Mitachi Katsuhiko, Yun Hyun Gi, Gillman Cody D, Skorupinska-Tudek Karolina, Swiezewska Ewa, Clemons William M, Kurosu Michio
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, Tennessee 38163, United States.
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd., Pasadena, California 91125, United States.
ACS Infect Dis. 2020 Jun 12;6(6):1501-1516. doi: 10.1021/acsinfecdis.9b00242. Epub 2019 Dec 11.
MurG (uridine diphosphate--acetylglucosamine/-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol -acetylglucosamine transferase) is an essential bacterial glycosyltransferase that catalyzes the -acetylglucosamine (GlcNAc) transformation of lipid I to lipid II during peptidoglycan biosynthesis. Park's nucleotide has been a convenient biochemical tool to study the function of MraY (phospho-MurNAc-(pentapeptide) translocase) and MurG; however, no fluorescent probe has been developed to differentiate individual processes in the biotransformation of Park's nucleotide to lipid II via lipid I. Herein, we report a robust assay of MurG using either the membrane fraction of a strain or a thermostable MraY and MurG of as enzyme sources, along with Park's nucleotide or Park's nucleotide--C-dansylthiourea and uridine diphosphate (UDP)-GlcN-C-FITC as acceptor and donor substrates. Identification of both the MraY and MurG products can be performed simultaneously by HPLC in dual UV mode. Conveniently, the generated lipid II fluorescent analogue can also be quantitated via UV-Vis spectrometry without the separation of the unreacted lipid I derivative. The microplate-based assay reported here is amenable to high-throughput MurG screening. A preliminary screening of a collection of small molecules has demonstrated the robustness of the assays and resulted in rediscovery of ristocetin A as a strong antimycobacterial MurG and MraY inhibitor.
MurG（尿苷二磷酸 - 乙酰葡糖胺/-乙酰胞壁酰 -（五肽）焦磷酸化 - 十一异戊烯醇 - 乙酰葡糖胺转移酶）是一种必需的细菌糖基转移酶，在肽聚糖生物合成过程中催化脂质I向脂质II的乙酰葡糖胺（GlcNAc）转化。帕克核苷酸一直是研究MraY（磷酸 - 胞壁酰 -（五肽）转位酶）和MurG功能的便捷生化工具；然而，尚未开发出荧光探针来区分帕克核苷酸通过脂质I生物转化为脂质II的各个过程。在此，我们报道了一种使用菌株的膜组分或作为酶源的热稳定MraY和MurG进行MurG活性检测的方法，同时使用帕克核苷酸或帕克核苷酸 - C - 丹磺酰硫脲以及尿苷二磷酸（UDP） - GlcN - C - FITC作为受体和供体底物。通过高效液相色谱（HPLC）在双紫外模式下可同时鉴定MraY和MurG的产物。方便的是，可以通过紫外可见光谱法对生成的脂质II荧光类似物进行定量，而无需分离未反应的脂质I衍生物。本文报道的基于微孔板的检测方法适用于高通量的MurG筛选。对一系列小分子的初步筛选证明了该检测方法的可靠性，并重新发现瑞斯托菌素A是一种强效的抗分枝杆菌MurG和MraY抑制剂。
