Two Prenyltransferases Govern a Consecutive Prenylation Cascade in the Biosynthesis of Echinulin and Neoechinulin

Article abstract of DOI:10.1021/acs.orglett.7b02926

Two prenyltransferases from Aspergillus ruber control the echinulin biosynthesis via exceptional sequential prenylations. EchPT1 catalyzes the first prenylation step, leading to preechinulin. The unique EchPT2 attaches, in a consecutive prenylation cascade, up to three dimethylallyl moieties to preechinulin and its dehydro forms neoechinulins A and B, resulting in the formation of at least 23 2- to 4-fold prenylated derivatives. Confirming these products in fungal extracts unravels the unprecedented catalytic relevance of EchPT2 for structural diversity.

Full text of DOI:10.1021/acs.orglett.7b02926

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX-XXX
Two Prenyltransferases Govern a Consecutive Prenylation Cascade in
the Biosynthesis of Echinulin and Neoechinulin
Viola Wohlgemuth,† Florian Kindinger,† Xiulan Xie,‡ Bin-Gui Wang,§ and Shu-Ming Li*,†
†Institut fur Pharmazeutische Biologie und Biotechnologie, Philipps-Universitat Marburg, Robert-Koch-Straße 4, 35037 Marburg,
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Germany
‡Fachbereich Chemie, Philipps-Universitat Marburg, Hans Meerwein-Straße, 35032 Marburg, Germany
§Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the CAS, 266071 Qingdao, China
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S
* Supporting Information
ABSTRACT: Two prenyltransferases from Aspergillus ruber control the
echinulin biosynthesis via exceptional sequential prenylations. EchPT1
catalyzes the first prenylation step, leading to preechinulin. The unique
EchPT2 attaches, in a consecutive prenylation cascade, up to three
dimethylallyl moieties to preechinulin and its dehydro forms neo-
echinulins A and B, resulting in the formation of at least 23 2- to 4-fold
prenylated derivatives. Confirming these products in fungal extracts
unravels the unprecedented catalytic relevance of EchPT2 for structural
diversity.
ndole diketopiperazine (DKP) alkaloids have been established
as a steadily growing and reliable source for compounds of
number and position of additional DMA moieties. C-5 of the
I
indole ring seems to be the second preferred prenylation position
in echinulins and neoechinulins. A third DMA moiety is mainly
found at C-7 or at C-4.11,12 Various intriguing pharmacological
activities have been identified for echinulin and congeners,5 such
as protection against neuronal cell death13 and antiviral14 and
antitumor activities.15 In spite of over 40 years of studies on
structures, as well as biological and pharmacological activities of
this intriguing substance group, the enzymes involved in the
biosynthesis, especially those for the transfer of the different
prenyl moieties, have not been reported prior to this study.2,5
In nature, prenyl transfer reactions are catalyzed by
prenyltransferases (PTs). These enzymes employ isoprenic
precursors of various number of C5-units such as DMAPP
(C5).8 Known indole PTs from fungi belong to the dimethylallyl-
tryptophan synthase (DMATS) superfamily and usually catalyze
regio- and stereoselective regular or reverse prenylations. In most
cases, one PT only catalyzes one specific transfer reaction.8 For
example, fumitremorgin A from Neosartorya fischeri contains
three prenyl moieties, which are transferred from DMAPP by
three different PTs.16 Contrary examples of one PT involved in
more than one prenylation step are rare. Two membrane-bound
PTs, from Humulus lupulus, have been reported to be responsible
for three sequential prenylation steps in the biosynthesis of β-
bitter acid,17 and in the biosynthesis of the fungal metabolite
shearinine D, JanD from the DMATS superfamily catalyzes a
tandem diprenylation.18
significant biological activity.1,2 Their indole nucleus backbone,
as a privileged structure, has become a focus of fragment-based
drug discovery.1,3 This core ring system is commonly assembled
via one L-tryptophan and a second amino acid, usually catalyzed
by a nonribosomal peptide synthetase (NRPS),4 providing an
initial biosynthetic starting point in the form of an indole DKP
skeleton.5 Subsequent modification reactions including prenyla-
tions increase not only structural complexity but also biological
and pharmacological activities.4−8
Echinulins and neoechinulins derived from L-tryptophan and
L-alanine exemplify such DKPs and are highly decorated with
dimethylallyl (DMA) moieties.1,2,5 Their eponymous group
member echinulin (Figure 1) was first isolated from Aspergillus
Figure 1. Representatives of echinulin and neoechinulins.
amstelodami5 and later, together with congeners, from different
terrestrial and marine-derived fungi,5 e.g. Aspergillus cristatus9 and
Aspergillus glaucus.10 These prenylated cyclo-L-Trp-L-Ala deriva-
tives can be classified into the echinulin, neoechinulin A, and
neoechinulin B series (Figures 1 and S1 in Supporting
Information (SI)).11 They share a reverse C2-prenyl moiety
and differ from each other in exo double bounds at the DKP ring.
In turn, the members of each series differ from each other in the
A large number of echinulin congeners with different numbers
of prenyl moieties have been identified in and isolated from
Received: September 19, 2017
© XXXX American Chemical Society
A
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Figure 2. Putative echinulin gene clusters in Aspergillus strains. Genes with high sequence identities at the amino acid level are indicated by dotted lines.
Details of alignment and the putative gene functions can be found in Table S1.
Aspergillus ruber.12,14 In analogy to most biosynthetic pathways, it
could be speculated that three or even four PTs are necessary for
the attachment of the prenyl moieties in echinulin and its
congeners. Unexpectedly, mining the A. ruber CBS 135680
genome19 reveals just three DMATS PT genes. Two of them,
coding for EYE98742 (termed EchPT1 in this study) and
EYE98746 (EchPT2), build a cluster with an NRPS (EYE98744)
gene. The third one (EYE95342) is located in a separate cluster
with a polyketide synthase gene. Homologues of the putative
NRPS-containing cluster harboring only two PT genes are also
identified in the genomes of Aspergillus cristatus YKY807 and
Aspergillus glaucus CBS 516.65 (Figure 2, Table S1). This unusual
genetic organization prompted us to investigate the functions of
the three PTs from A. ruber.
Sequence analysis revealed the incorrect annotation of the
putative NRPS EYE98744 in the database by lacking 789 amino
acids at the N-terminus, which was corrected in this study (SI).
Figure 3. LC-MS analysis of the incubation mixtures of 1 with EchPT1,
EchPT2, and EYE95342 alone or in combinations.
The revised sequence of EYE98744 comprises 2113 amino acid
three PTs (Figure 3iv−vii). Assaying 1 with EchPT1 and EchPT2
residues (Table S1) and shares a sequence identity of 23.5% with
resulted in the formation of 1M1 and at least four additional
the known cyclo-L-Trp-L-Pro synthetase FtmPS.20 It can be
speculated that this enzyme, tentatively named EchPS, might be
products (iv). The same peaks were also detected in the assay
with all three enzymes (vii). In contrast, the combination of
responsible for the assembly of cyclo-L-Trp-L-Ala. The two
EchPT1 and EYE95342 (v) yields just the EchPT1 product 1M1,
putative echinulin prenyltransferases EchPT1 and EchPT2 are
while EchPT2 and EYE95342 catalyze no conversion of 1 at all
(vi). These results prove that EYE95342 is likely not involved in
polypeptides of 417 and 408 amino acids, respectively. They
share a clear sequence similarity with the members of the
the biosynthesis of echinulins and EchPT2 catalyzes the further
DMATS superfamily (Figure S2).8 It seems unbelievable that
metabolism of 1M1.
To gain detailed insights into the reaction mechanism of 1 with
EchPT1 and EchPT2 (Figure 3iv), we assayed 1M1 with EchPT2
in the presence of DMAPP. LC-MS analysis reveals six product
these two PTs should catalyze three or four prenylations deduced
from the number and positions of DMA moieties in echinulins
and congeners.
To investigate the possible involvement of the three PTs in the
peaks on three consecutive prenylation levels (Figures 4a and
biosynthesis of echinulin, the coding sequences of EchPT1,
S6a), i.e. two di-, tri-, and tetraprenylated products each, which
was confirmed by detection of their exact [M + H]+ ions (Table
EchPT2, and EYE95342 were PCR amplified from cDNA of the
endophytic fungus A. ruber QEN-0407-G212 and cloned into
pQE9 and pQE70 for heterologous expression in E. coli (SI,
Figures S3−S5). All three purified proteins were incubated
separately with the putative product of EchPS, cyclo-L-Trp-L-Ala
(1), in the presence of DMAPP. LC-MS analysis revealed product
formation in the reaction mixture with EchPT1, but with neither
EchPT2 nor EYE95342 (Figure 3i−iii). The [M + H]+ ion of the
single EchPT1 product 1M1 indicates a monoprenylation of 1.
For a better understanding, we use M, D, T, and Q after the
substrate number for mono-, di-, tri-, and tetraprenylation,
respectively. The number after these letters refers to the order of
the identified products. Formation of 1M1 is strictly dependent
on the presence of 1, DMAPP, and active EchPT1. NMR and MS
analyses of the isolated product confirmed 1M1 to be
preechinulin. This proved unequivocally that EchPT1 catalyzes
the first prenylation in the biosynthesis of echinulin in A. ruber
(for structure elucidation and kinetic parameters see SI, Tables S2
and S3, Figures S9−11, S16, and S18).
S3). The [M + H]+ ions of the first two products in ascending
order of retention times (Figure S6a) are 68 Da larger than that of
1M1, proving the presence of an additional prenyl moiety in their
structures. These putative diprenylated products are termed 1D1
and 1D2. The [M + H]+ ions of the following two triprenylated
products, 1T1 and 1T2, indicate the attachment of two prenyl
residues to 1M1, and the [M + H]+ ions of the last two products
1Q1 and 1Q2 indicate even three additional prenyl units attached
to the already monoprenylated substrate 1M1. Notably, while a
large number of echinulin-related structures were described in
the literature,5,12 no derivative with four prenyl moieties has been
reported to date. From Figure 4a, it is obvious that 1T2 is the
main product of the EchPT2 reaction. To the best of our
knowledge, six products across three consecutive prenylation
levels from an incubation mixture with only one enzyme have not
been reported prior to this study. These fascinating results
prompted us to investigate the relationships between these
products and their dependence on reaction time, DMAPP and
protein concentrations (Table S2, Figures S12−15 and S17).
HPLC analysis of the reaction mixtures revealed 1T2 as the
To verify the roles of the two other PTs in the biosynthesis of
echinulins, we carried out incubations containing either two or all
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B with an additional regular prenyl moiety at C-5 and C-7,
respectively, compared to that of 1M1. The two triprenylated
products 1T1 and 1T2 bear prenyl moieties at C-2 and C-5 and
differ from each other in the position of the third prenyl residue,
which is located at C-4 in 1T1 and C-7 in the case of 1T2. 1T1
and 1T2 can therefore be unequivocally identified as variecolorin
L and echinulin. Due to the low amount, the structure of 1Q1
cannot be determined in this study. 1Q2 was isolated from fungal
extracts (Figure S8) and identified as a reversely C2-, regularly
C4-, C5-, and C7-tetraprenylated derivative. Judging by
incubation results described below, 1Q1 must be a reversely
C2-, regularly C4-, C5-, and C6-tetraprenylated derivative
(Scheme 1; Tables S3−S7; Figures S19−29).
The single EchPT2 products were then incubated independ-
ently with EchPT2 (Figures 4a and S6a). LC-MS analysis showed
further conversion of 1D1 by EchPT2 to 1T1, 1T2, 1Q1, and
1Q2 (ii). The last two products were also detected in the assay of
1T1 with EchPT2 (iv). Conversely, 1D2, 1T2, 1Q1, and 1Q2
underwent no further conversion by EchPT2 (iii, v, and vi).
These confirmed the hypothesis that 1D2 and 1T2 are end
products of different branches of the EchPT2 reaction and C7-
prenylation serves as a termination step in a consecutive
prenylation cascade as shown in Scheme 1. To reach the
tetraprenylated products, the putative NRPS product 1 will be
reversely prenylated at C-2 by EchPT1. The resulting product
1M1 undergoes regular prenylation at C-5, then at C-4, and
finally at C-6 or C-7. No pentaprenylated derivative was detected
in the reaction mixtures of 1M1, 1D1, 1T1, 1Q1, or 1Q2 with
EchPT2 (Figure S6a), indicating that the tetraprenylated
derivatives 1Q1 and 1Q2 are the final products of the cascade.
1M1, 1D1, 1D2, 1T1, 1T2, and a mixture of 1Q1 and 1Q2 were
also incubated with EYE95342. LC-MS analysis revealed no
conversion of these substrates (Figure S7).
Figure 4. LC-MS analysis of EchPT2 assays with 1M1, 1D1, 1D2, 1T1,
1T2, and 1Q1/1Q2 (a), neoechinulins A (2M1) and B (3M1) (b).
predominant product in all enzyme assays. Most interestingly,
there are two divergent fate patterns for the enzyme products. In
all three dependency assays, the formation of 1D1 and 1T1
showed an initially rapid, yet short increase, followed by a
continuous decrease. In contrast, the yields of 1D2, 1T2, 1Q1,
and 1Q2 increased steadily in all assays (Figure S13). This
indicates that 1D1 and 1T1 could serve as intermediates in a
consecutive prenylation cascade, whereas 1D2 and 1T2 as well as
1Q2 and 1Q2 may represent end products of the (branch)
pathways.
Having explored the outstanding catalytic skill of EchPT2, we
wondered if the products of such a prenylation cascade, especially
the previously undescribed tetraprenylated derivatives 1Q1 and
1Q2, coexist in the fungal cultures. Based on previous results
To confirm our hypothesis, we isolated EchPT2 products for
structure elucidation. Interpretation of the spectra and literature
search confirmed 1D1 and 1D2 to be tardioxopiperazines A and
Scheme 1. Proposed Biosynthetic Pathway of Echinulin and Neoechinulin Series via Consecutive Multiprenylations by EchPT1
and EchPT2 in A. ruber
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reporting a strong influence of salt concentration on metabo-
lism,19 we cultivated A. ruber QEN-0407-G2 under different
conditions (SI). The fungal cultures were extracted and analyzed
by LC-MS (Figure S1). The enzyme products described above,
i.e. 1M1 of the EchPT1 reaction, as well as 1D1, 1D2, 1T1, 1T2,
and 1Q2 of the EchPT2 reaction, were detected by LC-MS
analysis, corresponding to the enzyme reactions with EchPT1
and EchPT2 (Figures S1, S6a, and S8).
Detailed experimental procedures including structural
elucidation, kinetic parameters, MS and NMR data, and
NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: shuming.li@staff.uni-marburg.de.
ORCID
As previously mentioned, neoechinulins with one (A series) or
two exo double bonds (B series) at the DKP ring are also
frequently identified in echinulin producers.14 LC-MS analysis of
the obtained fungal extract indeed revealed the presence of
members of both series with up to three prenylation levels
(Figure S1). In total, one mono- (neoechinulin A, 2M1), two di-,
and two triprenylated derivatives from the A series were detected.
In the case of the B series, one mono- (neoechinulin B, 3M1), two
di-, and four triprenylated derivatives were observed. In contrast
to the triprenylated 1T2 as the main metabolite of the echinulin
series (Figure S1), the monoprenylated derivatives 2M1 and
3M1 are found to be the major products of the neoechinulin
series A and B. Furthermore, cyclo-L-Trp-L-Ala (1), but not its di-
or tetradehydrogenated derivatives, i.e. the unprenylated
precursors of 2M1 and 3M1, was detected in the fungal cultures.
This could indicate that the first prenylation catalyzed by EchPT1
takes place before dehydrogenation, probably catalyzed by the
cytochrome P450 enzyme EchP450 (Scheme 1). We speculated
that both series of neoechinulins are also EchPT2 products of
sequential prenylations and, thus, isolated the monoprenylated
2M1 and 3M1 from the extracts (SI for structure elucidation,
Tables S3 and S8, Figures S30 and S31). LC-MS analysis of the
reaction mixture of 2M1 and 3M1 with EchPT2 indeed
demonstrated the clear acceptance of both substrates (Figures
4b, S6b) and the formation of eight and nine products with two to
four prenyl residues, respectively. In comparison to the fungal
extract, tetraprenylated products 2Q1 and 3Q1 were also clearly
detected in the EchPT2 assays with 2M1 and 3M1. This proves
that EchPT2 also catalyzes a prenylation cascade with 1M1
analogs bearing exo double bonds and that the biosynthetic
pathway illustrated in Scheme 1 can be expanded by neo-
echinulins. That is, conversion of 1M1 to neoechinulins A (2M1)
and B (3M1) by a putative cytochrome P450 enzyme
(EchP450)21 marks the starting point of the neoechinulin
formation. In analogy to 1M1, 2M1 and 3M1 undergo a
prenylation cascade catalyzed by EchPT2, resulting in the
formation of products with different prenylation grades.
Bin-Gui Wang: 0000-0003-0116-6195
Shu-Ming Li: 0000-0003-4583-2655
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
S.-M.L. acknowledges the Deutsche Forschungsgemeinschaft for
funding of the Bruker micrOTOF QIII mass spectrometer (INST
160/620-1). We thank S. Newel and R. Kraut (University
Marburg) for acquiring NMR and MS spectra and D. Jochheim
(University Marburg) for reading the manuscript.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b02926.
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DOI: 10.1021/acs.orglett.7b02926
Org. Lett. XXXX, XXX, XXX−XXX