Thompson J, Chassy B M
J Bacteriol. 1985 Apr;162(1):224-34. doi: 10.1128/jb.162.1.224-234.1985.
The bacterial phosphoenolpyruvate:sugar-phosphotransferase system (PTS) mediates the vectorial translocation and concomitant phosphorylation of sugars. The question arises of whether the PTS can also mediate the phosphorylation of intracellular sugars. To investigate this possibility in Streptococcus lactis 133, lactose derivatives have been prepared containing 14C-labeled 2-deoxy-glucose (2DG), 2-deoxy-2-fluoro-D-glucose (2FG), or alpha-methylglucoside as the aglycon substituent of the disaccharide. Two of the compounds, beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-D-glucopyranose (2'D-lactose) and beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-2'-fluoro-D-glucopyranose (2'F-lactose), were high-affinity substrates of the lactose-PTS. After translocation, the radiolabeled 2'F-lactose 6-phosphate (2'F-lactose-6P) and 2'D-lactose-6P derivatives were hydrolyzed by P-beta-galactoside-galactohydrolase to galactose-6P and either [14C]2FG or [14C]2DG, respectively. Thereafter, the glucose analogs appeared in the medium, but the rates of sugar exit from mannose-PTS-defective mutants were greater than those determined in the parent strain. Unexpectedly, the results of kinetic studies and quantitative analyses of intracellular products in S. lactis 133 showed that initially (and before exit) the glucose analogs existed primarily in phosphorylated form. Furthermore, the production of intracellular [14C]2FG-6P and [14C]2DG-6P (during uptake of the lactose analogs) continued when the possibility for reentry of [14C]2FG and 2DG was precluded by addition of mannose-PTS inhibitors (N-acetylglucosamine or N-acetylmannosamine) to the medium. By contrast, (i) only [14C]2DG, [14C]2FG, and trace amounts of [14C]2FG-6P were found in cells of a mannose-PTS-defective mutant, and (ii) only [14C]2FG and [14C]2DG were present in cells of a double mutant lacking both mannose-PTS and glucokinase activities. We conclude from these data that the mannose-PTS can effect the intracellular phosphorylation of glucose and its analogs in S. lactis 133.
糖磷酸转移酶系统（PTS）介导糖的向量转运和伴随的磷酸化。于是产生了一个问题，即PTS是否也能介导细胞内糖的磷酸化。为了在乳酸链球菌133中研究这种可能性，已制备了乳糖衍生物，其中含有14C标记的2-脱氧葡萄糖（2DG）、2-脱氧-2-氟-D-葡萄糖（2FG）或α-甲基葡萄糖苷作为二糖的糖苷配基取代基。其中两种化合物，β-O-D-吡喃半乳糖基-（1,4'）-2'-脱氧-D-吡喃葡萄糖（2'D-乳糖）和β-O-D-吡喃半乳糖基-（1,4'）-2'-脱氧-2'-氟-D-吡喃葡萄糖（2'F-乳糖），是乳糖-PTS的高亲和力底物。转运后，放射性标记的2'F-乳糖6-磷酸（2'F-乳糖-6P）和2'D-乳糖-6P衍生物分别被β-半乳糖苷-半乳糖水解酶水解为半乳糖-6P和[14C]2FG或[14C]2DG。此后，葡萄糖类似物出现在培养基中，但来自甘露糖-PTS缺陷型突变体的糖输出速率大于在亲本菌株中测定的速率。出乎意料的是，乳酸链球菌133中动力学研究和细胞内产物定量分析的结果表明，最初（以及在输出之前）葡萄糖类似物主要以磷酸化形式存在。此外，当通过向培养基中添加甘露糖-PTS抑制剂（N-乙酰葡糖胺或N-乙酰甘露糖胺）排除[14C]2FG和2DG重新进入的可能性时，（在摄取乳糖类似物期间）细胞内[14C]2FG-6P和[14C]2DG-6P的产生仍在继续。相比之下，（i）在甘露糖-PTS缺陷型突变体的细胞中仅发现[14C]2DG、[14C]2FG和微量的[14C]2FG-6P，并且（ii）在缺乏甘露糖-PTS和葡萄糖激酶活性的双突变体的细胞中仅存在[14C]2FG和[14C]2DG。从这些数据我们得出结论，甘露糖-PTS可以影响乳酸链球菌133中葡萄糖及其类似物的细胞内磷酸化。
