Oh H, Park Y, Park C
National Creative Research Initiative Center for Behavioral Genetics, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-Ku, Taejon, Republic of Korea.
J Biol Chem. 1999 May 14;274(20):14006-11. doi: 10.1074/jbc.274.20.14006.
Mutations arose from an Escherichia coli strain defective in the high (Rbs/ribose) and low (Als/allose and Xyl/xylose) affinity D-ribose transporters, which allow cells to grow on D-ribose. Genetic tagging and mapping of the mutations revealed that two loci in the E. coli linkage map are involved in creating a novel ribose transport mechanism. One mutation was found in ptsG, the glucose-specific transporter of phosphoenolpyruvate:carbohydrate phosphotransferase system and the other in mlc, recently reported to be involved in the regulation of ptsG. Five different mutations in ptsG were characterized, whose growth on D-ribose medium was about 80% that of the high affinity system (Rbs+). Two of them were found in the predicted periplasmic loops, whereas three others are in the transmembrane region. Ribose uptakes in the mutants, competitively inhibited by D-glucose, D-xylose, or D-allose, were much lower than that of the high affinity transporter but higher than those of the Als and Xyl systems. Further analyses of the mutants revealed that the rbsK (ribokinase) and rbsD (function unknown) genes are involved in the ribose transport through PtsG, indicating that the phosphorylation of ribose is not mediated by PtsG and that some unknown metabolic function mediated by RbsD is required. It was also found that D-xylose, another sugar not involved in phosphorylation, was efficiently transported through the wild-type or mutant PtsG in mlc-negative background. The efficiencies of xylose and glucose transports are variable in the PtsG mutants, depending on their locations, either in the periplasm or in the membrane. In an extreme case of the transmembrane change (I283T), xylose transport is virtually abolished, indicating that the residue is directly involved in determining sugar specificity. We propose that there are at least two domains for substrate specificity in PtsG with slightly altered recognition properties.
突变源自一株大肠杆菌，该菌株在高亲和力（Rbs/核糖）和低亲和力（Als/阿洛糖和Xyl/木糖）D - 核糖转运蛋白方面存在缺陷，这些转运蛋白能使细胞在D - 核糖上生长。对这些突变进行基因标记和定位后发现，大肠杆菌连锁图谱中的两个位点参与创建了一种新的核糖转运机制。一个突变发生在ptsG基因，它是磷酸烯醇丙酮酸：碳水化合物磷酸转移酶系统中的葡萄糖特异性转运蛋白；另一个突变发生在mlc基因，最近报道该基因参与ptsG的调控。对ptsG中的五个不同突变进行了表征，它们在D - 核糖培养基上的生长能力约为高亲和力系统（Rbs +）的80%。其中两个突变位于预测的周质环中，另外三个位于跨膜区域。突变体中的核糖摄取受到D - 葡萄糖、D - 木糖或D - 阿洛糖的竞争性抑制，其摄取量远低于高亲和力转运蛋白，但高于Als和Xyl系统。对突变体的进一步分析表明，rbsK（核糖激酶）和rbsD（功能未知）基因参与通过PtsG的核糖转运，这表明核糖的磷酸化不是由PtsG介导的，并且需要RbsD介导的一些未知代谢功能。还发现，另一种不参与磷酸化的糖D - 木糖，在mlc阴性背景下可通过野生型或突变型PtsG有效转运。木糖和葡萄糖转运的效率在PtsG突变体中各不相同，这取决于它们在周质或膜中的位置。在跨膜变化的极端情况（I283T）下，木糖转运几乎完全丧失，这表明该残基直接参与确定糖的特异性。我们提出，PtsG中至少有两个底物特异性结构域，其识别特性略有改变。
