Fig. 4: Homology modeling and substrate docking experiments reveal the basis for substrate promiscuity of HYC3Os and dual catalytic activity of HYC3Rs for both 3-dehydro MIA and strictosidine aglycone substrates.

a Docking studies of rauwolscine, mitragynine, geissoschizine methyl ether, and tetrahydroalstonine (THA) at the active sites of RtHYC3O and HpHYC3O homology models. See Supplementary Figs. 73, 77, 79, and 83 for larger representations. The surface surrounding the active sites are shown. Hydrogen bond (white dashed lines) networks and π-stacking interactions orient the substrates (orange) for efficient H3 hydride abstraction by FAD (green). The distance between the substrate’s H3 and the N5 of FAD is indicated by a black line, with corresponding distances in angstroms (Å). b Docking studies of 3-dehydrorauwolscine, 3-dehydro-THA, and 4,21-dehydro-THA at the active sites of the RsHYC3R homology model and CrTHAS2 crystal structure demonstrate key differences in substrate accommodation. See Supplementary Figs. 87, 89, 91, and 92 for larger representations. The spacious active site in RsHYC3R supports the binding and reduction of both 3-dehydro-THA and 4,21-dehydro-THA (a form of strictosidine aglycone mixture), accounting for its dual catalytic activity. In contrast, the narrower active site of CrTHAS2 selectively facilitates the reduction of 4,21-dehydro-THA but not 3-dehydro-THA. NADPH is shown in green, and alkaloid substrates are in magenta. The distance between the substrate’s H3 or H21 and the hydride donor of NADPH is indicated by a black line, with corresponding distances in angstroms (Å). c Illustration of rauwolscine oxidation by FAD at the RtHYC3O active site. d Illustration of 3-dehydrorauwolscine reduction by NADPH at the RsHYC3R active site. e Illustration of 3-dehydro-THA binding and reduction by NADPH at the RsHYC3R active site. f LC-MS/MS MRM [M + H]⁺ (355 > 144 and 353 > 144) chromatograms show the in vivo reduction of strictosidine aglycone by strictosidine aglycone reductases and HYC3Rs. These reductases were expressed in yeast Saccharomyces cerevisiae strain AJM-dHYS engineered for de novo production of strictosidine aglycone. The reductases exhibited diverse product spectra, reflecting the structural diversity of strictosidine aglycone in equilibrium. An unknown m/z 353 peak is labeled with a red dot. Source data are provided as a Source Data file.