Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase - A potential target for antimicrobial agents against Pseudomonas aeruginosa
Laboratorio de Osmorregulación, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios, Tlalnepantla, Estado de México, 54090 México, Mexico.Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 04/2006; 341(2):408-15. DOI: 10.1016/j.bbrc.2006.01.003
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.
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- "The modification by DSF can in this way destabilize the quaternary structure of the enzyme. In agreement with the models, PaBADH remains in its tetrameric native state after complete inactivation by any of the (di)thiosulfoxides or (di) thiosulfones (results not shown), in contrast to the DSF-modified enzyme that dissociates into monomers at 23 C or irreversibly aggregates at 37 C. Given that the nucleotide is the first to bind to the active site in the ordered kinetic mechanism of PaBADH, and that the dissociation constant of the enzyme-nucleotide complexes are below the intracellular concentrations of the nucleotides, it is likely that a high proportion of the enzyme has the nucleotide bound in vivo. "
ABSTRACT: Betaine aldehyde dehydrogenase from the human opportunistic pathogen Pseudomonas aeruginosa (PaBADH) catalyzes the irreversible, NAD(P)(+)-dependent oxidation of betaine aldehyde, producing glycine betaine, an osmoprotectant. PaBADH participates in the catabolism of choline and likely in the defense against the osmotic and oxidative stresses to which the bacterium is exposed when infecting human tissues. Given that choline or choline precursors are abundant in infected tissues, PaBADH is a potential drug target because its inhibition will lead to the build up of the toxic betaine aldehyde inside bacterial cells. We tested the thiol reagents, disulfiram (DSF) and five DSF metabolites-diethyldithiocarbamic acid (DDC), S-methyl-N,N-diethyldithiocarbamoyl sulfoxide (MeDDTC-SO) and sulfone (MeDDTC-SO(2)), and S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) and sulfone (MeDTC-SO(2))-as inhibitors of PaBADH and P. aeruginosa growth. As in vitro PaBADH inhibitors, their order of potency was: MeDDTC-SO(2)>DSF>MeDTC-SO(2)>MeDDTC-SO>MeDTC-SO. DDC did not inactivate the enzyme. PaBADH inactivation by DSF metabolites (i) was not affected by NAD(P)(+), (ii) could not be reverted by dithiothreitol, and (iii) did not affect the quaternary structure of the enzyme. Of the DSF metabolites tested, MeDTC-SO(2) and MeDDTC-SO produced significant in situ PaBADH inactivation and arrest of P. aeruginosa growth in choline containing media, in which the expression of PaBADH is induced. They had no effect in media lacking choline, indicating that PaBADH is their main intracellular target, and that arrest of growth is due to accumulation of betaine aldehyde. The in vitro and in situ kinetics of enzyme inactivation by these two compounds were very similar, indicating no restriction on their uptake by the cells. MeDDTC-SO(2) and DSF have no inhibitory effects in situ, probably because their high reactivity towards intracellular nonessential thiols causes their depletion. Our results support that PaBADH is a promising target to treat P. aeruginosa infections, and that some DSF metabolites might be of help in this aim.
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ABSTRACT: Initial attempts to express a choline oxidase from Arthrobacter pascens (APChO-syn) in Escherichia coli starting from a synthetic gene only led to inactive protein. However, activity was regained by the systematic exchange of individual segments of the gene with segments from a choline oxidase-encoding gene from Arthrobacter globiformis yielding a functional chimeric enzyme. Next, a sequence alignment of the exchanged segment with other choline oxidases revealed a mutation in the APChO-syn, showing that residue 200 was a threonine instead of an asparagine, which is, thus, crucial for confering enzyme activity and, hence, provides an explanation for the initial lack of activity. The active recombinant APChO-syn-T200N variant was biochemically characterized showing an optimum at pH 8.0 and at 37 degrees C. Furthermore, the substrate specificity was examined using N,N-dimethylethanolamine, N-methylethanolamine and 3,3-dimethyl-1-butanol.
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ABSTRACT: Pharmaceuticals are typically found in very low concentrations in the aquatic environment. Accordingly, environmental effects clearly assigned to residual drugs are consistent with high affinity interactions with conserved targets in affected wildlife species rather than with a general toxic effect. Thus, evolutionarily well-conserved targets in a given species are associated with an increased risk. In this study orthologs for 1318 human drug targets were predicted in 16 species of which several are relevant for ecotoxicity testing. The conservation of different functional categories of targets was also analyzed. Zebrafish had orthologs to 86% of the drug targets while only 61% were conserved in Daphnia and 35% in green alga. The predicted presence and absence of orthologs agrees well with published experimental data on the potential for specific drug target interaction in various species. Based on the conservation of targets we propose that aquatic environmental risk assessments for human drugs should always include comprehensive studies on aquatic vertebrates. Furthermore, individual targets, especially enzymes, are well conserved suggesting that tests on evolutionarily distant organisms would be highly relevant for certain drugs. We propose that the results can guide environmental risk assessments by improving the possibilities to identify species sensitive to certain types of pharmaceuticals or to other contaminants that act through well defined mechanisms of action. Moreover, we suggest that the results can be used to interpret the relevance of existing ecotoxicity data.
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