Fig. 3

In vitro biochemical characterization of successive functional enzymes (OpLAMT, OpSLS, OpSTR) involved in CPT biosynthesis in O. pumila. a Loganic acid methyltransferase HPLC assay with purified recombinant OpLAMT enzyme. (i) OpLAMT assay with loganic acid and SAM, (ii) boiled OpLAMT assay with loganic acid and SAM, (iii) OpLAMT assay with loganic acid, (iv) loganin (L) standard, (v) loganic acid (LA) standard, and (vi) S-adenosyl methionine (SAM) standard. b OpSTR LC-MS assay with recombinant OpSTR enzyme. Purified recombinant protein was assayed for strictosidine activity in reaction mixtures with secologanin/secologanic acid and tryptamine. (i) boiled STR assay with secologanin, (ii) STR assay with secologanin, (iii) boiled STR assay with secologanic acid, and (iv) STR assay with secologanic acid. c Time-course assay of STR activity. OpSTR assay with secologanin (producing strictosidine) and secologanic acid (producing strictosidinic acid) (three repeats). The assays were quenched at 2 min, 4 min, 6 min, 8 min, and 10 min and then measured by LC-MS. Curve fitting was performed by GraphPad Prism 8. d Six OpSLS assays with loganic acid (LA). The solid lines in black indicate samples from the microsome assay and secologanic acid standard (SLA). The dashed lines in grey indicate samples from the boiled microsome assay (control) and loganic acid standard. (i) Loganic acid and secologanic acid standards, (ii) OpSLS6, (iii) OpSLS5, (iv) OpSLS4, (v) OpSLS3, vi) OpSLS2, and (vii) OpSLS1. Wavelength, 254 nm. e Six OpSLSs assay with loganin (L). The solid lines in black indicate samples from the microsome assay and secologanin standard (SL). The dashed lines in grey indicate samples from the boiled microsome assay (control) and loganin standard. (i) Loganic acid and secologanic acid standards, (ii) OpSLS6, (iii) OpSLS5, (iv) OpSLS4, (v) OpSLS3, (vi) OpSLS2, and (vii) OpSLS1. Wavelength, 254 nm. f Summary of the CPT biosynthesis pathway in O. pumila. Loganic acid is converted into loganin in a reaction catalyzed by OpLAMT, and then loganin is converted into secologanin by OpSLSs. Secologanin and tryptamine are then condensed into strictosidine carried out by OpSTR, which is involved in CPT biosynthesis in O. pumila