Angewandte
Communications
Chemie
and, more recently, has unveiled novel opportunities for the
mimic 13 was synthesized in five steps from 4a (Supporting
generation of unnatural polyketide derivatives.[21c] We ini-
tially used our intermediate-capturing probes for in vivo
studies on fungal and bacterial strains harboring 6-MSAS
genes, including the natural 6-MSA producer P. patulum, an
engineered E. coli host strain heterologously expressing P.
patulum 6-MSAS (E. coli BAP1 pKOS007-109),[22] and S.
antibioticus DSM40725 (producer of chlorothricin).[14a] Each
strain was grown in the presence of substrates 4–8, which are
hydrolyzed in vivo to the corresponding carboxylates 9–12
(Figure 1A; Supporting Information, Figure 2S).[20]
Information) and tested as a substrate for both 6-MSAS and
its H958A mutant. In neither case was 13 enzymatically
dehydrated (Figure 2A; Supporting Information, Fig-
ure 47S); rather, we found that purified 13 dehydrated over
The overall outcome of these in vivo experiments is
illustrated in Figure 1 (for details, see the Supporting Infor-
mation, Tables 1S–3S and following figures). In most of the
ethyl acetate extracts from both fungal and bacterial hosts,
a series of trapped intermediates, including diketides, trike-
tides, reduced triketides, and a range of putative hydroxy,
dehydrated, and aromatized tetraketides, were identified by
HR-LC-MS: these would directly reflect the nature of ACP-
bound substrates in 6-MSA assembly. Besides, putative
hydroxy, dehydrated, and aromatized pentaketides arising
from the off-loading of 6-MSAS-bound tetraketides were also
identified (Figure 1C and the Supporting Information). All
the captured intermediates, absent in control samples, were
characterized by MSn analysis, showing diagnostic peaks
resulting from the loss of N-acyl chains and subsequent cyclic
imine formation (Figure 1C and the Supporting Information).
For the majority of the putative tetraketides and pentaketides,
multiple peaks were observed: these may arise from isomer-
ization, cyclization and dehydration events which can be
spontaneous or enzyme-catalyzed. A distinction between
hydroxy, dehydrated, and aromatized advanced species was
made on the basis of variable LC retention times as well as of
detected accurate masses. On the same basis, distinct species
with masses corresponding to dehydrated triketides could not
be identified. From the lack of direct evidence for dehydrated
triketides and the identification of the putative hydroxy
tetraketides and pentaketides, it appears that, whether the
PKS is of fungal or of bacterial origin, route b) of Scheme 1 is
followed.
Nonetheless, to seek additional confirmation of these
insights and further dissect 6-MSAS catalytic activities, we
also utilized recombinant P. patulum 6-MSAS from heterol-
ogous E. coli BAP1 host strain,[23] as well as an additional
mutant form of the enzyme (6-MSAS H958A) bearing an
alanine in place of a histidine in the THID active site for in
vitro assays.[22] The capture of biosynthetic intermediates in
vitro proved much more challenging than in vivo. Using
probes 9a–b (generated from pig liver esterase- assisted
hydrolysis of 4a–b),[19b] only intermediates from two rounds of
chain extension were consistently identified in the ethyl
acetate extracts of 6-MSAS assays (Supporting Information,
Figure 43S). When recombinant 6-MSAS was primed with
acetoacetyl-CoA instead of acetyl-CoA in the attempt to
improve advanced intermediate capture, the accumulation of
a possibly dehydrated triketide was observed (Supporting
Information, Figure 46S).
Figure 2. Analytical HPLC analyses showing that: A) the synthetic
triketide mimic 13 is not dehydrated by 6-MSAS nor by its mutant 6-
MSAS H958A and B) the thioester substrates 14 and 15 are not
hydrolyzed by 6-MSAS nor by its mutant 6-MSAS H958A.
long-term storage. Surprisingly, when 6-MSAS and the
H958A mutant were incubated with the N-acetylcysteamine
thioester of 6-MSA 14, previously utilized to probe the THID
function in ATX,[12] no free 6-MSA was generated (Fig-
ure 2B; Supporting Information, Figure 48S). An N-decanoyl
thioester analogue 15 was synthesized as an additional
substrate, with the idea of utilizing a long acyl chain to
mimic the phosphopantetheine cofactor of ACP. However, 15
was also resistant to hydrolysis by either enzyme (Supporting
Information, Figure 49S). This unexpected outcome suggests
that 6-MSAS differs from ATX in that covalent attachment of
tetraketide intermediates to the ACP or coenzyme A might
be necessary for their processing. Alternatively, this may
indicate that readily aromatized thioesters are not true
substrates for THID domains. The results reported herein
strongly point towards no enzymatic dehydration taking place
at a triketide stage of 6-MSA assembly, so loss of water must
occur at the tetraketide stage. If this is enzyme-catalyzed, the
configuration of the resulting alkene (from an R-alcohol, as
recently established for the 6-MSA-like mellein synthase)[24]
would likely be trans. On the basis of multiple peaks observed
for dehydrated tetraketides (for example, Supporting Infor-
mation, Figures 24S), it is tempting to speculate that, along
with final thioester hydrolysis, the THID might act as
a template domain to aid trans to cis double bond isomer-
ization, and/or cyclization and aromatization; however, this
remains undetermined. Amongst iPKSs with significant
homology to 6-MSAS and leading to 6-MSA related products,
the THID domain is highly conserved (Supporting Informa-
tion, Figure 50S). Its presence in non-reducing iPKSs such as
the orsellinic acid synthase supports its role in product
cyclization and release.[14a] Intriguingly, a THID is not present
in MicC, an iPKS responsible for the formation of 6-
pentasalicylic acid in micacocidin biosynthesis, whose assem-
bly allegedly proceeds similarly to that of 6-MSA.[7b, 25]
Although bioinformatic analysis usefully pinpoints the sim-
To investigate whether this species could have been
enzymatically formed, a racemic 3-hydroxytriketide substrate
Angew. Chem. Int. Ed. 2016, 55, 3463 –3467
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3465