González-Segura Lilian, Mújica-Jiménez Carlos, Muñoz-Clares Rosario A
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México DF, México.
Chem Biol Interact. 2009 Mar 16;178(1-3):64-9. doi: 10.1016/j.cbi.2008.10.049. Epub 2008 Nov 5.
Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NAD(P)H. In the opportunistic pathogen Pseudomonas aeruginosa, this enzyme (PaBADH) could be an antimicrobial target. Several aldehyde dehydrogenases (ALDHs) are inactivated by arsenite in the presence of a low molecular thiol, a finding that was interpreted as a demonstration of the existence of vicinal thiols in these enzymes. As part of our studies on the susceptibility to chemical modification of the catalytic cysteine (C286) of PaBADH, we treated the enzyme with two arsenical reagents widely used to inhibit enzymes that have vicinal thiols: sodium m-arsenite plus 2,3-dimercaptopropanol (arsenite-BAL) and phenylarsine oxide (PAO). Here we report that they readily and reversibly inactivate PaBADH, even though the four cysteine residues of this enzyme (C286, C353, C377, and C439) are far from each other in the three-dimensional structure. Modification of PaBADH by both reagents was reversible by an excess of a dithiol (dithiothreitol), but only the PAO-modified enzyme could be reactivated by a monothiol (2-mercaptoethanol). C286 is the reactive residue as indicated by the following findings: (i) betaine aldehyde and NADP(+) afforded full protection against enzyme inactivation; (ii) the mutant proteins C353A, C377A, and C439A showed similar inactivation kinetics that the wild-type enzyme, and (iii) pretreatment of PaBADH with arsenite-BAL prevented irreversible inactivation by N-ethylmaleimide. Our results confirm previous findings on other ALDHs, and indicate that these vicinal thiol-specific reagents readily react with certain monothiols, such as the one of the catalytic cysteinyl residue of ALDHs. As arsenicals are being recently used to treat certain cancers, human ALDHs, even those not having conformationally vicinal thiols, may be unsuspected targets in these treatments.
甜菜碱醛脱氢酶（BADH）催化甜菜碱醛不可逆地氧化为甘氨酸甜菜碱，同时将NAD(P)(+)还原为NAD(P)H。在机会致病菌铜绿假单胞菌中，这种酶（PaBADH）可能是一个抗菌靶点。在低分子硫醇存在的情况下，几种醛脱氢酶（ALDHs）会被亚砷酸盐灭活，这一发现被解释为这些酶中存在邻位硫醇的证据。作为我们对PaBADH催化半胱氨酸（C286）化学修饰敏感性研究的一部分，我们用两种广泛用于抑制具有邻位硫醇的酶的砷试剂处理该酶：间亚砷酸钠加2,3-二巯基丙醇（亚砷酸盐-BAL）和苯砷酸氧化物（PAO）。在此我们报告，尽管该酶的四个半胱氨酸残基（C286、C353、C377和C439）在三维结构中彼此相距甚远，但它们能轻易且可逆地使PaBADH失活。两种试剂对PaBADH的修饰可通过过量的二硫醇（二硫苏糖醇）逆转，但只有PAO修饰的酶可被单硫醇（2-巯基乙醇）重新激活。C286是反应性残基，如下发现表明：（i）甜菜碱醛和NADP(+)能完全保护酶不被灭活；（ii）突变蛋白C353A、C377A和C439A显示出与野生型酶相似的失活动力学，以及（iii）用亚砷酸盐-BAL预处理PaBADH可防止N-乙基马来酰亚胺导致的不可逆失活。我们的结果证实了之前关于其他ALDHs的发现，并表明这些邻位硫醇特异性试剂能轻易与某些单硫醇反应，比如ALDHs催化半胱氨酰残基中的一种。由于最近砷试剂被用于治疗某些癌症，人类ALDHs，即使是那些没有构象邻位硫醇的，在这些治疗中可能是未被怀疑的靶点。
