From the Department of Chemistry, University of Wisconsin, Oshkosh, Wisconsin 54901 and.
the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894.
J Biol Chem. 2018 Apr 13;293(15):5715-5730. doi: 10.1074/jbc.M117.790717. Epub 2018 Feb 13.
Manganese (Mn) is an essential trace nutrient for organisms because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016, and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and inductively coupled plasma-mass spectrometry (ICP-MS) to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negatively charged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch and found that the UPF0016 family of proteins has manganese export activity. We provide here the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, but rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand the available knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mn-responsive riboswitch can be used to identify new components of the manganese homeostasis machinery.
锰（Mn）是生物体必需的痕量营养元素，因为它在酶的辅助因子和提供对活性氧物质（ROS）的保护方面发挥作用。许多细菌需要锰才能与真核宿主细胞形成致病或共生相互作用。然而，过量的锰是有毒的，需要细胞具有锰输出机制。目前已知细菌具有两种广泛分布的锰输出蛋白家族，MntP 和 MntE，但可能存在其他类型的转运蛋白。此外，MntP 的结构和功能还不太清楚。在这里，我们研究了三种结构相关的蛋白在细菌中的作用，这些蛋白已知或预测与锰的运输有关，分别属于 MntP、UPF0016 和 TerC 家族。这些研究使用计算分析来分析系统发育和结构，使用生理测定来测试对高水平锰和 ROS 的敏感性，以及使用电感耦合等离子体质谱（ICP-MS）来测量金属水平。我们发现 MntP 改变了细胞对 ROS 的抗性。此外，我们使用广泛的计算分析和表型测定来鉴定 MntP 活性所需的氨基酸。这些带负电荷的残基可能直接结合锰并将其从细胞质穿过膜运输。我们进一步研究了另外两种与锰感应核糖开关相关的潜在锰转运蛋白，发现 UPF0016 家族的蛋白具有锰输出活性。我们在这里提供了 Alx（TerC 家族的成员）在锰稳态中的作用的第一个表型和生化证据。它似乎不输出锰，而是令人好奇地促进了细胞内锰浓度的增加。这些发现扩展了关于细菌中锰稳态蛋白的身份和机制的可用知识，并表明接近 Mn 响应性核糖开关可用于鉴定锰稳态机制的新组件。
