HPLC analyses of WelI1-WelI3 catalyzed indole-isonitrile formation. A) Biosynthetic steps catalyzed by WelI1 and WelI3 respectively. In vitro reconstitution assay for indole-isonitrile biosynthesis using cell lysates of E. coli BL21(DE3) heterologously expressing WelI1 and WelI3. Models of WelI1 and WelI3 were built based on homology to PvcA and PvcB X-ray structures  using Phyre2.0. B) HPLC was analyzed at 310 nm with a UV detector. X-axis – retention time in minutes (min). Y-axis - intensity in arbitrary units. Presented as a stacked Y-plot and is drawn to relative intensity units. B1) Synthesized cis indole-isonitrile only (tR = 8.8 min). B2) Synthesized trans indole-isonitrile only (tR = 13.1 min). B3) Co-injection of synthetic standards of cis and trans indole-isonitrile. B4) Control for enzyme assay where cell lysates of E. coli BL21(DE3) were subjected to assay conditions without WelI1 and WelI3. B5) WelI1 and WelI3 enzyme assay after 16 h incubation at 25°C. B6) Control sample (4) spiked with cis indole-isonitrile after 3 h incubation. B7) Control sample (4) spiked with cis indole-isonitrile after 16 h incubation. B8) Co-injection of cis and trans indole-isonitrile with enzyme assay mixture. Peaks show only relative intensities and are not normalized for concentration of metabolites.