Figure 5.

Enzyme assays of recombinant CHS proteins. A, RP-TLC analysis of products extracted from enzyme assays of recombinant proteins (CalCHS1, SbCHS2, SbCHS8, and RtSTS1). Assays were performed with 1.0 μg of purified protein, radiolabeled malonyl-CoA, and either cinnamoyl-CoA or p-coumaroyl-CoA. Positions of flavanones (Pc, pinocembrin; N, naringenin), stilbenes (Ps, pinosylvin; R, resveratrol), and the BNY-type and CTAL-type pyrone by-products (BNY-P and CTAL-P) are indicated. Inset, SDS-PAGE analysis of recombinant proteins visualized with Coomassie Brilliant Blue R250. Lane 1, SbCHS2 crude cell lysate; lane 2, purified SbCHS2; lane 3, SbCHS8 crude cell lysate; lane 4, purified SbCHS8. B, Ratios of flavanone-to-stilbene products in the assay reactions. ¹⁴C-labeled products were quantified after phosphoimaging and ratios were calculated based on average values from three independent assays. C, LC-ESI-SRM analysis of reaction products. Flavanone and stilbene products were confirmed by LC-MS/MS in SRM mode. RT, Retention time; CE, collision energy. Structures of the starter CoAs, flavanones, and stilbenes are shown in Figure 1.