PBP1 inhibits the synergistic hydrolysis of cell wall by RipA-RpfB complex.

N-terminal GST fusion proteins were expressed and purified from E. coli. Equal amounts of individual or combinations of proteins were incubated with insoluble FITC-labeled substrate: M. luteus cell wall material (A) or Streptomyces peptidoglycan (B). The extent of hydrolysis was determined by measuring the amount of soluble FITC remaining after centrifugation, and thus released during hydrolysis of the insoluble substrate. GST and buffer alone were used to determine background release of FITC and were subtracted from final values. Data are from representative experiments, each done in triplicate. Data are represented as mean +/− SEM. p-values for one-tailed, unpaired t-tests: (A) 1 vs. 2: 0.027 significant, 2 vs. 4: 0.016 significant, 1 vs. 3: 0.074 not significant, 1 vs. 4: 0.188 not significant, (B): 1 vs. 2: 0.009 significant, 2 vs. 4: 0.018 significant, 1 vs. 3: 0.279 not significant, 1 vs. 4: 0.240 not significant (significant to p<0.05). (C) Schematic diagram of the basic structure of mycobacterial peptidoglycan, indicating where RpfB and RipA are predicted to have hydrolytic activity and where PBP1 is predicted to have synthetic activity. Lines connecting NAG to NGM represent β-1,4-glycosidic bonds, while lines connecting NGM to NGM represent peptide cross-linkages. NAG: N-acetyl glucosamine, NGM: N-glycolyl muramic acid, NAM: N-acetyl muramic acid (note that mycobacteria have a mixture of NGM and NAM, with the NGM structure shown here).

N端谷胱甘肽S-转移酶（GST）融合蛋白均从大肠杆菌（E. coli）中表达并纯化获得。将等量的单一蛋白或蛋白组合与不溶性异硫氰酸荧光素（FITC）标记底物进行孵育，底物包括藤黄微球菌（M. luteus）细胞壁材料(A)或链霉菌肽聚糖(B)。通过离心后检测上清液中可溶性FITC的含量，可反映不溶性底物水解过程中释放的可溶性荧光标记物总量，以此表征水解反应程度。设置仅添加GST蛋白及仅添加缓冲液的对照组以测定FITC的本底释放量，并将该本底值从最终检测结果中扣除。所有数据均来自重复三次的代表性独立实验，结果以平均值±标准误（SEM）表示。单尾非配对t检验的p值如下：(A) 组1与组2：0.027，具有显著性差异；组2与组4：0.016，具有显著性差异；组1与组3：0.074，无显著性差异；组1与组4：0.188，无显著性差异。(B) 组1与组2：0.009，具有显著性差异；组2与组4：0.018，具有显著性差异；组1与组3：0.279，无显著性差异；组1与组4：0.240，无显著性差异（显著性阈值设为p<0.05）。(C) 分枝杆菌肽聚糖基本结构示意图，标注了RpfB与RipA的预测水解活性位点，以及PBP1的预测合成活性位点。连接N-乙酰葡糖胺（NAG，N-acetyl glucosamine）与N-羟乙酰胞壁酸（NGM，N-glycolyl muramic acid）的线条代表β-1,4-糖苷键，而连接NGM与NGM的线条代表肽交联键。缩写说明：NAG即N-acetyl glucosamine（N-乙酰葡糖胺），NGM即N-glycolyl muramic acid（N-羟乙酰胞壁酸），NAM即N-acetyl muramic acid（N-乙酰胞壁酸）（注：分枝杆菌细胞壁同时含有NGM与NAM，本图展示的为NGM结构）。