Velasco-García Roberto, Chacón-Aguilar Victor M, Hervert-Hernández Deisy, Muñoz-Clares Rosario A
Laboratorio de Osmorregulación, FES Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios No. 1, Tlalnepantla, 54090, Estado de México, Mexico.
Chem Biol Interact. 2003 Feb 1;143-144:149-58. doi: 10.1016/s0009-2797(02)00199-0.
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 1 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.
甜菜碱醛脱氢酶（BADH）活性对于人类病原体铜绿假单胞菌在感染条件下的生长可能至关重要，因此似乎是抗菌剂的合适靶点。作为寻找BADH抑制剂的第一步，我们测试了已知的醛脱氢酶抑制剂双硫仑（DSF）对铜绿假单胞菌和苋属植物（苋菜）叶片BADHs活性的影响。DSF以时间和剂量依赖性方式完全灭活了这两种酶。对于假单胞菌酶，失活动力学是单相的，在pH 6.9时二级失活速率常数为4.9±0.4 M⁻¹ s⁻¹，而植物酶在双相过程中失活，在pH 7.5时二级失活速率常数为6.8±0.6和0.33±0.04 M⁻¹ s⁻¹。在pH 8.8时，细菌酶失活的二级速率常数为1×10³ M⁻¹ s⁻¹，这与人类肝脏线粒体醛脱氢酶（ALDH2）的报道值相当，ALDH2是酒精中毒厌恶疗法中DSF抑制的靶点。在有NAD(P)⁺存在时，两种BADHs的失活速度比不存在时更快，而NAD(P)H和甜菜碱醛保护了细菌酶，但增加了植物酶的失活速率。失活的酶可被二硫苏糖醇重新激活，但不能被高浓度的生理性还原剂谷胱甘肽重新激活。铜绿假单胞菌BADH对DSF的高体外敏感性，特别是在有NAD(P)⁺存在时，以及谷胱甘肽对DSF修饰缺乏可逆性，使得这种抑制剂成为一种潜在的抗菌剂，并表明在细菌生长需要BADH活性的培养条件下，值得在体内测试其及其代谢产物的作用。
