Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
FEBS Open Bio. 2021 May;11(5):1332-1342. doi: 10.1002/2211-5463.13136. Epub 2021 Apr 4.
Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd ) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg ) and ferredoxin (Fd; 27.2 U·mg ) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein , consistent with the presence of three predicted [4Fe-4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd.
铁氧还蛋白氧化还原酶（PFOR）是细菌厌氧代谢中的关键酶。由于使用低电位铁氧还蛋白（Fd）作为电子载体，PFOR 允许在异养生长期间进行氢的释放以及在自养生长期间进行丙酮酸的合成。嗜热产乙酸模型细菌 Thermoanaerobacter kivui 可以使用这两种生活方式，但该生物体中的 PFOR 的性质以前尚未确定。在这里，我们已经从以葡萄糖生长的细胞中分离出明显均一的 PFOR。肽质量指纹图谱显示它由 pfor1 编码。PFOR 以丙酮酸作为电子供体，以亚甲基蓝（1.8 U·mg）和铁氧还蛋白（Fd；27.2 U·mg）作为电子受体，该反应依赖于硫胺素焦磷酸、丙酮酸、辅酶 A 和 Fd。最适 pH 和温度分别为 7.5 和 66°C。我们检测到 13.6 mol 的铁·mol 的蛋白质，与存在三个预测的[4Fe-4S]簇一致。提供还原型 Fd 的能力使 PFOR 成为酶测定的一种有趣的辅助酶。为了简化和加快纯化程序，我们在 T. kivui 中建立了同源蛋白生产的方案。因此，pfor1 被克隆并在 T. kivui 中表达，编码的蛋白含有经过基因工程改造的 His 标签，仅通过两步即可纯化至明显均一。同源产生的 PFOR1 具有与来自 T. kivui 的酶相同的性质。该酶可作为需要还原型 Fd 作为电子供体的酶测定中的辅助酶，例如电子分叉酶，以保持还原型 Fd 的恒定水平。
