Inactivation of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves by disulfiram

Betaine aldehyde dehydrogenase (BADH) activity might be crucial for the growth of the human pathogen Pseudomonas aeruginosa under conditions of infection and therefore appears to be a suitable target for antimicrobial agents. As a first step in the search for BADH inhibitors, we have tested the effects of the known aldehyde dehydrogenase inhibitor disulfiram (DSF) on the activity of P. aeruginosa and Amaranthus hypochondriacus (amaranth) leaf BADHs. DSF totally inactivated both enzymes in a time- and dose-dependent manner. In the case of the Pseudomonas enzyme, inactivation kinetics were monophasic with a second-order inactivation rate constant at pH 6.9 of 4.9+/-0.4 M-1 s(-1), whereas the plant enzyme was inactivated in a biphasic process with second-order inactivation rate constants at pH 7.5 of 6.8+/-0.6 and 0.33+/-0.04 M-1 s(-1). At pH 8.8, the second-order rate constants for inactivation of the bacterial enzyme was I x 10(3) M-1 s(-1), which compare well with that reported for human liver mitochondrial aldehyde dehydrogenase (ALDH2), the target of DSF inhibition in the aversion therapy of alcoholism. Both BADHs were inactivated faster in the presence of NAD(P)(+) than in its absence, whereas NAD(P)H and betaine aldehyde protected the bacterial, but increased the inactivation rate of the plant enzyme. The inactivated enzymes were reactivated by dithiothreitol, but not by a high concentration of the physiological reductant glutathione. The high in vitro sensitivity of the Pseudomonas BADH to DSF, particularly in the presence of NAD(P)(+), together with the lack of reversibility of DSF modification by glutathione, makes this inhibitor a potential antimicrobial agent and suggests that it might be worth testing its effects and those of its metabolites in vivo, under culture conditions in which the activity of BADH is required for growth of the bacteria. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.