Anion Exchange Mechanisms in Bacteria

本项目涉及革兰氏阴性厌氧菌--产状草酸杆菌中的一种新的转运蛋白OxlT。OxlT催化外部二价草酸盐与内部单价甲酸盐的交换。这种生电交换，以及细胞内脱羧化过程中的质子消耗，构成了一个间接的质子泵，当细胞从草酸中提取“代谢能量”时，该泵产生质子动力。这种模式似乎被其他细胞中的其他反应所复制，间接质子泵在微生物学中可能很常见。为了在分子水平上理解这一事件，我们建议研究反向转运蛋白OxlT。目前，我们已经有了纯化和重组OxlT的方法，我们已经提出了抗OxlT的多克隆抗体，这将有助于我们克隆oxlT基因。利用纯化的蛋白和克隆的基因，我们将产生多肽特异性抗体并构建基因融合，以帮助确定运输蛋白的结构。定点突变将被用来进一步表征酶的结构/功能性质，以及生化和生物物理方法来确定蛋白质的最小功能单位(单体或二聚体)、螺旋含量和结合性质。%我们已经证明了一种新的机制，从环境中获取能量的微生物，草酸杆菌。这种细胞使用一种特殊的膜蛋白，称为OxlT，将二价草酸分子带入细胞，以换取单价甲酸盐，甲酸盐同时被泵出细胞。完成交换循环所需的甲酸盐是在分子进入细胞后降解草酸而产生的。负电荷离子的不对称交换导致跨细胞膜形成膜电势，然后细胞可以用来制造额外的能量和生长。这种新的能量形成机制的核心是膜蛋白OxlT。我们将使用分子生物学(克隆、诱变)、免疫学(特异性抗体)等工具。生物化学(纯化和重建)和生物物理(光谱学)，研究酶的结构和功能特性，以帮助我们了解这种不寻常的膜“泵”蛋白质，如这一种，是如何为细胞提供额外能量的。

This project concerns OxlT, a novel transport protein in Oxalobacter formigenes, a gram-negative anaerobic bacterium. OxlT catalyzes the exchange of external divalent oxalate for internal monovalent formate. This electrogenic exchange, along with proton consumption during intracellular decarboxylation, constitutes an indirect proton pump that generates a proton-motive force as the cell extracts "metabolic energy" from oxalate. It appears that this pattern is replicated by other reactions in other cells, and that indirect proton pumps may be common in microbiology. To understand this event at the molecular level, we propose to study OxlT, the antiport protein. Presently, we have methods to purify and reconstitute OxlT, and we have raised anti-OxlT polyclonal antibodies that will aid us in cloning the oxlT gene. With purified protein and a cloned gene, we will raise peptide-specific antibodies and construct gene fusions to aid in the determination of transport protein structure. Site-directed mutagenesis will be used to further characterize the structure/function properties of the enzyme together with biochemical and biophysical methods to determine the minimal functional unit (monomeric or dimeric), .- helical content and the binding properties of the protein. %%% We have shown that the microorganism, Oxalobacter formigenes, has a novel mechanism to harvest energy from its environment. This cell uses a specific membrane protein, termed OxlT, to bring divalent oxalate molecules into the cell in exchange for monovalent formate, which is simultaneously pumped out of the cell. The formate, which is required to complete the exchange cycle is generated by degradation of oxalate after the molecule is brought into the cell. The asymmetric exchange of negatively charged ions results in the formation of a membrane potential across the cell membrane, that the cell can then use to make additional energy and grow. At the heart of this new mechanism of energy formation is the membrane protein, OxlT. We will use the tools of molecular biology (cloning, mutagenesis), immunology (specific antibodies). biochemistry (purification and reconstitution) and biophysics (spectroscopy) to study the structural and functional properties of the enzyme to help us understand how such an unusual membrane "pump" protein, such as this one, operates to provide extra energy for the cells.