In vitro biochemical characterization of LobP1
For in vitro biochemical characterization, LobP1 was produced as
soluble His6-tagged proteins in E. coli (Figure
S9). The purified LobP1 was shown to convert 6 , the major
product from the ∆lobP1 mutant of Streptomyces sp. SCSIO
01127 (Figure 2), to the C-32 hydroxylated product 2 in the
presence of ferrodoxin (Fdx) and ferrodoxin reductase (FdR) from
cyanobacterium Synechococcus elongatusPCC7942,[24] in contrast, 6 remained
unchanged in the absence of LobP1 (Figure 3, Figure S10). Subsequently,
another five intermediates 3 ‒5 , 7 ‒8from the ∆lobP1 mutant of Streptomyces sp. SCSIO 01127
were assayed with LobP1. Interestingly, LOBs 3 ‒5 and7 could be converted by LobP1 to their putative hydroxylated
products LOBs CR5 (19 ), CR1
(20 ),[7] CR6 (21 ) and A
(1 ) (Figure 3), respectively, upon comparison with the standard
or by LC-ESI-HRMS analyses (Figure S10-S11). However, no reaction of
LobP1 with 8 was detected (Figure S12). Cumulatively, thesein vitro biochemical assays further confirmed LobP1 as the LOB
C-32 hydroxylase.
To probe the substrate scope of LobP1, 13 more LOBs,9 ‒15 (Figure 2) and 22 ‒27 (Figure 3)
were also assayed with LobP1. LOBs F (22 ) and G2-1
(23 ), previously isolated from the mutant
∆lobG2 /Streptomyces sp. SCSIO
01127,[9] could be converted by LobP1 to putative
hydroxylated products LOBs L2 (28 ) and L
(29 )[14] (Figure 3, Figure S10),
respectively, by analyzing the LC-ESI-HRMS (Figure S11). However, LOBs9 ‒15 and 24 ‒27 , previously isolated
from S. coelicolor M1154/pCSG5560 (carrying the lob BGC)
and S. coelicolor M1154/pCSG5561 (carrying the lob BGC
with ∆lobG1 ),[13] could not react with
LobP1 (Figure S12). Conclusively, LobP1 recognizes LOBs containing a
sugar at C-17 and a tri- or di-saccharide chain at C-9
(3 ‒7 , 22 and 23 ), while does not
recognize LOBs with no sugar at C-17 (8 and24 ‒27 ), or LOBs with a monosaccharide at C-9
(11 ‒15 ). Apparently, LobP1 displays relatively higher
catalytic efficiency towards substrates with a trisaccharide at C-9
(e.g. 3 ‒7 ) than those with a disaccharide at
C-9 (e.g. 22 and 23 ) (Figure 3), indicating
that the C-32 hydroxylation by LobP1 most likely occurs after the
terminal digitoxosylation by LobG2 (Figure 1). Intriguingly, biochemical
characterization showed that LobP1 failed to recognize LOBs with a
monosaccharide at C-9, such as 11 , 13 and 14 ,
which was inconsistent with the facts that their corresponding C-32
hydroxylated counterparts 18 , 16 and 17 were
quantitatively produced in S. coelicolor M1154/pCSG5560 (carrying
the lob BGC) (Figure 2). This apparent in vivo andin vitro functional discrepancy of LobP1 led to the assumption
that LobP1 might require native redox partners from S. coelicolorM1154 to perform in vivo C-32 hydroxylation on 11 ,13 and 14 , since different redox partners may exert
distinct effects on P450 enzyme reactions.[25]
We have previously shown that the methyltransferase LobS1 catalyzed the
installation of the 7c-O -methyl group at the terminal
L-digitoxose moiety of LOBs.[8] It was unclear
about the reaction timing of LobS1 and LobP1 in the LOB biosynthetic
pathway. For a better understanding, we compared kinetic parameters of
LobP1 toward different substrates (Table 1 and Figure S13). It was shown
that LobP1 displayed 100 times higher affinity
(K m) toward 6 than 7 , with thek cat/K m value toward6 30 times greater than 7 , suggesting a preference of
LobP1 to LOBs with a nitrosugar over an amino sugar at C-17. LobP1
displayed a slightly higherk cat/K m value of3 (7C-OMe) than 6(7C-OH), indicating that
7C-O -methylation by LobS1 might occur after the
LobP1-catalyzed C-32 hydroxylation.
Table 1 Kinetic parameters of LobP1 toward different substrates