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