Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase--a potential target for antimicrobial agents against Pseudomonas aeruginosa.
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.
Velasco-García
Roberto
R
Laboratorio de Osmorregulación, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios, Tlalnepantla, Estado de México, 54090 México, Mexico.
Zaldívar-Machorro
Víctor J
VJ
Mújica-Jiménez
Carlos
C
González-Segura
Lilian
L
Muñoz-Clares
Rosario A
RA
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
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