Substrate Tolerance of Bacterial Glycosyltransferase MurG: Novel Fluorescence-Based Assays.
Substrate Tolerance of Bacterial Glycosyltransferase MurG: Novel Fluorescence-Based Assays.
复制标题
细菌糖基转移酶 MurG 的底物耐受性：新型荧光检测方法。
DOI：
10.1021/acsinfecdis.9b00242
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发表时间：
2020
影响因子：
5.3
通讯作者：
Kurosu,Michio
中科院分区：
文献类型：
--
作者：
Mitachi,Katsuhiko;Yun,HyunGi;Gillman,CodyD;Skorupinska-Tudek,Karolina;Swiezewska,Ewa;ClemonsJr,WilliamM;Kurosu,Michio
MurG （uridine diphosphate-N-acetylglucosamine/N-acetylmuramyl-（五肽）pyrophospyl - decaprenoln -acetylglucosamine transferase）是一种重要的细菌糖基转移酶，它在肽聚糖生物合成过程中催化non -acetylglucosamine （GlcNAc）将脂质I转化为脂质II。Park’s nucleotide已成为研究MraY （phospho-MurNAc-（五肽）转位酶）和MurG功能的便捷生化工具；然而，还没有开发出荧光探针来区分Park 's核苷酸通过脂质i生物转化为脂质II的单个过程。在这里，我们报告了使用aM的膜部分对MurG进行的稳健测定。smgmatisstrain或耐热的MraY和MurG ofHydrogenivirga sp.作为酶源，与Park 's核苷酸或Park 's核苷酸- nε - c6 -丹硫脲和尿苷二磷酸(UDP)-GlcN-C6-FITC作为受体和供体底物。在双紫外模式下，可以同时对MraY和MurG产品进行鉴定。方便的是，生成的脂质II荧光类似物也可以通过紫外可见光谱法定量，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分析适用于高通量MurG筛选。对一组小分子的初步筛选证明了检测方法的稳健性，并重新发现了瑞斯托司汀A作为一种强效抗细菌MurG和MraY抑制剂。
MurG (uridine diphosphate-N-acetylglucosamine/N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenolN-acetylglucosamine transferase) is an essential bacterial glycosyltransferase that catalyzes theN-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 aM. smegmatisstrain or a thermostable MraY and MurG ofHydrogenivirga sp.as enzyme sources, along with Park’s nucleotide or Park’s nucleotide-Nε-C6-dansylthiourea and uridine diphosphate (UDP)-GlcN-C6-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.