Scott C, Guest J R, Green J
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Mol Microbiol. 2000 Mar;35(6):1383-93. doi: 10.1046/j.1365-2958.2000.01799.x.
The commercially important bacterium Lactococcus lactis contains two FNR-like proteins (FlpA and FlpB) which have a high degree of identity to each other and to the FLP of Lactobacillus casei. FlpA was isolated from a GST-FlpA fusion protein produced in Escherichia coli. Like FLP, isolated FlpA is a homodimeric protein containing both Zn and Cu. However, the properties of FlpA were more like those of the E. coli oxygen-responsive transcription factor FNR than the FLP of L. casei. As prepared FlpA recognized an FNR site (TTGAT-N4-ATCAA) but not an FLP site (CCTGA-N4-TCAGG) in band-shift assays. In contrast to FLP, DNA binding by FlpA did not require the formation of an intramolecular disulphide bond. However, despite containing only two cysteine residues per monomer, FlpA was able to acquire an FNR-like, oxygen-labile [4Fe 4S] cluster. But, whereas the incorporation of a [4Fe 4S] cluster into FNR enhances interaction with target DNA, it abolished DNA binding by FlpA. An FlpA variant (FlpA') with an N-terminal region designed to be more FLP-like failed to incorporate an iron-sulphur cluster but could now form an intramolecular disulphide. This simple example of protein engineering, converting an oxygen-labile [4Fe 4S] containing FNR-like protein into a dithiol-disulphide FLP-like redox sensor demonstrates the versatility of the basic CRP structure. Attempts to demonstrate an FlpA-based aerobic-anaerobic switch in the heterologous host E. coli were unsuccessful. However, studies with a series of FNR-dependent lac reporter fusions in strains of E. coli expressing flpA or flpB revealed that both homologues were able to activate expression of FNR-dependent promoters in vivo but only when positioned 61 base pairs upstream of the transcription start.
具有商业重要性的乳酸乳球菌含有两种FNR样蛋白（FlpA和FlpB），它们彼此之间以及与干酪乳杆菌的FLP具有高度同源性。FlpA是从在大肠杆菌中产生的GST-FlpA融合蛋白中分离出来的。与FLP一样，分离出的FlpA是一种同时含有锌和铜的同二聚体蛋白。然而，FlpA的特性更类似于大肠杆菌的氧响应转录因子FNR，而不是干酪乳杆菌的FLP。在凝胶迁移实验中，制备好的FlpA能识别FNR位点（TTGAT-N4-ATCAA），但不能识别FLP位点（CCTGA-N4-TCAGG）。与FLP不同，FlpA与DNA的结合不需要分子内二硫键的形成。然而，尽管每个单体仅含有两个半胱氨酸残基，FlpA仍能够获得一个FNR样的、对氧不稳定的[4Fe 4S]簇。但是，虽然将[4Fe 4S]簇掺入FNR会增强与靶DNA的相互作用，但它却消除了FlpA与DNA的结合。一种N端区域设计得更像FLP的FlpA变体（FlpA'）无法掺入铁硫簇，但现在可以形成分子内二硫键。这个简单的蛋白质工程实例，即将一个含有对氧不稳定的[4Fe 4S]的FNR样蛋白转化为一个二硫醇-二硫键的FLP样氧化还原传感器，证明了基本CRP结构的多功能性。在异源宿主大肠杆菌中尝试证明基于FlpA的好氧-厌氧开关未成功。然而，对在表达flpA或flpB的大肠杆菌菌株中一系列依赖FNR的lac报告基因融合体的研究表明，这两种同源物在体内都能够激活依赖FNR的启动子的表达，但前提是要位于转录起始点上游61个碱基对处。
