Tjaden J, Winkler H H, Schwöppe C, Van Der Laan M, Möhlmann T, Neuhaus H E
Pflanzenphysiologie, Universität Osnabrück, D-49069 Osnabrück, Germany.
J Bacteriol. 1999 Feb;181(4):1196-202. doi: 10.1128/JB.181.4.1196-1202.1999.
The genome of Chlamydia trachomatis, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1Ct and Npt2Ct (nucleoside phosphate transporters 1 and 2 of C. trachomatis), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium Rickettsia prowazekii at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in Escherichia coli to study their biochemical properties. His10-Npt1Ct catalyzed ATP and ADP transport in an exchange mode. The apparent Km values were 48 (ATP) and 39 (ADP) microM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His10-Npt2Ct transported all four ribonucleoside triphosphates with apparent Km values of 31 microM (GTP), 302 microM (UTP), 528 microM (CTP), and 1,158 microM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m-chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2Ct. This observation indicated that His10-Npt2Ct acts as a nucleosidetriphosphate/H+ symporter energized by the proton motive force across the Escherichia coli cytoplasmic membrane. We conclude that Npt1Ct provides chlamydiae with energy whereas Npt2Ct catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.
沙眼衣原体是最主要的人类病原体之一，其基因组包含两个编码蛋白质的结构基因，本文称为Npt1Ct和Npt2Ct（沙眼衣原体的核苷磷酸转运蛋白1和2），在推导的氨基酸水平上，它们与细胞内细菌普氏立克次体的ATP/ADP转运蛋白的相似性分别为68%和61%。亲水性分析和序列比对表明这两种蛋白质都有12个跨膜结构域。为了研究它们的生化特性，将推定的转运蛋白在大肠杆菌中表达为组氨酸标签蛋白。His10-Npt1Ct以交换模式催化ATP和ADP的转运。表观Km值分别为48（ATP）和39（ADP）微摩尔。ATP和ADP的转运具有特异性，因为AMP、GTP、CTP、UTP、dATP、dCTP、dGTP和dTTP不抑制摄取。相比之下，His10-Npt2Ct转运所有四种核糖核苷三磷酸，表观Km值分别为31微摩尔（GTP）、302微摩尔（UTP）、528微摩尔（CTP）和1158微摩尔（ATP）。核糖核苷二磷酸和单磷酸以及脱氧核糖核苷酸不是底物。质子载体间氯羰基亚胺基苯腙消除了Npt2Ct对所有核苷三磷酸的摄取。这一观察结果表明，His10-Npt2Ct作为一种核苷三磷酸/H+同向转运体，由跨大肠杆菌细胞质膜的质子动力提供能量。我们得出结论，Npt1Ct为衣原体提供能量，而Npt2Ct催化合成代谢反应所需的核糖核苷三磷酸的净摄取。
