S1 Supporting Information In Vitro Stepwise Reconstitution of Amino Acid Derived Vinyl Isocyanide Biosynthesis: Detection of an Elusive Intermediate Wei-chen Chang,* a Dev Sanyal, a Jhih-Liang Huang, a Kuljira Ittiamornkul, b Qin Zhu b and Xinyu Liu* b a Department of Chemistry, North Carolina State University, Raleigh, NC 27695, and b Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260 Contents: General……………………………………………………………………….…………………..S2 Overexpression and purification of AmbI1, AmbI2, AmbI3 and IsnB.. ……………………..S2-S3 Synthesis of (2S)-3-(1H-indol-3-yl)-2-isocyanopropanoic acid 1………………...................S3-S5 Biochemical assay and LCMS study of AmbI1, AmbI2, AmbI3 and IsnB catalyzed reaction……………………...……………………………………………………………......S5-S6 Supporting Figures S1-S4…..………………………………………………...………………S6- S9 References………………………………………………………………………………………S10