MurG (uridine diphosphate-N-acetylglucosamine/N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase) is an essential bacterial glycosyltransferase that catalyzes the N-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 M. smegmatis strain or a thermostable MraY and MurG of Hydrogenivirga sp. as enzyme sources, along with Park’s nucleotide or Park’s nucleotide-N^ε-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.

中文翻译：

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细菌糖基转移酶MurG的底物耐受性：新的基于荧光的分析。

MURG（尿苷diphosphate- Ñ乙酰氨基葡萄糖/ Ñ -acetylmuramyl-（五肽）焦磷-undecaprenol Ñ乙酰氨基葡萄糖转移酶）是一个重要的细菌糖基转移酶，其催化Ñ肽聚糖的生物合成过程中的脂质I的乙酰氨基葡萄糖（GlcNAc的）转化为脂质II。Park的核苷酸已成为研究MraY（磷酸-MurNAc-（五肽）转位酶）和MurG的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核苷酸通过脂质I转化为脂质II的生物转化中的各个过程。在此，我们报道了使用耻垢分枝杆菌的膜级分进行健壮的MurG测定的方法。Hydrogenivirga sp。的菌株或热稳定的MraY和MurG 。作为酶源，随着公园的核苷酸或公园的核苷酸- ñ ^ε -C ₆ -dansylthiourea和尿苷二磷酸（UDP）-GlcN-C ₆-FITC作为受体和供体的底物。可通过HPLC在双UV模式下同时进行MraY和MurG产品的鉴定。方便地，生成的脂质II荧光类似物也可以通过UV-Vis光谱进行定量，而无需分离未反应的脂质I衍生物。此处报道的基于微孔板的测定适用于高通量MurG筛选。对小分子集合物的初步筛选已证明了该测定的稳健性，并导致重新发现了瑞斯托霉素A作为强抗分枝杆菌MurG和MraY抑制剂。