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
(3 ‒5 ) 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 3 ‒8 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 16 ‒18 ,[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.