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 35 , 78from the ∆lobP1 mutant of Streptomyces sp. SCSIO 01127 were assayed with LobP1. Interestingly, LOBs 35 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,915 (Figure 2) and 2227 (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, LOBs915 and 2427 , 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 (37 , 22 and 23 ), while does not recognize LOBs with no sugar at C-17 (8 and2427 ), or LOBs with a monosaccharide at C-9 (1115 ). Apparently, LobP1 displays relatively higher catalytic efficiency towards substrates with a trisaccharide at C-9 (e.g. 37 ) 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