Chemoenzymatic syntheses of water-soluble lipid I fluorescent probes
Peptidoglycan (PG) is unique to bacteria, and thus, the enzymes responsible for its biosynthesis are promising antibacterial drug targets. The membrane-embedded enzymes in PG remain significant challenges in studying their mechanisms due to the fact that preparations of suitable enzymatic substrates require time-consuming biological transformations or chemical synthesis. Lipid I (MurNAc(pentapeptide)-pyrophosphoryl prenol) is an important PG biosynthesis intermediate to study the central enzymes, translocase I (MraY/MurX) and MurG. Lipid I isolated from nature contains the C_(50)- or C_(55)-prenyl unit that shows extremely poor water-solubility that renders studies of translocase I and MurG enzymes difficult. We have studied biological transformation of water soluble lipid I fluorescent probes using bacterial membrane fractions and purified MraY enzymes. In our investigation of the minimum structural requirements of the prenyl phosphates in MraY-catalyzed lipid I synthesis, we found that (2Z,6E)-farnesyl phosphate (C_(15)-phosphate) can be recognized by Escherichia coli MraY to generate the water-soluble lipid I fluorescent probe in high-yields. Under the optimized conditions, the same reaction was performed by using the purified MraY from Hydrogenivirga spp. to afford the lipid I analog with high-yields in a short reaction time.
© 2015 Elsevier Ltd. Received 5 December 2014; Revised 6 January 2015; Accepted 6 January 2015; Available online 12 January 2015. The National Institutes of Health is greatly acknowledged for financial support of this work (AI084411 to MK and DP1GM105385 to WMC). We also thank University of Tennessee for generous financial support. NMR data were obtained on instruments supported by the NIH Shared Instrumentation Grant. The following reagent was obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, Strain H37Rv, and Gamma-Irradiated Mycobacterium tuberculosis, NR-14819.
Accepted Version - nihms653295.pdf
Supplemental Material - mmc1.pdf