Substrate Tolerance of Bacterial Glycosyltransferase MurG: Novel Fluorescence-based Assays
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-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.
© 2019 American Chemical Society. Received 2 July 2019. Published online 26 November 2019. Published in issue 12 June 2020. The National Institutes of Health is greatly acknowledged for financial support of this work (Grant GM114611). M.K. thanks UTRF (University of Tennessee Health Science Center) for generous financial support (Innovation award R079700292). We thank Miss Kendal G. Crawley (UTHSC Research Scholar Program), Miss Shakiba Eslamimehr, and Mr. Stewart J. Clayton (UTHSC) for preparing membrane fractions from the bacteria and for performing the cytotoxicity assays. NMR and HR-MS data were obtained on instruments supported by the NIH Shared Instrumentation Grant. The following microbes were obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, strain H37Rv, and gamma-irradiated Mycobacterium tuberculosis, NR-14819. The authors declare no competing financial interest.
Supplemental Material - id9b00242_si_001.pdf
Accepted Version - nihms-1059256.pdf