AstPT catalyses both reverse N1- and regular C2 prenylation of a methylated bisindolyl benzoquinone

Article abstract of DOI:10.1002/cbic.201300610

Prenylated bisindolyl benzoquinones exhibit interesting biological activities, such as antidiabetic or anti-HIV activities. A number of these compounds, including asterriquinones, have been isolated from Aspergillus terreus. In this study, we identified two putative genes by genome mining, ATEG-09980 and ATEG-00702, which share high sequence similarity with the known bisindolyl benzoquinone prenyltransferase TdiB from Aspergillus nidulans. The coding sequences were cloned and overexpressed in E. coli. The overproduced recombinant proteins were purified to near homogeneity and used for enzyme assays with asterriquinone D in the presence of dimethylallyl diphosphate. HPLC analysis showed that product formation was only detected in enzyme assays with EAU29429 encoded by ATEG-09980, not in those with EAU39348 encoded by ATEG-00702. Product isolation and structure elucidation by NMR and MS analyses led to identification of N1-reversely and C2-regularly monoprenylated derivatives, as well as N1′,N1′′reversely, N1′-reversely, C2′′-regularly diprenylated derivatives. This proved that EAU29429 functions as an asterriquinone prenyltransferase (AstPT) and indicated the involvement of EAU29429 rather than EAU39348 in the biosynthesis of methylated asterriquinones. Furthermore, incubation of monoprenylated enzyme products with AstPT resulted in the formation of the diprenylated derivatives. Copyright

Full text of DOI:10.1002/cbic.201300610

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DOI: 10.1002/cbic.201300610
AstPT Catalyses both Reverse N1- and Regular C2
Prenylation of a Methylated Bisindolyl Benzoquinone
Sylwia Tarcz, Lena Ludwig, and Shu-Ming Li*[a]
Prenylated bisindolyl benzoquinones exhibit interesting biolog-
ical activities, such as antidiabetic or anti-HIV activities. A
number of these compounds, including asterriquinones, have
been isolated from Aspergillus terreus. In this study, we identi-
fied two putative genes by genome mining, ATEG_09980 and
ATEG_00702, which share high sequence similarity with the
known bisindolyl benzoquinone prenyltransferase TdiB from
Aspergillus nidulans. The coding sequences were cloned and
overexpressed in E. coli. The overproduced recombinant pro-
teins were purified to near homogeneity and used for enzyme
assays with asterriquinone D in the presence of dimethylallyl
diphosphate. HPLC analysis showed that product formation
was only detected in enzyme assays with EAU29429 encoded
by ATEG_09980, not in those with EAU39348 encoded by
ATEG_00702. Product isolation and structure elucidation by
NMR and MS analyses led to identification of N1-reversely and
C2-regularly monoprenylated derivatives, as well as N1’,N1’’-
reversely, N1’-reversely, C2’’-regularly diprenylated derivatives.
This proved that EAU29429 functions as an asterriquinone
prenyltransferase (AstPT) and indicated the involvement of
EAU29429 rather than EAU39348 in the biosynthesis of methy-
lated asterriquinones. Furthermore, incubation of monopreny-
lated enzyme products with AstPT resulted in the formation of
the diprenylated derivatives.
Introduction
Bisindolyl benzoquinones are fungal secondary metabolites de-
rived from two molecules of l-tryptophan and are therefore
considered prenylated indole alkaloids.[1] They are formed
through dimerisation of indolyl pyruvate catalysed by nonribo-
somal peptide synthetase (NRPS)-like proteins.[2,3] Most of
these compounds carry prenyl moieties derived from dimethyl-
allyl diphosphate (DMAPP).[1] Derivatisation of bisindolyl benzo-
quinones by prenyl residues at different positions leads to
increased structural diversity as well as biological activities. A
number of prenylated bisindolyl benzoquinones have been iso-
lated from fungi[4–7] and can be divided in two main groups
depending on the substitution of the benzoquinone ring. One
group carries hydroxy moieties, for example, terrequinone A
from Aspergillus nidulans[8] and Aspergillus terreus,[9] asterriqui-
none[10] and asterriquinone CT5 from A. terreus,[5] and cochliodi-
nol from Chaetomium sp.[11] The second group bears methoxy
groups instead, for example, asterriquinones A1, A3, B3, B4, C1
and F from A. terreus (Scheme 1).[12,13] The two unprenylated
substances, didemethyl asterriquinone D (DDAQ D)[14] and as-
terriquinone D (AQ D; Scheme 1),[12] are also worth mentioning.
Many bisindolyl benzoquinones exhibit interesting biological
activities such as cytotoxic,[15–17] antifungal, or antibacterial
activities.[18–20] It has been also shown that these compounds
can act as insulin receptor agonists[21–23] or HIV protease inhibi-
tors.[6]
In contrast to the large number of known structures of pre-
nylated bisindolyl benzoquinones, biochemical investigations
were carried out only for the biosynthesis of terrequinone A in
A. nidulans.[2,3,24] A putative biosynthetic gene cluster and path-
way were proposed for asterriquinones (see the Discussion).[25]
In ascomycetous fungi, the prenyl moieties of the prenylated
indoles are transferred from DMAPP by prenyltransferases of
the dimethylallyltryptophan synthase (DMATS) superfamily.[26]
Enzyme TdiB from this group is involved in the biosynthesis of
terrequinone A and catalyses a reverse C2 prenylation at the
indole ring and a regular prenylation at the benzoquinone
ring.[2] Blasting the genome sequence of A. terreus NIH2624
with TdiB revealed the presence of ten putative genes, which
share clear sequence similarity with known indole prenyltrans-
ferases of the DMATS superfamily.[27] Two of these, ATEG_09980
and ATEG_00702, showed significantly higher sequence similar-
ity with TdiB than with other enzymes. Their encoded enzymes
could be involved in the biosynthesis of asterriquinones. In
this study, we report the cloning and overexpression of ATEG_
09980 and ATEG_00702, as well as their role in the prenylation
of AQ D (1, Scheme 1) in the presence of DMAPP.
Results and Discussion
[a] S. Tarcz, L. Ludwig, Prof. Dr. S.-M. Li
Philipps-Universitꢀt Marburg
Sequence analysis and gene cloning
Institut fꢁr Pharmazeutische Biologie und Biotechnologie
Deutschhausstrasse 17A, 35037 Marburg (Germany)
E-mail: shuming.li@staff.uni-marburg.de
Sequence analyses showed that ATEG_09980 and ATEG_00702
in A. terreus NIH2624 have very similar exon-intron structures
to tdiB from A. nidulans[24] and consist of three exons, interrupt-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300610.
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A1156 (equivalent to A. terreus NIH2624) as the template for
PCR amplification in a similar way. The PCR product was
cloned into the expression vector pHIS8 to obtain the expres-
sion construct pST37.
Overexpression and purification
For overproduction of the recombinant protein EAU29429
(AstPT-His6), E. coli XL1 Blue MRF’ cells harbouring pST24 were
cultivated at 308C without induction by isopropyl-b-d-thioga-
lactopyranoside (IPTG), as addition of IPTG led only to high
protein amounts of insoluble inclusion bodies (data not
shown). One-step purification on Ni-NTA agarose resin resulted
in a major protein band that was slightly larger than the
45 kDa size marker on SDS-PAGE (Figure S1A). This also coin-
cided with the calculated molecular mass of AstPT-His6 of
48.9 kDa. A yield of 3 mg of purified protein was calculated for
1 L of bacterial culture. The molecular mass of the native re-
combinant protein was determined by size-exclusion chroma-
tography as 48.9 kDa, thus indicating that AstPT-His6 presuma-
bly acts as a monomer.
EAU39348 was overproduced by cultivating E. coli BL21
(DE3) pLysS cells harbouring pST37 and induction with 0.1 mm
IPTG at 228C for 16 h. After two-step purification on Ni-NTA
agarose and HisPur cobalt resin, a protein band above the
45 kDa size marker on SDS-PAGE was obtained (Figure S1B). A
yield of 0.1 mg of purified protein was obtained from 1 L of
bacterial culture.
Enzyme activity and substrate specificity
AstPT was assayed with AQ D (1), which was synthesised from
indole and 2,5-dichlorobenzoquinone in three steps, in the
presence of DMAPP. After incubation at 378C for 1 h with
20 mg of recombinant enzyme in 100 mL assays, ethyl acetate
extracts of the reaction mixtures were analysed by HPLC. Four
additional peaks (2–5) were clearly observed in the HPLC chro-
matogram (Figure 1A). No product formation was detected in
the incubation mixtures with heat-inactivated protein or in the
absence of the prenyl donor DMAPP (data not shown). The
ratios of the four product peaks were obviously time-depen-
dent (Figure 2). Peaks 2 and 3 were detected as main enzyme
products in the initial phase and increased rapidly until they
reached their maxima at 25 min. They subsequently decreased.
In contrast, products 4 and 5 were detected somewhat later
and increased slowly but continuously. After 2 h incubation, 3,
4 and 5 reached comparable amounts. These results indicated
that 2 and 3 could be intermediates for 4 and 5. Considering
the fact that mono-, di- and even triprenylated bisindolyl ben-
zoquinones have been isolated from A. terreus,[12,25] it can be
assumed that 2 and 3 were consumed by AstPT, leading to the
formation of 4 and 5.
Scheme 1. Examples of naturally occurring bisindolyl benzoquinones.
ed by two introns (Table S1). The resulting proteins EAU29429
and EAU39348 have polypeptide chains of the same length
(424 amino acids) and share an amino acid sequence identity
of 45 and 48%, respectively, with TdiB from A. nidulans.[24] The
sequence identity between EAU29429 and EAU39348 was
found to be 71% on the amino acid level.
Based on the fact that the genes of interest and tdiB consist
of the same number of introns and exons in similar size, the
coding region of ATEG_09980 was directly amplified from ge-
nomic DNA of A. terreus DSM1958 by two-round PCR (see the
Experimental Section) and used for gene cloning and protein
overproduction. The resulting PCR product without intron se-
quences was cloned, by using the pGEM-T Easy vector, into
the expression vector pQE70, resulting in the expression con-
struct pST24. After unsuccessful attempts to amplify the se-
quence of ATEG_00702 from the same strain, we used genom-
ic DNA from the genome reference strain A. terreus FGSC
To prove this hypothesis, we isolated and incubated 2 and 3
with AstPT in two parallel assays. As shown in Figure 1B, 2 was
converted to 4 and an unknown product with a slightly larger
retention time than 3 after incubation at 378C for 16 h. In the
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presence of geranyl diphosphate (GPP) and farnesyl diphos-
phate (FPP), proving its high specificity for DMAPP in the pres-
ence of 1. Incubation of 1 with purified EAU39348 in the pres-
ence of DMAPP, GPP, or FPP did not result in the formation of
any enzyme product.
Structural elucidation of enzyme products
The four products, 2–5, of the incubation mixtures of AstPT
with 1 were isolated by preparative HPLC from combined
assays of two and 16 h incubation mixtures and were subject-
ed to spectroscopic analyses. Molecular masses in positive HR-
EI-MS data confirmed the monoprenylation of 2 and 3 (68 Da
larger than that of 1) and diprenylation of 4 and 5 (136 Da
1
larger than that of 1; Table S2). H NMR data clearly showed
the presence of one regular prenyl moiety in each of the struc-
tures of 2 and 4 with signals at 5.39 (t sept, J=7.3, 1.4 Hz; H-
9’), 3.46 or 3.47 (m; H-8’) and 1.70 ppm (s or d, J=1.2 Hz; H-
11’+H-12’; see the Supporting Information for spectra). The
chemical shifts of H-8’ at about 3.5 ppm indicated the attach-
ment of the prenyl moiety to a carbon atom. Signals of reverse
prenyl moieties at 6.26 (dd, J=17.5 or 17.4, 10.7 Hz; H-9’), 5.28
(dd, J=10.7, 0.8 or 0.7 Hz; H-10’), 5.25 (dd, J=17.5 or 17.4, 0.8
or 0.7 Hz; H-10’) and 1.86 ppm (s; H-11’+H-12’) were observed
in the spectra of 3, 4 and 5 (Table 1). The chemical shifts of the
two methyl groups indicated the attachment of the prenyl
1
moiety to a hetero atom. H NMR spectra of 2–5 showed the
presence of signals for the four coupling aromatic protons of
the indole ring (H-4’–H7’ as well as H4’’–H7’’) in the range of
7.00–7.60 ppm, indicating that prenylation did not take place
at these positions. Significant differences were found between
signals of NH-1’ and H-2’ and NH-1’’ and H-2’’. The 1H NMR
spectrum of 2 showed two signals for NH-1’’ and NH-1’’ at
10.87 (brs) and 10.32 ppm (brs), respectively, but only a signal
for H-2’’ at 7.68 ppm (d, J=2.6 Hz). The appearance as a dou-
blet with a coupling constant of 2.6 Hz proved that H-2’’ still
had a coupling partner at position NH-1’’. Therefore, prenyla-
tion of 2 must occur at position C-2’. For 3, only one NH signal
was observed at 10.80 ppm (brs), together with two signals for
H-2’ (7.74 ppm, s) and H-2’’ (7.69 ppm, d, J=2.7 Hz). The ap-
pearance of H-2’ as a singlet and H-2’’ as a doublet provided
a strong evidence for the attachment of the prenyl moiety to
position N-1’. This compound has already been reported and
Figure 1. HPLC analysis of enzyme assays of AstPT with A) 1, B) 2 and C) 3.
Assays with 1 were incubated for 1 h; assays with 2 and 3 were incubated
for 16 h at 378C. Detection was carried out with a diode array detector with
absorption at 277 nm. The asterisk in B) marks an unknown product that
was not identified in this study due to low product quality.
1
named AQB3 by Arai and co-workers.[12] The H NMR spectrum
of 4 showed signals for regular and reverse prenylation. Only
one NH signal (10.33 ppm, brs) and one H-2’’ signal (7.73 ppm,
s) were observed, indicating that regular prenylation had oc-
curred at position C-2’ and reverse prenylation at position N-
1’’. This structure has already been synthesised in the context
of cytotoxicity investigations.[28] Although a different solvent
was used for NMR spectroscopy, good conformity was found
between 4 and substance “6b” in the published data. Because
of the fact that half of the number of signals was found in the
1H NMR spectrum of 5 only, we concluded that this molecule
must be symmetric. No NH signals were observed, and H-2’/H-
2’’ appeared at 7.74 ppm as a singlet. This proved that both ni-
trogen atoms were reversely prenylated in 5. A reference spec-
Figure 2. Dependence of product formation of AstPT reactions on incuba-
tion time. Two replicates with error bars are illustrated.
assay for 3, compounds 4 and 5 were clearly detected (Fig-
ure 1C).
Some of the known prenyltransferases of the DMATS super-
family were found to accept a broad spectrum of substrates,
such as aromatic amino acids and cyclic dipeptides thereof, as
well as flavonoids and hydroxynaphthalenes.[26] Therefore, we
assayed AstPT with such aromatic substances in the presence
of DMAPP. No product formation was found after incubation of
AstPT with the aforementioned substances. Similarly, no prod-
uct was detected in incubation mixtures of AstPT with 1 in the
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Table 1. 1H NMR data of 1 and its enzyme products in (CD3)2CO (500 MHz).
Compound
AQ D (1)
2
3
4
5
proton
dH, mult., J
dH, mult., J
dH, mult., J
dH, mult., J
dH, mult., J
7
8
1’
3.81, s
3.81, s
10.82, brs
7.69, d, 2.7
3.79, s
3.72, s
10.32, brs
–
3.83, s
3.81, s
–
3.80, s
3.72, s
10.33, brs
–
3.82, s
3.82, s
–
2’
7.74, s
7.74, s
4’
5’
6’
7’
8’
9’
10’
7.49, dt, 8.1, 1.1
7.08, ddd, 8.1, 7.0, 1.1
7.16, ddd, 8.1, 7.0, 1.1
7.53, ddt, 8.1, 1.1, 0.7
–
–
–
7.31, ddd, 8.0, 1.2, 0.7
7.00, ddd, 8.0, 7.1, 1.1
7.09, ddd, 8.1, 7.1, 1.2
7.49, ddd, 8.1, 1.1, 0.7
3.47, m
7.51, ddd, 8.2, 1.4, 0.7
7.09, ddd, 8.2, 6.9, 1.2
7.12, ddd, 8.3, 6.9, 1.4
7.60, ddd, 8.3, 1.2, 0.7
–
6.26, dd, 17.5, 10.7
5.28, dd, 10.7, 0.8
5.25, dd, 17.5, 0.8
1.86, s
7.30, ddd, 8.0, 1.2, 0.7
7.00, ddd, 8.0, 6.9, 1.2
7.08, ddd, 7.9, 6.9, 1.2
7.49, ddd, 7.9, 1.2, 0.7
3.46, m
7.50, ddd, 7.9, 1.4, 0.7
7.08, ddd, 7.9, 7.0, 1.5
7.12, ddd, 8.3, 7.0, 1.4
7.60, ddd, 8.3, 1.5, 0.7
–
6.26, dd, 17.4, 10.7
5.28, dd, 10.7, 0.7
5.25, dd, 17.4, 0.7
1.86, s
5.39, t sept, 7.3, 1.4
–
5.39, t sept, 7.3, 1.4
–
11’
12’
1’’
–
–
1.70, s
1.70, s
10.87, brs
1.70, d, 1.2
1.70, d, 1.2
–
1.86, s
10.80, brs
1.86, s
–
10.82, brs
2’’
7.69, d, 2.7
7.68, d, 2.6
7.69, d, 2.7
7.73, s
7.74, s
4’’
5’’
6’’
7’’
8’’
9’’
10’’
7.49, dt, 8.1, 1.1
7.08, ddd, 8.1, 7.0, 1.1
7.16, ddd, 8.1, 7.0, 1.1
7.53, ddt, 8.1, 1.1, 0.7
–
–
–
7.35, ddd, 8.1, 1.2, 0.8
7.06, ddd, 8.1, 7.0, 1.3
7.16, ddd, 8.1, 7.0, 1.2
7.52, ddd, 8.1, 1.3, 0.8
–
–
–
7.49, dt, 8.1, 0.9
7.08, ddd, 8.1, 7.0, 1.1
7.16, ddd, 8.2, 7.0, 1.2
7.53, ddt, 8.2, 1.1, 0.9
–
7.35, ddd, 8.1, 1.2, 0.9
7.06, ddd, 8.1, 7.0, 1.1
7.12, ddd, 8.3, 7.0, 1.2
7.60, ddd, 8.3, 1.1, 0.9
–
6.26, dd, 17.5, 10.7
5.28, dd, 10.7, 0.7
5.25, dd, 17.5, 0.7
1.86, s
7.50, ddd, 7.9, 1.4, 0.7
7.08, ddd, 7.9, 7.0, 1.5
7.12, ddd, 8.3, 7.0, 1.4
7.60, ddd, 8.3, 1.5, 0.7
–
6.26, dd, 17.4, 10.7
5.28, dd, 10.7, 0.7
5.25, dd, 17.4, 0.7
1.86, s
–
–
11’’
12’’
–
–
–
–
–
–
1.86, s
1.86, s
Chemical shifts (d) are given in ppm and coupling constants (J) in Hz.
trum is given for asterriquinone A1 and, in spite of a different
solvent, the signals of 5 and those of the dimethyl ether of as-
terriquinone prepared by Yamamoto and co-workers harmo-
nise well.[10,12] Identification of enzyme products proved un-
equivocally that AstPT functions as an asterriquinone prenyl-
transferase and catalyses mono- and diprenylation at nitrogen
and carbon atoms in the presence of DMAPP (Scheme 2).
In order to determine the affinity of AstPT towards its sub-
strate 1, kinetic parameters including the Michaelis–Menten
constant (Km) and turnover number (kcat) were calculated from
Eadie–Hofstee, Hanes–Woolf and Lineweaver–Burk plots. The
reaction catalysed by AstPT apparently followed Michaelis–
Menten kinetics (Figure S9). We calculated a Km value of
463 mm with a kcat of 0.16 sÀ1 for 1. This Km value was almost at
the upper boundary of the measured concentrations. Unfortu-
nately, due to low solubility of 1, it was not possible to mea-
sure reaction rates at higher substrate concentrations. By using
1 at a final concentration of 0.5 mm as an aromatic substrate,
Km and kcat were determined for DMAPP at 33.5 mm and
0.02 sÀ1, respectively (Figure S10).
Biochemical properties and determination of kinetic
parameters of AstPT
Most members of the DMATS superfamily catalyse their reac-
tions in the absence of metal ions as well. Divalent cations
such as Ca2+ and Mg2+ usually enhance their enzyme activi-
ties.[26,29] We assayed the dependence of the AstPT reaction on
metal ions at final concentrations of 5 mm in the presence of
0.5 mm 1 and 2 mm DMAPP. Incubation mixtures without addi-
tives or with EDTA were used as controls. Our results showed
that AstPT also catalyses its reaction in the presence of the
chelating agent EDTA. However, addition of Ca2+ slightly in-
creased the activity of AstPT (Figure S8).
Cultivation of A. terreus and screening for methylated and
unmethylated bisindolyl benzoquinones, as well as their
prenylated derivatives
A. terreus DSM1958 and FGSCA1156 were cultivated as de-
scribed in the Experimental Section. Culture filtrates and myce-
lia of both strains were analysed by LC/MS for the presence of
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Scheme 2. Prenylation of 1 catalysed by AstPT.
methylated and unmethylated bisindolyl benzoquinones as
well as prenylated derivatives thereof. [M+H]+ ions at m/z
467.1966 for monoprenylated and at m/z 535.2588 for dipre-
nylated derivatives of 1 were only detected in the extracts of
mycelia of A. terreus DSM1958 (Figure S11), but not in cultures
of A1156 (Figures S12 and S14). Even trace amounts of nonpre-
nylated 1 were observed in the mycelia of DSM1958 (Fig-
ure S11D). [M+H]+ ions of a diprenylated unmethylated bisin-
dolyl benzoquinone (DDAQ D) at m/z 507.2288 or 507.2281
were clearly detected in the extracts of mycelia of A1156 and
the filtrate of DSM1958 (Figures S12 and S13). An [M+H]+ ion
of monoprenylated DDAQ D at m/z 439.1658 was also detect-
ed in the extracts of the culture filtrate of A. terreus DSM1958
(Figure S13).
lans, TdiB, is also involved in two distinct prenylation steps in
the biosynthesis of terrequinone A.[2,24] On its own, it catalyses
a reverse prenylation at C-2 of the indole ring[24] and, in the
presence of TdiC and TdiE, prenylation at the benzoquinone
ring.[2] It was proposed that bisindolyl dihydroxybenzoquinone
is first reduced by TdiC to tetrahydroxybenzene, and one of
the hydroxy groups is replaced by a regular prenyl moiety in
the presence of TdiB and TdiE. The monoprenylated intermedi-
ate with a prenyl moiety at the benzoquinone ring is convert-
ed by TdiB to terrequinone A. In contrast to TdiB, AstPT intro-
duces two prenyl moieties in either regular or reverse manner
to two distinct sites of AQ D in the presence of DMAPP with-
out additional enzymes. Structural analysis of enzymes of the
DMATS superfamily has revealed that these enzymes share
very similar structures, and only one active site was found in
their PT-barrel folds.[31–33] It could be speculated that AstPT has
a similar structure to other known homologues, and the sub-
strates are placed in AstPT in such positions that the C-1’ of
DMAPP is close to the C-2 of 1 and the C-3’ of DMAPP to N1
of 1. This orientation would make a nucleophilic attack possi-
ble at both sites, N1 or C-2, and results in reverse N1 prenyla-
tion and regular C2 prenylation. A final explanation of the
mechanism of the AstPT reaction will be possible when a crys-
tal structure of this enzyme becomes available.
Discussion
In the last decade, a large number of prenyltransferases, in-
cluding the members of the DMATS superfamily, have been
characterised biochemically.[26,30] Most of the known enzymes
from the DMATS superfamily used tryptophan or tryptophan-
containing cyclic dipeptides as natural or best substrates.[26]
Despite the fact that a number of prenylated bisindolyl benzo-
quinones have been isolated from different fungi, only one
prenyltransferase (TdiB) was characterised biochemically. In this
study, we demonstrated the successful cloning, overexpression,
and purification of the methylated bisindolyl benzoquinone,
prenyltransferase AstPT from A. terreus DSM1958. By isolation
and structure elucidation of the enzyme products, AstPT was
shown to be responsible for the transfer of prenyl moieties
from DMAPP to positions N-1 or C-2 of 1 to form mono- and
diprenylated products 2, 3, 4 and 5. Products 3 and 5 have
been isolated from A. terreus.[10,12]
Incubation of the two monoprenylated products, 2 and 3,
with AstPT in the presence of DMAPP led to the formation of 4
and 5. One uncharacterised compound was detected in the
HPLC chromatogram of the incubation mixture of AstPT with 2
(marked with an asterisk in Figure 1B). However, due to the
low quality, structure elucidation was not possible in this
study. It is considerable that this peak contained a substance
with two regular C2 prenylations. Taking into account the rela-
tionship of structures and retention times of the enzyme prod-
ucts, the retention time of this substance was consistent with
a presumed C2’,C2’’ diprenylation, as N-prenylation led to
higher hydrophobicity and therefore to longer retention times.
A remarkable feature of AstPT is the introduction of two
prenyl moieties at two distinct positions of 1 with both regular
and reverse prenylations. Its homologous protein from A. nidu-
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Analysis of the genes near to ATEG_09980 did not indicate
the presence of any biosynthetic gene cluster for secondary
metabolites. ATEG_09980 has only one neighbouring gene
that shows 48% identity on the amino acid level to tdiC, which
acts as NADH-dependent oxidoreductase in the biosynthesis of
terrequinone A.[2] The second tdiB homologue, ATEG_00702
from A. terreus, is likely located in a similar putative biosynthet-
ic gene cluster to that of terrequinone A, with four homolo-
gous genes. ATEG_00702 is separated from a NRPS-like tdiA ho-
mologue (ATEG_00700) by a tdiC homologue (ATEG_00701;
Figure S2). Inactivation of ATEG_00700 abolished the produc-
tion of asterriquinone CT5 (AQ CT5), a regularly C2’,C2’’-dipre-
nylated bisindolyl benzoquinone.[25] Therefore, it is plausible
that the putative prenyltransferase EAU39348 encoded by
ATEG_00702 would catalyse the prenylation reaction in the
biosynthesis of AQ CT5. However, incubation of 1 with purified
EAU39348 did not result in the formation of any enzyme prod-
ucts. One reason might be that 1 carries two methoxy groups
instead of hydroxy groups at the benzoquinone ring and
therefore is a poor substrate for EAU39348. Support for this hy-
pothesis was obtained from experiments with TdiB, which was
overproduced and purified by using a successfully expressed
construct as described.[24] No enzyme activity was detected
with 1 and TdiB in the presence of DMAPP (data not shown). It
can be assumed that EAU39348 and TdiB both show similar
substrate specificities for unmethylated substances. Attempts
to remove the methyl groups of 1 chemically under different
conditions remained unsuccessful. The high substrate specifici-
ty of AstPT is unusual for enzymes of the DMATS superfamily,
which usually show high flexibility towards their aromatic sub-
strates. For example, the known tryptophan and tryptophan-
containing cyclic dipeptide prenyltransferases also accept a
large number of indole derivatives and cyclic dipeptides.[26] In
contrast, incubation of AstPT with tryptophan or tryptophan-
containing cyclic dipeptides did not result in the formation of
any enzyme product. The high substrate specificity of AstPT
cannot be explained by its sequence similarities to other
known prenyltransferases. It is known that enzymes of the
DMATS superfamily share low sequence similarity with each
other, usually in the range of 30–35% on the amino acid level,
even for those with same substrates. For example, the trypto-
phan C4 prenyltransferase FgaPT2[34] shows a sequence identi-
ty of only 31% to 7-DMATS,[35] which catalyses the prenylation
of tryptophan at C-7. On the other hand, enzymes from this
group with distinct substrates show comparable sequence sim-
ilarities to each other. Examples are SirD, XptB and NscD,
which catalyse prenylation reactions of tyrosine, a xanthone
and an anthrachene derivative, respectively.[36–38] Therefore, it is
difficult to predict the substrates of an unknown prenyltrans-
ferase or its prenyltransfer reaction, or to explain its substrate
specificity by sequence comparison with other known en-
zymes. An explanation could be provided by analysis of the
crystal structure of the enzyme.
tives of both methylated and unmethylated bisindolyl benzo-
quinones in cultures of A. terreus DSM1958 (Figures S11 and
S13), although the coding sequence of EAU39349 was not am-
plified from genomic DNA of this strain. (Reasons for the unex-
pected result could be the unsuitable primers or conditions
used for the PCR amplification). The two TdiB homologues
AstPT and EAU39348 share a sequence identity of 71% on the
amino acid level, which is extremely high for members of the
DMATS superfamily. It can therefore be speculated that they
accept very similar substrates and catalyse similar reactions.
The results on the putative cluster of AQ CT5 reported by Guo
and co-workers[25] and the fact that AstPT but not EAU39348,
can convert 1 to prenylated derivatives lead to the hypothesis
that AstPT is responsible for the prenylation of O-methylated
benzoquinone 1 and EAU39349 for the prenylation of its de-
methylated form. Formation of 1 could be catalysed by an O-
methyltransferase. Evidence for this hypothesis is also provided
by the work of Arai and Yamamoto, who detected methylation
activity using DDAQ D (Scheme 1) as the substrate for crude
enzyme extracts from A. terreus.[39] The structure gene for this
activity could be ATEG_00703, coding for EAU39349 from the
AQ CT5 cluster (Figure S2),[25] as no methylation is involved in
its biosynthesis. The homologue of EAU39349 from the tdi
cluster TdiE also shows homology to methyltransferases but
did not function as a methyltransferase in the biosynthesis of
terrequinone A, as no methylation is necessary in that case.[2]
From the LC/MS analyses, the ratio of the total peak areas of
prenylated methylated derivatives (Figure S11F and G) to
those of unmethylated ones (Figure S13C and D), obtained
from samples corresponding to same amounts of fungal cul-
tures, was found to be 1:1.3. As shown in this study, AstPT has
a high Km value and low turnover number. The slow conversion
of 1 by AstPT might lead to accumulation of 1, which could in-
hibit the activity of the putative O-methyltransferase. As a con-
sequence, the unmethylated bisindolyl benzoquinone would
be consumed less for methylation but more for prenylation by
EAU39348 so that prenylated derivatives of both substrates are
produced by the strain.
Experimental Section
Chemicals: Dimethylallyl diphosphate (DMAPP), geranyl diphos-
phate, and farnesyl diphosphate were prepared according to the
method described for geranyl diphosphate by Woodside.[40] Asterri-
quinone D (AQ D, 1) was prepared through a three-step procedure.
2,5-Dichloro-3-indolyl-1,4-benzoquinone was synthesised from
indole and 2,5-dichlorobenzoquinone according to the method by
Pirrung et al.[41] and was used to obtain 3,6-dichloro-2,5-bis-(3-in-
dolyl)-1,4-benzoquinone.[42] Conversion of 3,6-dichloro-2,5-bis-(3-in-
dolyl)-1,4-benzoquinone to 1 by methoxylation was then per-
formed according to a method described by Tanoue et al.[43] For
detailed synthetic procedures, see the Supporting Information.
NMR data for 1 are given in Table 1.
As mentioned in the Introduction, prenylated bisindolyl ben-
zoquinones with hydroxy and methoxy groups have been iso-
lated from A. terreus (Scheme 1). LC/MS analyses in this study
also revealed the presence of mono- and diprenylated deriva-
Computer-assisted sequence analysis: The software packages
DNASIS (version 2.1; Hitachi Software Engineering, San Bruno, CA,
USA) and FGENESH (Softberry, Inc; http://www.softberry.com/ber-
ry.phtml) were used for intron prediction and sequence analysis, re-
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spectively. Alignments of amino acid sequences were carried out
by using the program BLAST (http://blast.ncbi.nlm.nih.gov).
amplicon by sequencing (Eurofins MWG Operon), pST35 was di-
gested with BamHI and NotI (Jena Bioscience). The resulting
1283 bp fragment was cloned into BamHI and NotI restriction sites
of pHIS8 to give plasmid pST37.
Bacterial strains, plasmids and culture conditions: E. coli XL1-
Blue MRF’ (Stratagene) and E. coli BL21 (DE3) pLysS (AMS Biotech-
nology, Abingdon, UK) were used for gene cloning and expression.
pGEM-T Easy and pQE70 vectors were obtained from Promega and
Qiagen, respectively. pHIS8 was obtained from J. Noel.[44] Bacteria
harbouring these vectors were cultivated at 378C in liquid or on
solid lysogeny broth (LB) with agar (1.5%, w/v), supplemented
with carbenicillin (50 mgmLÀ1) for selection of recombinant E. coli
strains.[45] For E. coli BL21 (DE3) pLysS, kanamycin (25 mgmLÀ1) was
also used.
Overproduction and purification of recombinant proteins: For
overproduction of AstPT-His6, E. coli XL1 blue MRF’ cells harbouring
pST24 were cultivated in LB medium supplemented with carbeni-
cillin (50 mgmLÀ1). An overnight culture (2%, v/v) was used for in-
oculation of 1000 mL LB medium supplemented with carbenicillin
(50 mgmLÀ1) in a 2000 mL flask. After growing at 378C and
220 rpm for 6 h, the culture was cooled to 308C and subsequently
incubated at 308C for additional 16 h. Cells were harvested by cen-
trifugation (4000g, 10 min, 48C) and resuspended in lysis buffer
(50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole, pH 8.0) at 2–
Cultivation of A. terreus for DNA isolation: A. terreus DSM 1958
was purchased from the German Collection of Microorganisms and
Cell Cultures (Leibniz Institute DSMZ, Braunschweig, Germany).
A. terreus FGSCA1156 (identical to the genome reference strain NIH
2624) was kindly provided by Dr. Matthias Brock (Hans-Knçll-Insti-
tut, Jena, Germany). The strains were cultivated in 300 mL Erlen-
meyer flasks containing 100 mL liquid or on plates with solid YME
medium consisting of yeast extract (4.0 gLÀ1), malt extract
(10.0 gLÀ1), glucose (4 gLÀ1) and agar (20.0 gLÀ1) at 308C in dark-
ness.
5 mL per gram wet weight. After addition of lysozyme (1 mgmLÀ1
)
and incubation on ice for 30 min, cells were sonicated six times for
10 s each at 200 W. To remove cellular debris from the soluble pro-
tein, the lysate was centrifuged at 20000g and 48C for 30 min. Pu-
rification of the recombinant His6-tagged fusion protein by affinity
chromatography with Ni-NTA agarose resin (Macherey–Nagel,
Dꢁren, Germany) was carried out according to the manufacturer’s
instructions. AstPT-His6 was subsequently eluted with NaH2PO4
(50 mm), NaCl (300 mm) and imidazole (250 mm, pH 8.0). The re-
sulting fraction was passed through a PD-10 desalting column (GE
Healthcare) that had been previously equilibrated with Tris·HCl
(50 mm) and glycerol (15%, v/v, pH 7.5) to perform buffer ex-
change. AstPT-His6 was eluted with the same buffer and stored at
À808C.
DNA propagation, PCR amplification and gene cloning: Standard
procedures for DNA isolation and manipulation in E. coli were per-
formed as described previously.[45] Genomic DNA of A. terreus was
isolated from five-day-old mycelia grown by using a stationary
method on liquid YME medium according to the chloroform/iso-
amyl alcohol method. A MiniCycler from Bio-Rad was used for PCR
amplification. Amplification of ATEG_09980 was carried out by
a two-step PCR reaction with an Expand High Fidelity Kit (Roche
Diagnostics) and genomic DNA of A. terreus DSM1958. In the first
step, the three exons of ATEG_09980 were amplified in separate re-
actions, with primer pairs ATEG_09980_1 and ATEG_09980_2 for
exon 1, ATEG_09980_3 and ATEG_09980_4 for exon 2 and ATEG_
09980_5 and ATEG_09980_6 for exon 3 (see Table S3 for primer se-
quences). The primers ATEG_09980_2, 3, 4 and 5 contained over-
lapping regions with the adjacent exons, which were used for
fusion of the exons in the second step of amplification. The entire
coding sequence was then propagated by using primer pair ATEG_
09980_1 and ATEG_09980_6 and cloned into pGEM-T Easy vector
to give plasmid pST22. After confirmation of the sequence integrity
of the cloned amplicon by sequencing (Eurofins MWG Operon,
Eberberg, Germany), pST22 was digested with BamHI and BglII
(Jena Bioscience, Jena, Germany). The resulting 1280 bp fragment
was cloned into the BamHI and BglII restriction sites of pQE70 to
give plasmid pST24 after confirmation of the correct orientation.
For overproduction of His8-EAU39348, E. coli BL21 (DE3) pLysS cells
harbouring pST37 were cultivated in LB medium supplemented
with carbenicillin (50 mgmLÀ1) and kanamycin (25 mgLÀ1). An over-
night culture (2%, v/v) was used to inoculate LB (1 L) medium sup-
plemented with carbenicillin (50 mgmLÀ1
)
and kanamycin
(25 mgLÀ1) in a 2000 mL flask. Cells were grown at 378C to an A600
of 0.6. Gene expression was induced by addition of IPTG to a final
concentration of 0.1 mm. The bacterial culture was cultivated for
a further 16 h at 228C and then centrifuged to harvest the cells.
Protein purification was carried out as described above, with an
additional purification step on HisPur cobalt resin (Thermo Fisher
Scientific) after affinity chromatography with Ni-NTA agarose resin.
Protein analysis and determination of the molecular mass of
active AstPT-His6: Purity of AstPT-His6 and His8-EAU39348 was ana-
lysed by SDS-PAGE according to the method of Laemmli[46] with
12% polyacrylamide gels by using Coomassie Brilliant Blue G-250
for staining. Protein quantification was carried out according to
the method of Bradford.[47] The molecular mass of the recombinant
AstPT-His6 was determined by size-exclusion chromatography on
a HiLoad 16/60 Superdex 200 column (GE Healthcare, Freiburg,
Germany) that had been equilibrated with Tris·HCl buffer (50 mm,
pH 7.5) containing NaCl (150 mm). The column was calibrated with
Blue Dextran 2000 (2000 kDa), ferritin (440 kDa), aldolase (158 kDa),
conalbumin (75 kDa), carbonic anhydrase (29 kDa) and ribonuclea-
se A (13.7 kDa; GE Healthcare). Separation was performed on an
ꢂKTA Purifier FPLC system (GE Healthcare) at a flow rate of
The same strategy was used for amplification of the coding se-
quence of ATEG_00702. PCR amplification was carried out by using
genomic DNA of A. terreus FGSCA1156 with addition of 5% (v/v)
DMSO, due to the high GC content of the genomic sequence.
Primer pairs ATEG_00702_1_pHis and ATEG_00702_2 were used for
the amplification of the first exon, ATEG_00702_3 and ATEG_
00702_4 for the amplification of the second exon and ATEG_
00702_5 and ATEG_00702_6_pHis for the amplification of the third
exon (see Table S3 for primer sequences). The primers ATEG_
00702_2, 3, 4 and 5 contained overlapping regions with the adja-
cent exons, which were used for fusion of the exons in the second
step of amplification. The entire coding sequence was subsequent-
ly propagated by using primer pair ATEG_00702_1 and ATEG_
00702_6 and cloned into pGEM-T Easy vector to give plasmid
pST35. After confirmation of the sequence integrity of the cloned
1 mLminÀ1
.
Enzyme assays with AstPT: The enzyme reaction mixtures (100 mL)
contained Tris·HCl (50 mm, pH 7.5), CaCl2 (5 mm), 1 (0.5 mm),
DMAPP (2 mm), 0.8% (v/v) glycerol, and purified recombinant
AstPT-His6 (20 mg, 4.1 mm). The reaction mixtures were incubated at
378C for 1 or 16 h. For determination of the kinetic parameters of
1, the assays contained DMAPP (2 mm), 1 (0.02, 0.05, 0.08, 0.1, 0.2,
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0.3, 0.4 and 0.5 mm), and purified recombinant AstPT-His6 (10 mg).
The kinetic parameters of DMAPP were determined with
1 (0.5 mm) and DMAPP (0.02, 0.05, 0.1, 0.3, 0.5, 1.0, 1.5 and
2.0 mm). Incubation mixtures for determination of the kinetic pa-
rameters were incubated for 20 min. The enzymatic reaction was
stopped by three extractions with EtOAc (200 mL). After evapora-
tion of the combined organic phases to dryness, the residue was
dissolved in DMSO (20 mL), diluted with CH3CN (100 mL), and ana-
lysed by HPLC as described below. Two independent incubations
were carried out routinely. Kinetic parameters were calculated as
average values from Eadie–Hofstee, Hanes–Woolf, and Lineweaver–
Burk plots. Conversion yields and velocities were calculated from
the amount of consumed substrate by measuring the decrease in
the peak area of 1 in the HPLC chromatogram.
over Na2SO4 and subsequently evaporated in vacuo to dryness. For
LC/MS analyses, the extracts were dissolved in CH3CN. LC/MS anal-
ysis was carried out on a 1100 HPLC system (Agilent Technologies)
coupled to an LTQ-FT Ultra ESI-MS instrument (Thermo Fischer Sci-
entific) by using a linear ion trap (MS) and an ICR cell (HR-MS), op-
erated in positive ionisation mode. Water (solvent A) and CH3CN
(solvent B) were used at a flow rate of 1 mLminÀ1. A linear gradient
of 10–100% (v/v) solvent B in 20 min was used. The column was
then washed with 100% solvent B for 5 min and equilibrated with
10% (v/v) solvent B for 5 min.
Acknowledgements
This work was supported by a grant from the Deutsche For-
schungsgemeinschaft (Li844/4-1 to S.M.L.). We thank Dr. Matthias
Brock (Jena, Germany) for providing A. terreus FGSCA1156 and
Prof. Dr. Dirk Hoffmeister (Jena, Germany) for providing tdiB
expression construct pPS1. We thank Dr. Uwe Linne and Jan
Bamberger (Marburg, Germany) for LC/MS analyses.
For isolation and structure elucidation of enzyme products, incuba-
tions were carried out at 378C for 2 or 16 h. The incubation mix-
tures (20 mL) contained Tris·HCl (50 mm, pH 7.5), CaCl2 (5 mm),
1 (0.5 mm), DMAPP (2 mm) and purified recombinant protein
(4.8 mg).
Enzyme assays with EAU39349: The enzyme reaction mixtures
(100 mL) contained Tris·HCl (50 mm, pH 7.5), CaCl2 (5 mm),
1 (0.5 mm), DMAPP (2 mm), 4.5% (v/v) glycerol and purified re-
combinant His8-EAU39349 (6.3 mg). The reaction mixtures were
incubated at 378C for 16 h, extracted three times with EtOAc
(200 mL), and analysed by HPLC, as described below.
Keywords: alkaloids
· Aspergillus terreus · biosynthesis ·
bisindolyl benzoquinones · prenyltransferase
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Published online on December 2, 2013
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