Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 341, Issue 2, Pages 408-415Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2006.01.003
Keywords
betaine aldehyde dehydrogenase; Pseudomonas aeruginosa; chemical modification; enzyme inactivation; enzyme dissociation; enzyme aggregation; disulfiram; antimicrobial agent
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In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors-abundant at infection sites-and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys -> Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocal-bamyl] disulfide (disulfiram) induced enzyme dissociation-at 23 degrees C-and irreversible aggregation-at 37 degrees C. Of the four Cys -> Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa. (c) 2006 Elsevier Inc. All rights reserved.
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