J Am Chem Soc. 2020 Mar 18;142(11):5034-5048. doi: 10.1021/jacs.9b08822. Epub 2020 Mar 2.
Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of β-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on β-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of β-lactam acylation.
青霉素结合蛋白（PBPs）催化转肽反应，稳定细菌细胞壁的肽聚糖成分，是β-内酰胺类抗生素的作用靶点，β-内酰胺类抗生素是迄今为止最成功的临床抗生素。然而，由于历史上缺乏具有生理相关底物的动力学能力检测，以及以前未被重视的蛋白辅因子对 PBP 活性的贡献，PBP-转肽酶的酶学分析一直难以进行。通过对肽聚糖合成的重新设计，我们构建了一个连续分光光度测定法，用于 PBP1B 对天然或近乎天然的肽聚糖前体和片段的转肽反应，使我们能够：（a）确定转肽酶底物的识别元件；（b）揭示肽聚糖转肽过程中立体化学编辑的新机制；（c）评估肽聚糖底物对转肽反应中β-内酰胺类抗生素靶向的影响；（d）证明转肽反应发生之前，底物都必须先结合。这些结果使我们能够将高分子量 PBPs 作为酶进行特征描述，而不仅仅是β-内酰胺酰化的靶点。
