Biological Sciences, The University of Auckland, Auckland, 1010, New Zealand.
Institute for Molecular Bioscience (IMB), The University of Queensland, St. Lucia, Brisbane, 4072, Australia.
Biochem Biophys Res Commun. 2018 Nov 30;506(3):660-667. doi: 10.1016/j.bbrc.2018.10.113. Epub 2018 Oct 27.
Entamoeba histolytica, the causative agent of amoebic dysentery, liver abscess and colitis, exploits its vesicular trafficking machinery for survival and virulence. Rab family of small GTPases play a key role in the vesicular transport by undergoing the GTP/GDP cycle which is central to the biological processes. Amoebic genome encodes several atypical Rab GTPases which are unique due to absence of conserved sequence motif(s) or atypical residues in their catalytic site [Saito-Nakano et al., 2005 ]. Previously, EhRab21 has been reported to involve in amoebic invasion and migration [Emmanuel et al., 2015 ]. The conserved Glutamine of switch-II region is universally accepted to be crucial for GTP hydrolysis. Mutations that reduce the sidechain polarity of Glutamine render the protein GTPase activity deficient [Krengel et al., 1990]. Here, we report a catalytic role of atypical switch-I Arginine (R36) in intrinsic GTP hydrolysis catalysed by EhRab21. Unlike the GTPase activity deficient QL mutants, the GTPase activity of EhRab21Q64L was found to be marginally enhanced compared to the wild-type protein. Although EhRab21R36L mutant showed normal GTPase activity, the double mutant (R36L/Q64L) was found to be GTPase deficient. Thus, EhRab21 is a unique member of small GTPase family in which an atypical switch-I Arginine is capable of driving GTP hydrolysis independent of the conserved switch-II Glutamine.
溶组织内阿米巴，引起阿米巴痢疾、肝脓肿和结肠炎的病原体，利用其囊泡运输机制来生存和致病。小 GTP 酶 Ras 家族在囊泡运输中起着关键作用，经历 GTP/GDP 循环，这是生物过程的核心。阿米巴基因组编码几种非典型的 Ras GTP 酶，由于其催化部位缺乏保守序列基序或非典型残基而具有独特性 [Saito-Nakano 等人，2005]。先前已经报道 EhRab21 参与了阿米巴的侵袭和迁移 [Emmanuel 等人，2015]。普遍认为，开关-II 区域的保守谷氨酰胺对于 GTP 水解至关重要。降低谷氨酰胺侧链极性的突变会使蛋白 GTP 酶活性缺失 [Krengel 等人，1990]。在这里，我们报告了非典型开关-I 精氨酸（R36）在 EhRab21 催化的固有 GTP 水解中的催化作用。与 GTP 酶活性缺失的 QL 突变体不同，EhRab21Q64L 的 GTP 酶活性被发现比野生型蛋白略有增强。虽然 EhRab21R36L 突变体显示出正常的 GTP 酶活性，但双突变体（R36L/Q64L）被发现 GTP 酶活性缺失。因此，EhRab21 是小 GTP 酶家族中的一个独特成员，其中非典型的开关-I 精氨酸能够独立于保守的开关-II 谷氨酰胺驱动 GTP 水解。
