Fig. 7: In vitro enzymatic conversions of compound 7 and proposed biosynthetic pathway of MDB formation in streptovaricins.

a LC–ESI–HRMS analysis of enzymatic conversion by StvM1 and/or StvA2 and/or StvP2 using 7 as substrate. (i) 7 in reaction buffer with Fdx, FdR, and NADPH. (ii) 7 and StvM1 in reaction buffer without Fdx, FdR, and NADPH. (iii) 7 and StvM1 in reaction buffer with Fdx, FdR, and NADPH. (iv) 7 and StvA2 in reaction buffer without Fdx, FdR, and NADPH. (v) 7 and StvA2 in reaction buffer with Fdx, FdR and NADPH. (vi) 7, StvM1 and StvA2 in reaction buffer without Fdx, FdR, and NADPH. (vii) 7, StvM1 and StvA2 in reaction buffer with Fdx, FdR, and NADPH. (viii) 7, StvM1, StvA2, and StvP2 in reaction buffer without Fdx, FdR, and NADPH. (ix) 7, StvM1, StvA2, and StvP2 in reaction buffer with Fdx, FdR, and NADPH. All the enzymatic reactions were incubated at 28 °C for 3 h. Pure compounds (1, 2, 7, and 8) identified by NMR were used as standards or substrate in assay. The traces were extracted at calcd. m/z for [M + H]⁺: 770.33823 (1, red peak), 772.35388 (2, blue peak), 728.32767 (8, purple peak), 714.31202 (7, pink peak), 814.36445 (yellow peak indicated the compound with double acetylations at C-4 and C-21, or C-25), and 812.34880 (green peak indicated the MDB-contained compound with double acetylations at C-4 and C-21 or C-25). b A proposed biosynthetic pathway of MDB formation in streptovaricins. Experiments in a are representative of three independent experiments.