Conclusions

In summary, the P450 monooxygenase LobP1 was functionally characterized as the C-32 hydroxylase in the biosynthesis of LOBs by in vivogenetic disruption and in vitro biochemical assays. Inactivation of lobP1 afforded three new LOBs (3 ‒5 ) and three known LOBs (6 ‒8 ). Notably, the major derivative6 from the ∆lobP1 mutant showed moderate cytotoxic activity, better than its hydroxylated derivative 2 . Investigations on the substrate scope reveal that LobP1 could recognize LOBs with di- or tri- saccharide chain at C-9 and a sugar at C-17 (3 ‒7 , 22 , 23 ), but could not recognize LOBs with a monosaccharide at C-9 (such as 11 ,13 , and 14 ) in in vitro biochemical assays. Thein vivo hydroxylation of compounds 16 ‒18 may be achieved by LobP1 with the help of native redox partner. Further studies are warranted to explain their generation. The kinetic parameters of LobP1 toward different substrates indicated that the C-32 hydroxylation may occur before sugar-O -methylation. Up to now, the enzymes responsible for tailoring modifications in LOBs A and B, including sugar-O -methylation (LobS1) and C-32 hydroxylation (LobP1) have been biochemically characterized.

Experimental