title: Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase--a potential target for antimicrobial agents against Pseudomonas aeruginosa.
abstract: 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[diethylthiocarbamyl] 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.
authors.affiliation: Laboratorio de Osmorregulación, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios, Tlalnepantla, Estado de México, 54090 México, Mexico.
authors.forename: Roberto
authors.initials: R
authors.lastname: Velasco-García
authors.forename: Víctor J
authors.initials: VJ
authors.lastname: Zaldívar-Machorro
authors.forename: Carlos
authors.initials: C
authors.lastname: Mújica-Jiménez
authors.forename: Lilian
authors.initials: L
authors.lastname: González-Segura
authors.forename: Rosario A
authors.initials: RA
authors.lastname: Muñoz-Clares
mesh_terms: D000409:Alanine / Q000737:chemistry; D000890:Anti-Infective Agents / Q000494:pharmacology*; D050728:Betaine-Aldehyde Dehydrogenase / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002845:Chromatography; D003545:Cysteine / Q000737:chemistry; D004221:Disulfiram / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000737:chemistry; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D013696:Temperature; D013997:Time Factors
chemical_list: D000890:Anti-Infective Agents; D004791:Enzyme Inhibitors; D011506:Proteins; D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; D009249:NADP; D004228:Dithionitrobenzoic Acid; D008741:Methyl Methanesulfonate; D050728:Betaine-Aldehyde Dehydrogenase; D005978:Glutathione; D003545:Cysteine; D000409:Alanine; D004221:Disulfiram