Characterization of the outer membrane receptor ShuA from the heme uptake system of Shigella dysenteriae -: Substrate specificity and identification of the heme protein ligands
Characterization of the outer membrane receptor ShuA from the heme uptake system of Shigella dysenteriae -: Substrate specificity and identification of the heme protein ligands
复制标题
DOI：
10.1074/jbc.m611121200
复制
发表时间：
2007-05-18
影响因子：
4.8
通讯作者：
Wilks, Angela
中科院分区：
文献类型：
--
作者：
Burkhard, Kimberly A.;Wilks, Angela
痢疾志贺氏菌，像许多细菌性病原体一样，已经进化出外膜受体介导的途径来吸收和利用血红素作为铁源。作为了解血红素摄取机制的第一步，我们对痢疾杆菌的外膜受体shu进行了定点诱变、光谱和动力学分析。纯化后的外膜受体在SDS-PAGE上的分子质量为73 kDa。对洗涤剂胶束或脂质小束中的蛋白质进行初步光谱分析，发现与受体结合的残余血红素，Soret最大值在413 nm处。用外源血红素对蛋白质进行滴定，在洗涤剂胶束437 nm处有一个Soret峰，在脂质胶束402 nm处有一个Soret峰。然而，血红蛋白中血红素的转移在413 nm处产生与分离蛋白相同的Soret最大值。进一步的光谱和动力学分析表明，氧化状态的血红蛋白最有可能是joshua的生理底物。此外，保守组氨酸H86A或H420A的突变导致受体从血红蛋白中有效提取血红素的能力丧失。而双突变体H86A/H420A则不能从血红蛋白中提取血红素。这些发现共同证实了His-86和His-420对底物识别、血红素协调和转移都是必不可少的。此外，全长TonB被证明与载脂蛋白-舒亚H86A/H420A或野生型舒亚形成1:1的复合物。这些研究结果为进一步研究tonb依赖性外膜受体对血红素的协调和转运提供了基础。
Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1: 1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.