Macheboeuf Pauline, Lemaire David, Teller Nathalie, Martins Alexandre Dos Santos, Luxen André, Dideberg Otto, Jamin Marc, Dessen Andréa
Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075 (CEA, CNRS, UJF, PSB), 41 rue Jules Horowitz, F-38027 Grenoble, France.
J Mol Biol. 2008 Feb 15;376(2):405-13. doi: 10.1016/j.jmb.2007.10.066. Epub 2007 Nov 1.
Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of beta-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a "trapped" covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-D-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the D-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of beta-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of beta-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.
A类青霉素结合蛋白（PBPs）催化肽聚糖生物合成的最后两步，肽聚糖是细菌细胞壁的关键组成部分。这两个反应，即糖基转移（聚糖链的聚合）和转肽作用（茎肽的交联），对于肽聚糖的稳定性和细胞分裂过程都是必不可少的，但人们对它们的了解仍然很少。PBP催化的转肽反应是β-内酰胺抗生素的作用靶点，但它们在全球范围内的广泛使用促使了高耐药菌株的出现，因此需要共同努力来了解转肽反应，以期开发出更好的抗菌药物。然而，由于PBP底物会迅速脱酰化，这一目标一直难以实现。在这项工作中，我们提供了A类PBP与一种假底物N-苯甲酰-D-丙氨酰巯基乙酸硫酯反应的“捕获”共价中间体的结构快照，该假底物部分模拟了天然的、与膜相关的底物脂II中所含的茎肽。该结构表明，共价中间体（N-苯甲酰-D-丙氨酸）的D-丙氨酰部分通过氢键网络稳定在裂隙中，使羰基靠近氧负离子孔，从而模拟了β-内酰胺抗生素在PBP活性位点内的空间排列。这种排列使目标键处于供体肽攻击的最佳位置，并且与β-内酰胺抗生素在PBP裂隙中的结构布局相似。这些信息有助于更好地理解PBP催化作用，并可能为新型PBP抑制剂的设计提供关键见解。
