Angewandte
Chemie
by the a-NH2 group. The subsequent decarboxylation then
leads to the formation of 10 (Scheme 2). This conversion may
be aided similarly by the catalase CnsD, as is the case during
chanoclavine biosynthesis.[8b] Taken together, our knockout
and chemical-complementation experiments revealed the
divergent synthesis of two different indole-containing build-
ing blocks, tryptamine and 10, from l-Trp.
We then turned to the downstream enzymes that may
modify the core 9 through methylation, acylation, and
epoxidation. The N15-methylation and N16-acylation reac-
tions take place on the secondary amines on opposite poles of
the core and may be important in stabilizing the otherwise
labile aminal linkages. Deletion of cnsE, which encodes the
sole methyltransferase in the cluster, led to the disappearance
encodes a highly reducing PKS that has ketoreductase and
dehydratase domains, but no enoylreductase domain. As
expected, knockout of cnsI led to the exclusive formation of
1 (Figure 2B). The gene cnsK encodes an enzyme belonging
to the transferase family (PFAM02458), and may play a role
in transferring the polyketide chain to N16 of the core.
Deletion of cnsK abolished the production of both 1 and 2.
Instead, a single product 12 (lmax = 267 nm, m/z 415 [M+H]+)
was produced (Figure 2D). The new compound was structur-
ally characterized as the non-acylated version of 1 and 2 (see
Table S9 and Figures S22 and S23). Production of 12 (com-
munesin I) confirmed the role of CnsK as the N16 acyltrans-
ferase. It is possible that the hexadienyl product is first
hydrolytically removed from the PKS by the serine hydrolase
CnsH, converted into hexadienyl-CoA by the CoA ligase
CnsG, and then transferred to 12 by CnsK. Surprisingly, the
simultaneous loss of 1 in this mutant strain suggests that CnsK
may also be a promiscuous acyltransferase that can tolerate
a range of acyl groups, including acetyl-, propionyl-, and
butyryl-CoA, which lead to 1, 7, and 8, respectively.
Exploration of different acyl groups could lead to the
production of additional non-natural communesin ana-
logues.
of 1 and 2, and the emergence of two compounds 3 (lmax
=
268 nm, m/z 495 [M+H]+) and 5 (lmax = 269 nm, m/z 443
[M+H]+), which are the desmethyl versions of 2 and 1,
respectively (Figure 2A). The structure of 3 was confirmed by
The gene cnsJ encodes a putative oxidative enzyme that
displays 40% sequence similarity to FtmF found in the
fumitremorgin pathway.[12] CnsJ may therefore install the
C21–C22 epoxide in the prenylated portion of 1 and 2.
Indeed, deletion of cnsJ led to the production of 6 (m/z 441
[M+H]+), 13 (m/z 493 [M+H]+), and 14 (m/z 399 [M+H]+)
(Figure 2C), all of which were structurally characterized
(see Tables S6, S10, and S11 and Figures S24–S32). The
predominant product 14 (communesin K) is a new com-
pound that is not acylated at N16 and non-oxidized at C21–
C22. The two minor products 6 and 13 are communesin F
and
a
new analogue (communesin J), respectively
(Scheme 2). The accumulation of non-acylated 14 as the
major product suggests that the downstream acyl-transfer
reaction catalyzed by CnsK may be sluggish in the absence
of the C21–C22 epoxide and most likely takes place after
the actions of CnsJ (Scheme 2). Communesin K (14) is the
simplest characterized communesin that contains the
heptacyclic core. We also constructed a DcnsJ/DcnsE
double mutant in an attempt to isolate 9. Although
selective ion monitoring showed the presence of a new
compound with UV and mass spectra consistent with those
of 9, its isolation was unsuccessful owing to rapid degrada-
tion of the molecule. Although we could not pinpoint the
timing of the methylation at N15 with the knockout studies,
the structure of 14 coupled with its significantly improved
stability as compared to that of 9 suggests that the
methylation step takes place immediately after the forma-
tion of 9 to yield 14.
Figure 2. Mapping of the late stages of the communesin biosynthetic
pathway. Confirmation of the roles of enzymes encoded by A) cnsE
(methyltransferase), B) cnsI (PKS), C) cnsJ (epoxidase), and D) cnsK (acyl-
transferase). E) The role of the enzyme encoded by cnsC (P450) was also
investigated. In each case, the selected ion monitoring MS trace with the
masses indicated in different colors is shown below the UV trace
(l=270 nm).
NMR spectroscopy to be communesin C (see Table S5 and
Figures S15 and S16).
Oxidative coupling of 10 and tryptamine was proposed to
be a possible route to the formation of the challenging vicinal
quaternary centers of the communesins.[5] We therefore
investigated the role of CnsC, a cytochrome P450 encoded
in the cluster. Protein-sequence analysis suggests CnsC may
be an atypical P450 with low sequence homology (< 25%) to
those in the P450 database.[13] Genetic inactivation of cnsC led
The co-isolation of 1 and 2 suggests that N16-acylation by
acetyl-CoA and the hexadienyl unit should be in competition.
Acylation to introduce the hexadienyl chain in 2 is reminis-
cent of the acylation of fumagillol by a pentaene polyketide in
the biosynthesis of fumagillin.[11] In the cns pathway, cnsI
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
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