Fig. 5.

Extracted ion chromatograms from LC/MS analysis of PagF assays, showing substrates (blue), singly prenylated products (orange), and doubly prenylated products (green). Extracted ion chromatograms for the substrate mass (blue) are shown at each time point for comparison. Some peaks in the blue chromatograms at each time point come from fragmentation of the prenylated products in the MS. The drawing of the YLY portion of the peptides shows where the first and second prenyl groups are attached. MS/MS fragmentation shows that the first prenylation of the linear peptides is on the N-terminal tyrosine (SI Appendix, Fig. S9), whereas the position of the first prenyl group in the cyclic peptide is inferred from the natural product prenylagaramide B. The cyclic[INPYLYP] assay contained DMAPP (5 mM), peptide substrate (40 μM), and PagF (0.2 μM). Linear peptide assays contained YLYEIAR (0.5 mM) or YLYQWLGAPV (0.5 mM), DMAPP (5 mM), and PagF (3 μM). Singly prenylated YLYQWLGAPV eluted from HPLC as a double peak. All assays' components are described in SI Appendix, Materials and Methods.