Background and Originality Content
Lobophorins (LOBs) belong to a large group of spirotetronate antibiotics, which possess a pentacyclic aglycon embedded in the macrocyclic core scaffold and feature a tetronate moietyspiro -linked with a cyclohexene ring, displaying potential antibacterial and antitumor activities.[1]Different modifications, such as glycosylation, methylation and hydroxylation on the core scaffold endow LOBs with significant structural and bioactive diversity. To date, more than 30 LOBs have been reported.[2-16]
The structural complexity and pharmaceutical potency inspired a number of biosynthetic studies on LOBs and related spirotetronates to elucidate the formation of the pentacyclic aglycon[17-23]and the tailoring modification reactions,[8, 9, 13] such as the stepwise glycosylations and the sugar-O -methylation (Figure 1). LobG1 was characterized as theO -glycosyltransferase to attach a D-kijanose at C-17. LobG3 was demonstrated as a flexible glycosyltransferase that could not only attach the first two L-digitoxoses at C-9, but also add the D-kijanose or its variants at C-9. LobG2 was verified to attach the terminal digitoxose,[9, 13] while LobS1 was responsible for installing the methyl group at the terminal digitoxose[8] (Figure 1). However, the enzyme responsible for the C-32 hydroxylation has not been elucidated. Bioinformatic analysis shows that the P450 monooxygenase LobP1 is the most likely candidate responsible for catalyzing this reaction.
Figure 1 The structures of LOBs A (1 ) and B (2 ) and enzymes responsible for the glycosylation, methylation and possible hydroxylation in the post modifications.
Herein, we report (i) the isolation, structure elucidation and bioactivity evaluation of LOB derivatives from thelobP1 -inactivated mutant; (ii) the functional characterization of LobP1 as the C-32 hydroxylase by both in vivo genetic experiments and in vitro biochemical assays; (iii) the investigation of the substrate scope and kinetic parameters of LobP1 towards different substrates. This study characterizes the catalytic features of LobP1 and provides important implications for the reaction timing of tailoring steps in LOB biosynthesis.
Results and Discussion
Genetic characterization of lobP1
Bioinformatics analysis shows that lobP1 encodes a cytochrome P450 monooxygenase, sharing 70% identity with KijA3, an enzyme proposed to be responsible for the C-32 hydroxylation of kijanimicin.[18] Therefore, LobP1 was proposed to be the most likely candidate catalyzing the C-32 hydroxylation in LOB biosynthesis. To verify this hypothesis, lobP1 was inactivated by insertional mutation in Streptomyces sp. SCSIO 01127 (Figure S1). The resulting ∆lobP1 mutant abolished the production of LOBs A and B (1 and 2 ), but accumulated several LOB-related products (Figure 2). Subsequently, three new derivatives LOBs N1‒N3 (35 ) were isolated from the ∆lobP1 mutant (Figure 2, Figures S2‒S4 and Table S3), together with three known ones LOBs E (6 ),[4] N (7 )[15] and CR4 (8 )[9, 13, 16] determined by comparing their NMR and HRESIMS data with those previously reported, respectively (Figure 2, Figures S5‒S7).
LOB N1 (3 ) (C60H88N2O20,m /z 1155.5854 [M − H], calcd 1155.5857; Figure S2) was highly similar to6 .[4] The only difference was that the oxymethyl (δ C/δ H57.4/3.41, CH3-7C) in 6 was absent in 3 , accordingly, the carbon signal of C-4C was shielded from δ C 88.2 (in CDCl3) in 6 to δ C69.0 (in DMSO-d6 )) in 3 . Thus, 3 was determined to be desmethyl derivate of 6 . The molecular formula of LOB N2 (4 ) was established to be C61H91NO19(m /z 1140.6117 [M − H], calcd 1140.6112, Figure S3) by HRESIMS. Inspection of the1H, 13C, and 2D NMR data of4 (Table S3) revealed that 4 only differed from7 in the sugar D moiety: a 3-hydroxyl-D-kijanose (δ C 70.7, C-3D) in 4 , and a 3-amino-D-kijanose in 7 (δ C 54.2, C-3D). Therefore, 4 was characterized as 3-hydroxyl-D-kijanose derivative of 7 . The molecular formula of LOB N3 (5 ) was assigned as C63H94N2O20(m /z 1197.6325 [M − H], calcd 1197.6327, Figure S4) by HRESIMS. The NMR data of 5 were highly similar to those of 4 except for the signals of an additional carbonyl (δ C 172.6, C-10D) and methyl (δ C/δ H 26.3/2.00, CH3-11D) groups existed in 5 . The methyl and carbonyl were found to belong to aN -methylcarbamoyl group by the COSY correlation between H3-11D/10D-NH (δ H 9.57) and HMBC correlation from H3-11D to C-10D, which was linked to 3D-O by shielded C-3D in5 comparing to that in 4 and the NOESY correlation between H3-11D/H3-7D.
Compounds 38 were all confirmed as LOBs lacking the hydroxyl group at C-32. Besides, introduction of the BAC pCSG5661 (containing lob BGC with inactivated lobP1 ; Figure S8) into S. coelicolor M1154 also abolished the production of the C32-hydroxylated LOBs 1618 ,[13]but led to the non-hydroxylated LOBs (such as 10 , 12 ,13 , and 15 ) (Figure 2). These in vivo results confirmed LobP1 as the requisite C-32 hydroxylase.