Fig. 4. β (1–6) sugar transfer by OsUGT91C1 at the R1 end of steviol substrates STB and Reb E.

a, d LC-MS was used to monitor the reaction progress of STB (a) or Reb E (d) with OsUGT91C1. The HPLC traces represent the 18 h control reaction without the enzyme (black); the incubations for 2 h and 18 h with the enzyme at 0.15 mg ml⁻¹; repeated with the enzyme (5x) at 0.75 mg ml⁻¹ (blue). The yields of the products are related to the enzyme concentration and the reaction duration, consistent with enzymatic-catalyzed reaction. b, e Mass spectra of the new peak in LC-MS are consistent with a single glucose mass addition at the R1 end of the molecule. The products are arbitrarily named STB-X (b) and Reb E-X (e). The main negative derived ions are labeled in MS analyses. The negative parent ions [(STB-X)-H]⁻ with m/z at 803 and [(Reb E-X)-H]⁻ with m/z at 1127 were explicitly isolated and characterized by MS/MS. The insert notes where the ester bond breaks first during MS/MS fragmentation with a red dash line. c, f Reaction schemes catalyzed by OsUGT91C1 on substrate STB (c) and Reb E (f).