Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
J Biol Chem. 2021 Sep;297(3):101046. doi: 10.1016/j.jbc.2021.101046. Epub 2021 Aug 4.
Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5'-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B) ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.
细菌需要高效的摄取系统才能在营养有限的环境中生存和繁殖，例如在宿主生物中发现的环境。细菌质膜中的 ABC 转运蛋白提供了许多底物运输的机制。在这项研究中，我们研究了放线杆菌中包含周质结合蛋白的操纵子，以研究其在营养获取中的潜在作用。从周质结合蛋白晶体结构获得的 1.76 Å 分辨率的电子密度图与包含在蛋白质结合位点内的吡哆醛-5'-磷酸（P5P/吡哆醛磷酸/维生素 B 的活性形式）配体的分子模型最佳匹配。与该周质结合蛋白结合的 P5P 的身份通过等温滴定量热法、微量热泳动和质谱法得到验证，这使我们将该蛋白命名为 P5PA，并将该操纵子命名为 P5PAB。为了说明这种摄取系统的功能实用性，我们将来自胸膜肺炎放线杆菌的 P5PAB 操纵子引入缺乏 P5P 合成所需关键酶的大肠杆菌 K-12 菌株中。该菌株在低水平的 P5P 下的生长支持了该操纵子在 P5P 摄取中的功能作用。这是第一个专门用于 P5P 细菌摄取系统的报告，但通过生物信息学，我们在巴氏杆菌科的致病性代表中发现了同源物，这表明该操纵子在放线杆菌属之外更广泛地存在。
