Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Am Chem Soc. 2013 Mar 27;135(12):4632-5. doi: 10.1021/ja312510m. Epub 2013 Mar 13.
The bacterial cell wall precursor, Lipid II, has a highly conserved structure among different organisms except for differences in the amino acid sequence of the peptide side chain. Here, we report an efficient and flexible synthesis of the canonical Lipid II precursor required for the assembly of Gram-negative peptidoglycan (PG). We use a rapid LC/MS assay to analyze PG glycosyltransfer (PGT) and transpeptidase (TP) activities of Escherichia coli penicillin binding proteins PBP1A and PBP1B and show that the native m-DAP residue in the peptide side chain of Lipid II is required in order for TP-catalyzed peptide cross-linking to occur in vitro. Comparison of PG produced from synthetic canonical E. coli Lipid II with PG isolated from E. coli cells demonstrates that we can produce PG in vitro that resembles native structure. This work provides the tools necessary for reconstituting cell wall synthesis, an essential cellular process and major antibiotic target, in a purified system.
除了肽侧链氨基酸序列的差异外，细菌细胞壁前体脂质 II 在不同生物体中具有高度保守的结构。在这里，我们报告了一种有效的、灵活的合成革兰氏阴性肽聚糖 (PG) 组装所需的典型脂质 II 前体的方法。我们使用快速 LC/MS 分析来分析大肠埃希氏菌青霉素结合蛋白 PBP1A 和 PBP1B 的 PG 糖基转移 (PGT) 和转肽酶 (TP) 活性，并表明脂质 II 肽侧链中的天然 m-DAP 残基对于 TP 催化的肽交联在体外发生是必需的。用合成的典型大肠杆菌脂质 II 制备的 PG 与从大肠杆菌细胞中分离的 PG 的比较表明，我们可以在体外产生类似于天然结构的 PG。这项工作为在纯化系统中重新构建细胞壁合成这一重要的细胞过程和主要抗生素靶标提供了必要的工具。
