Padmanabhan Prasad K, Mukherjee Angana, Singh Sushma, Chattopadhyaya Swati, Gowri Venkataraman S, Myler Peter J, Srinivasan Narayanaswamy, Madhubala Rentala
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Biochem Biophys Res Commun. 2005 Dec 2;337(4):1237-48. doi: 10.1016/j.bbrc.2005.09.179. Epub 2005 Oct 7.
Glyoxalases are involved in a ubiquitous detoxification pathway. In pursuit of a better understanding of the biological function of the enzyme, the recombinant glyoxalase I (LdGLOI) protein has been characterized from Leishmania donovani, the most important pathogenic Leishmania species that is responsible for visceral leishmaniasis. A 24kDa protein was heterologously expressed in Escherichia coli. LdGLOI showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOI protein could detect a band of anticipated size approximately 16kDa in promastigote extracts. Several inhibitors of human GLOI showed that they are weak inhibitors of L. donovani growth. Overexpression of GLOI gene in L. donovani using Leishmania expression vector pspalpha hygroalpha, we detected elevated expression of GLOI RNA and protein. Comparative modelling of the 3-D structure of LDGLOI shows that substrate-binding region of the model involves important differences compared to the homologues, such as E. coli, specific to glutathione. Most notably a substrate-binding loop of LDGLOI is characterized by a deletion of five residues compared to the E. coli homologue. Further, a critical Arg in the E. coli variant at the substrate-binding site is replaced by Tyr in LDGLOI. These major differences result in entirely different shapes of the substrate-binding loop and presence of very different chemical groups in the substrate-binding site of LDGLOI compared to E. coli homologue suggesting an explanation for the difference in the substrate specificity. Difference in the substrate specificity of the human and LDGLOI enzyme could be exploited for structure-based drug designing of selective inhibitors against the parasite.
乙二醛酶参与了一条普遍存在的解毒途径。为了更好地理解该酶的生物学功能，已对来自杜氏利什曼原虫（Leishmania donovani）的重组乙二醛酶I（LdGLOI）蛋白进行了表征，杜氏利什曼原虫是导致内脏利什曼病的最重要致病利什曼原虫物种。一种24kDa的蛋白在大肠杆菌中进行了异源表达。LdGLOI对锥虫硫醇半硫代乙缩醛的底物特异性明显高于谷胱甘肽半硫代乙缩醛。抗重组LdGLOI蛋白的抗血清能在前鞭毛体提取物中检测到一条预期大小约为16kDa的条带。几种人GLOI抑制剂表明它们是杜氏利什曼原虫生长的弱抑制剂。利用利什曼原虫表达载体pspalpha hygroalpha在杜氏利什曼原虫中过表达GLOI基因，我们检测到了GLOI RNA和蛋白表达的升高。LDGLOI三维结构的比较建模表明，该模型的底物结合区域与同系物（如大肠杆菌，其对谷胱甘肽具有特异性）相比存在重要差异。最显著的是，与大肠杆菌同系物相比，LDGLOI的底物结合环有五个残基缺失。此外，大肠杆菌变体在底物结合位点的一个关键精氨酸在LDGLOI中被酪氨酸取代。这些主要差异导致底物结合环的形状完全不同，并且与大肠杆菌同系物相比，LDGLOI的底物结合位点存在非常不同的化学基团，这为底物特异性的差异提供了解释。人和LDGLOI酶底物特异性的差异可用于针对该寄生虫的基于结构的选择性抑制剂药物设计。
