Urbach Carole, Fastrez Jacques, Soumillion Patrice
Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Place Croix du Sud 4-5, bte3, 1348 Louvain la-Neuve, Belgium.
J Biol Chem. 2008 Nov 21;283(47):32516-26. doi: 10.1074/jbc.M805375200. Epub 2008 Sep 18.
It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and beta-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a beta-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A beta-lactamases. Interestingly, the Omega-loop, which, in the beta-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate beta-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter Omega-loop.
人们普遍认为，丝氨酸β-内酰胺酶是由一些参与细菌肽聚糖生物合成和维持的祖先DD-肽酶进化而来的。DD-肽酶也被称为青霉素结合蛋白（PBPs），因为它们与β-内酰胺抗生素（如青霉素）形成稳定的酰基酶。另一方面，β-内酰胺酶与这些抗生素的反应类似，但酰基酶不稳定且迅速水解。此外，所有已知的PBPs和β-内酰胺酶的序列相似性都非常低，因此很难理解PBP如何进化成β-内酰胺酶。在这项研究中，我们鉴定了一个新的蓝藻PBP家族，其与最广泛存在的A类β-内酰胺酶具有最高的序列相似性。有趣的是，在β-内酰胺酶中携带参与脱酰基过程的必需谷氨酸的Ω环短了六个氨基酸，并且不包含任何谷氨酸残基。从这个新的蛋白质家族中，我们对嗜热栖热菌的PBP-A进行了表征，并发现它对通常是DD-肽酶良好底物的合成硫酯具有水解活性。青霉素降解途径以及酰化和脱酰化速率具有PBPs的特征。在首次尝试产生β-内酰胺酶活性时，通过在较短的Ω环中引入谷氨酸，脱酰化速率提高了90倍。
