FIG 5.

Biochemical characterization of PrfT and ForT as β-RFA-P synthase-like enzymes for C-glycosidic bond construction. (A) HPLC traces of the PrfT-catalyzed reaction. Complete-PrfT, complete reaction of PrfT with DCOP and PRPP as the substrates; −DCOP, PrfT reaction without DCOP; −PRPP, PrfT reaction without PRPP; -Mg²⁺, PrfT reaction without Mg²⁺; N.C, PrfT reaction lacking an enzyme as the negative control; Complete-ForT, complete reaction of ForT with DCOP and PRPP as the substrate. (B) HPLC traces of the ForT-catalyzed reaction. Complete-ForT, complete reaction of ForT using ADCP and PRPP as the substrates; −ADCP, ForT reaction without ADCP; −PRPP, ForT reaction without PRPP; −Mg²⁺, ForT reaction without Mg²⁺; N.C, ForT reaction without an enzyme as a negative control; Complete-PrfT, complete reaction of PrfT with ADCP and PRPP as the substrates; Coinjection, coinjection of the ForT reaction mixture and the PrfT reaction mixture. (C) Scheme of the tautomerization reaction between DCOP and HDCP (4-hydroxy-3,5-dicarboxypyrazole). (D) Proposed mechanism for the PrfT/ForT-catalyzed reaction. When the R group is OH (NH₂), the reaction is from HDCP to compound 19 (ADCP to compound 12).