Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute of Antimicrobial Research, University of Oxford, Oxford, UK.
Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, Cluj-Napoca, Romania.
Commun Biol. 2024 Sep 18;7(1):1173. doi: 10.1038/s42003-024-06785-3.
The essential L,D-transpeptidase of Mycobacterium tuberculosis (Ldt) catalyses the formation of 3 3 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit Ldt have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on Ldt We determined a crystal structure of wild-type Ldt reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the Ldt active site. The results will be useful in design of Ldt inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.
结核分枝杆菌（Ldt）的必需 L，D-转肽酶催化细胞壁肽聚糖中 3 个 3 交叉连接的形成，是开发抗结核治疗药物的靶点。由于缺乏其结合底物的知识，抑制 Ldt 的努力受到了阻碍。为了解决这一差距，我们通过过度生产肽聚糖囊来优化从棒状杆菌属细胞壁中分离天然二糖四肽单体。这些四肽肽被用于结合/周转测定和 Ldt 的生物物理研究。我们确定了与天然底物反应的野生型 Ldt 的晶体结构，即肽聚糖层的四肽单体。该结构显示了催化半胱氨酸和供体底物之间形成硫酯键，反映了转肽酶反应的中间产物；它说明了供体底物进入 Ldt 活性位点的方式。这些结果将有助于设计 Ldt 抑制剂，包括基于底物结合相互作用的抑制剂，这是一种成功应用于其他亲核半胱氨酸酶的策略。
