Takamatsu ◽  
Yoshitaka Nakajima ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Chemical Modification ◽  
Cysteine Protease ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Protease Gene ◽  
Active Site Residues ◽  
Protein Determination ◽  
35 Kda Protein

Abstract In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

scholarly journals Chemical Modification of the Active Site of the Acetylcholine Receptor

1969 ◽  
Vol 54 (1) ◽  
pp. 245-264 ◽  
Author(s):  
Arthur Karlin
Keyword(s):  
Chemical Modification ◽  
Acetylcholine Receptor ◽  
Disulfide Bond ◽  
Active Site ◽  
Quaternary Ammonium ◽  
Competitive Inhibitor ◽  
Affinity Labels ◽  
Electrophorus Electricus ◽  
Depolarizing Agents ◽  
Monoquaternary Ammonium

The receptor for acetylcholine in the subsynaptic membrane of the electroplax of Electrophorus electricus is a protein with a disulfide bond in the vicinity of the active site. This disulfide can be reduced and reoxidized with concomitant inhibition and restoration of the response to acetylcholine and other monoquaternary ammonium-depolarizing agents. Conversely, the bisquaternary hexamethonium, normally a competitive inhibitor, causes depolarization, and the activity of decamethonium is increased following reduction of the disulfide. The reduced receptor can be alkylated by various maleimide derivatives and is then no longer reoxidizable. Some quaternary ammonium maleimide derivatives act as affinity labels of the reduced receptor, alkylating it at a rate three orders of magnitude faster then do uncharged maleimide derivatives. Other types of potential affinity labels also react only with the reduced receptor and the resulting covalently attached quaternary ammonium moieties interact with the active site, strongly activating the receptor. These results suggest a model for the active site and its transitions in which an activator such as acetylcholine bridges between a negative subsite and a hydrophobic subsite in the vicinity of the disulfide, causing an altered conformation around the negative subsite and a decrasee of a few angstroms in the distance between the two subsites.

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.

scholarly journals Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

Structure ◽  
2017 ◽  
Vol 25 (5) ◽  
pp. 697-707.e4 ◽  
Author(s):  
Kelli L. Hvorecny ◽  
Christopher D. Bahl ◽  
Seiya Kitamura ◽  
Kin Sing Stephen Lee ◽  
Bruce D. Hammock ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Virulence Factor ◽  
Active Site ◽  
Epoxide Hydrolase ◽  
Bacterial Virulence ◽  
Bacterial Virulence Factor
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