Figure 5.

HPLC analysis (254 nm) of in vitro biochemical conversions of bafilomycins. Trace i, the negative control for trace ii, in which Orf2 and Orf3 were boiling inactivated; trace ii, in vitro transformation from 1 to 2 by Orf2 and Orf3; trace iii, in vitro conversion from 1 to 4 by Orf2 and Orf3; trace iv, the negative control for trace v, in which BafX, BafY, and BafZ were boiling inactivated; trace v, in vitro transformation from 2 to 3 by BafX, BafY, and BafZ; trace vi, the negative control for trace vii, in which all five tailoring enzymes were boiling inactivated; trace vii, one-pot transformation from 1→2→3 by Orf2, Orf3, BafX, BafY, and BafZ. Trace viii, 3 authentic standard. Note: all reactions were carried out at 28 °C for 4 h. The concentration of all used enzymes was 10 μm.