gem-Diprenylation of Acylphloroglucinols by a Fungal Prenyltransferase of the Dimethylallyltryptophan Synthase Superfamily

Article abstract of DOI:10.1021/acs.orglett.6b03594

Aspergillus terreus aromatic prenyltransferase (AtaPT) catalyzes predominantly C-monoprenylation of acylphloroglucinols in the presence of different prenyl diphosphates. With dimethylallyl diphosphate (DMAPP) as prenyl donor, gem-diprenylated products 1D3, 2D3, and 3D3 were also detected. High conversion of 1D1 to 1D3, 2D1 to 2D3, and 3D1 to 3D3 was demonstrated by incubation with AtaPT and DMAPP. The first example of gem-diprenylation by a member of the dimethylallyltryptophan synthase superfamily is provided.

Full text of DOI:10.1021/acs.orglett.6b03594

Letter
pubs.acs.org/OrgLett
gem-Diprenylation of Acylphloroglucinols by a Fungal
Prenyltransferase of the Dimethylallyltryptophan Synthase
Superfamily
Kang Zhou,† Carsten Wunsch,† Jungui Dai,‡ and Shu-Ming Li*,†
†Institut fur Pharmazeutische Biologie und Biotechnologie, Philipps-Universitat Marburg, Robert-Koch-Strasse 4, 35037 Marburg,
̈
̈
Germany
‡State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Xian Nong Tan Street, Beijing 100050, China
S
* Supporting Information
ABSTRACT: Aspergillus terreus aromatic prenyltransferase (AtaPT) catalyzes predominantly C-monoprenylation of
acylphloroglucinols in the presence of different prenyl diphosphates. With dimethylallyl diphosphate (DMAPP) as prenyl
donor, gem-diprenylated products 1D3, 2D3, and 3D3 were also detected. High conversion of 1D1 to 1D3, 2D1 to 2D3, and 3D1
to 3D3 was demonstrated by incubation with AtaPT and DMAPP. The first example of gem-diprenylation by a member of the
dimethylallyltryptophan synthase superfamily is provided.
renylated acylphloroglucinols (APs) are characteristic
constituents of several plant families. Compounds of this
Scheme 1. Prenylation Steps in the Biosynthesis of β-Bitter
Acids in Humulus lupulus
P
class have fascinating chemical structures and intriguing
biological and pharmacological activities.1−5 Main structural
features of prenylated APs are highly oxygenated and densely
decorated with prenyl such as dimethylallyl and geranyl
moieties. Polyprenylated APs like α- and β-bitter acids (Figure
1) from Humulus lupulus (Cannabinaceae), commonly known
Scheme 1). Clusianone carries the skeleton of phlorbenzophe-
none (PBZP, 3, Scheme 2). The AP cores of these compounds
are tetraketides and formed by condensation of three malonyl-
CoA molecules with different start units, i.e., isobutyryl-CoA in
the case of 1, isovaleryl-CoA in the case of 2, and benzoyl-CoA
in the case of 3. The responsible polyketide synthases have
been characterized.10−12 In comparison, little is known about
the enzymes for the multiple prenylation steps. Only the
prenyltransferases involved in the biosynthesis of bitter acids in
H. lupulus have been reported.12,13 Tsurumaru et al.13 reported
the overproduction of the membrane-bound prenyltransferase
HIPT-1 from H. lupulus and its biochemical characterization. It
was found that this enzyme catalyzed the prenylation of 2 in the
presence of DMAPP and also accepted 1 as prenylation
acceptor. Recently, Li et al.12 identified two membrane-bound
prenyltransferases HIPT1L and HIPT2 from H. lupulus.
Figure 1. Examples of polyprenylated APs from plants.
as hops, are considered as multipotent bioactive compounds
including their sedative effects.4 Clusianone (Figure 1) and its
7-epimer from different plants such as Garcinia brasiliensis and
Clusia torresii, both from the family Clusiaceae, exhibit anti-HIV
and antitumor activities.6−9 An exceptional structure feature of
polyprenylated APs is the gem-diprenylation at C-atoms.
Biogenetically, cohumulone and colupulone are prenylated
phlorisobutyrophenone (PIBP, 1, Scheme 1), while humulone
and lupulone prenylated phlorisovalerophenone (PIVP, 2,
Received: December 1, 2016
© XXXX American Chemical Society
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sequence identity with the hypothetical protein EAU34068
encoded by ATEG_04999 from A. terreus NIH2624 and differs
at only three residues. Among the tested aromatic substrates,
AtaPT also consumed 3 in the presence of DMAPP, GPP, and
FPP, although no noteworthy sequence homology exits
between AtaPT and HIPT1L or HIPT2. These reactions
were not studied in detail.20
Scheme 2. Prenylations of 1−3 by AtaPT in the Presence of
DMAPP, GPP, and FPP
In this study, we investigate the behavior of AtaPT toward
the three APs 1−3, which serve as precursors of most
polyprenylated APs,1−3,5 in the presence of DMAPP, GPP,
and FPP. We hope to increase the structure diversity of
prenylated APs by high conversion yields with GPP and FPP as
well as by multiple prenylations, especially gem-diprenylations.
The coding sequence of AtaPT orthologue from A. terreus
DSM 1958 was amplified by PCR and cloned into the
expression vector pQE-70, resulting in the expression construct
pCaW7 (see the Supporting Information for details). Sequence
analysis revealed that differences at only four residues were
found between AtaPT and its orthologue from DSM 1958.
S230, T290, A292, and N373 in AtaPT were replaced by A230,
K290, E292, and S373 in that of strain DSM 1958, respectively.
Because these residues are not located in the active sites,20 it
can be expected that the orthologue from DSM 1958 will fulfill
the function of AtaPT, and we therefore use hereafter the name
AtaPT also for this enzyme. Gene expression in E. coli and
purification of the soluble protein resulted in a predominant
band on SDS-PAGE with a migration above the 45 kDa size
marker, corresponding well to the calculated mass of 48.7 kDa
for AtaPT-His6. The protein yield was calculated to be 29 mg of
purified protein per liter of culture (see the SI for details).
To compare the activities of AnaPT mentioned above and
AtaPT toward 1−3, incubations on a 100 μL scale, containing
20 μg of protein, DMAPP, GPP, or FPP, were carried out at 37
°C for 2 h. HPLC analysis revealed that 1−3 were much better
converted by AtaPT than by AnaPT in all enzyme assays. Total
conversion yields of 63.67 2.43, 23.4 1.26, and 27.35
0.25% were calculated for 1, 2, and 3 with AtaPT and DMAPP,
respectively. These values are significantly higher than those of
AnaPT, with conversion yields of 3.64 0.77, 4.33 0.52, and
7.66 0.49%, respectively (Scheme 2; see the SI for details).
The poor acceptance of GPP by AnaPT was confirmed in this
study, and product formation was only detected in the reaction
mixtures of 1 and 3 with GPP. In contrast, GPP served as a very
good prenyl donor for the AtaPT reactions, with product yields
of 32.85 2.58, 47.62 1.83, and 54.72 0.42% for 1, 2, and
3, respectively (Scheme 2; see the SI for details). FPP was
accepted by AtaPT with conversion yields of 7.19 1.57, 6.66
0.51, and 6.27 0.09% for 1, 2, and 3, respectively (Scheme
2; see the SI for details).
Coexpression of different genes in Saccharomyces cerevisiae
revealed that HIPT1L and HIPT2 catalyzed three sequential
prenylation steps in the ß-bitter acid pathway. HIPT1L was
confirmed to be an orthologue of HIPT1 identified by
Tsurumaru et al.13 Interestingly, HIPT2 was only active when
it was co-expressed with HIPT1L. This led to the hypothesis
that HIPT1L and HIPT2 form a metabolon as the catalytic
unit.12
In recent years, significant progress has been achieved for the
members of the dimethylallyltryptophan synthase (DMATS)
superfamily, mainly from ascomycetous fungi.14 These soluble
enzymes use predominantly tryptophan and other indole
derivatives as prenyl acceptors but also accept a broad spectrum
of aromatic compounds as substrates.14 Diprenylation of indole
derivatives by one DMATS enzyme was observed in several
cases.15−17 However, a gem-diprenylation has not been reported
for such enzymes or for other soluble prenyltransferases. The
sole example of gem-diprenylation of an aromatic substrate was
described for the metabolon of HIPT1L and HIPT2 mentioned
above.12
In a previous study,18 we demonstrated the prenylation of
APs 1−3 by the soluble fungal prenyltransferase AnaPT from
Neosartorya fischeri, which catalyzed the prenylation of (R)-
benzodiazepinedinone, a cyclic dipeptide of tryptophan, and
anthranilic acid.19 The observed activities of AnaPT toward
these substrates are much higher than that of a microsomal
fraction containing the overproduced prenyltransferase
HIPT1.13 However, only monoprenylated derivatives were
obtained in the presence of DMAPP, and the conversion yields
of 1−3 with GPP as prenyl donor were very low.18
Only one product each was detected in the AnaPT reactions
of 1−3 with DMAPP as well as those of 1 and 2 with GPP.
These products were identified as predominant ones in the
AtaPT reactions. Isolation and structure elucidation by NMR
and MS analyses (see the SI for details) proved the main
products of the AtaPT reactions to be monoprenylated
derivatives; that is 1D1−3D1 with DMAPP, being consistent
with the AnaPT products,18 1G1−3G1 with GPP, and 1F1−
3F1 with FPP (Scheme 2, see the SI for details).
Additional product peaks with longer retention times were
also detected in the reaction mixtures of AtaPT with 1−3 in the
presence of all three prenyl donors (see the SI for details). The
intensities of these peaks are clearly increased in the incubation
mixtures with 50 μg of protein at 37 °C for 2 h, which were
Very recently, a soluble prenyltransferase AtaPT from
Aspergillus terreus strain A8-4 was demonstrated to carry an
unprecedented promiscuity toward diverse aromatic acceptors
and prenyl donors including DMAPP, geranyl diphosphate
(GPP), and farnesyl diphosphate (FPP). AtaPT shares high
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detected by LC−MS analysis (Figure 2). In the incubation
mixtures of 1−3 with GPP and FPP, two monoprenylated
LC−MS analysis revealed clear conversion by detection of the
gem-diprenylated derivatives 1D3, 2D3, and 3D3 as predom-
inant products, with product yields of 4.88
0.15, 57.18
1.07, and 33.31 0.23%, respectively (Figure 3). Better
Figure 3. LC−MS analysis of AtaPT reactions (50 μg per 100 μL, 37
°C for 2 h) with DMAPP and 1D1, 2D1, or 3D1. Red lines are UV
absorptions, and black lines are extracted positive-ion chromatograms
(EIC).
conversion of 2D1 and 3D1 than 1D1 confirmed the higher
product yields of 2D3 and 3D3 in the incubation mixtures of 2
and 3 than 1D3 in that of 1 (Figure 2). A minor diprenylated
product each with larger retention times and product yields of
1.99 0.07, 8.24 0.87, and 4.16 0.20%, respectively, was
also detected. The second product 2D4 from the incubation
mixture of 2D1 was proven to be a C- and O-diprenylated
derivative, which was also isolated and identified from the
incubation mixture of 2 with DMAPP (Figures 2 and 3 and
Schemes 2 and 3; see the SI for details). The second
Scheme 3. Reactions of Monoprenylated Derivatives with
AtaPT
Figure 2. LC−MS analysis of the reaction mixtures of DMAPP, GPP,
or FPP with 1 (A), 2 (B), and 3 (C) after incubation with 50 μg of
AtaPT per 100 μL at 37 °C for 2 h. Red lines are UV absorptions, and
black lines are extracted positive-ion chromatograms (EIC).
derivatives each were detected. In addition to the C-prenylated
1G1−3G1 and 1F1−3F1, one O-prenylated derivative each,
1G2, 2G2, 3G2, 1F2, 2F2, or 3F2 ,was identified by NMR and
MS analyses as a minor product after isolation and structure
elucidation (Scheme 2 and Figure 2; see the SI for details).
In the incubation mixtures of 1 with DMAPP, two mono-
and two diprenylated derivatives were detected. In comparison,
three mono- and three diprenylated derivatives were found in
the reaction mixture of 2 with DMAPP and one mono- and one
diprenylated products of 3 with DMAPP. Isolation and
structure elucidation proved the presence of O-prenylated
1D2 and 2D2 in the reaction mixtures of 1 and 2, respectively.
Interestingly, gem-diprenylated derivatives 1D3, 2D3, and 3D3
were identified in the reaction mixtures of 1, 2, and 3,
respectively (Figure 2 and Scheme 2; see the SI for details).
These results proved the ability of AtaPT for a gem-
dipenylation of acylphloroglucinols and encouraged us to
investigate the conversion of the monoprenylated 1D1, 2D1,
and 3D1 by AtaPT. 1D1, 2D1, and 3D1 were then incubated in
the presence of DMAPP with 50 μg of AtaPT at 37 °C for 2 h.
diprenylated derivative of the reaction mixtures with 1D1 and
3D1 could not be isolated and identified. However, on the basis
of their retention times and UV spectra together with LC−MS
data, it can be speculated that these compounds are also C- and
O-diprenylated derivatives.
These results provide evidence for successive diprenylations
of 1, 2, and 3 by AtaPT, which was also confirmed by time
dependence of the product formation in the incubation
mixtures of 2 and 3 with DMAPP. The formation of 2D3
and 3D3 increased continuously, while 2D1 and 3D1 reached
their maxima in short time and decreased after that (see the SI
for details). No product formation was detected in the
incubation mixtures of 1D1, 2D1, and 3D1 with AnaPT and
DMAPP under the same conditions (data not shown).
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As mentioned above, no diprenylated product was detected
in the incubation mixtures of AtaPT with 1−3 and GPP or FPP
(Figure 2 and Scheme 2). This was confirmed by incubation of
their C-monoprenylated derivatives. Product formation was not
observed for the reaction mixtures of 1G1, 2G1, and 3G1 with
AtaPT in the presence of GPP or those of 1F1, 2F1, and 3F1
with FPP (Scheme 3). Incubation of 1D1, 2D1, and 3D1 with
AtaPT and GPP or FPP did not result in product formation. A
previous study showed that AtaPT also catalyzed C-
diprenylations of several aromatic acceptors in the presence
of GPP and FPP, although no gem-prenylated products were
detected.20 No formation of diprenylated 1−3 with these
donors resulted from their orientations in the reaction cavity.
No product formation was detected by LC−MS analysis after
incubation of 1D2 and 2D2 with 50 μg of AtaPT and DMAPP
at 37 °C for 2 h (Scheme 3), excluding the possible formation
of C- and O-diprenylated derivatives from O-monoprenylated
products.
To obtain more insights into the catalytic efficiency of the
tetrameric AtaPT, kinetic parameters including Michaelis−
Menten constants (KM) and turnover numbers (kcat), were
determined at pH 7.5 for 1, 2, and 3 in the presence of
DMAPP, GPP, and FPP as well as DMAPP with 1 and GPP
with 3 (see the SI for details). The catalytic efficiency of AtaPT
toward 1 is 32-fold of that of AnaPT in the presence of
DMAPP. compound 1 was better consumed by AtaPT in the
presence of DMAPP and FPP than 2 and 3. Compound 3 was
most efficiently consumed in the presence of GPP. The
determined kcat/KM values for 1−3 are in the range of 170−300
s−1 M−1 in the presence of GPP and between 17 and 37 s−1
M−1 in the presence of FPP. With 1 as acceptor, a 95-fold kcat/
KM value of that of AnaPT was determined for DMAPP with
AtaPT (see Table S8 for details).
donors and Rixa Kraut and Stefan Newel for assistance with the
MS and NMR spectra, respectively. The Bruker microTOF
QIII mass spectrometer was funded by the DFG (INST 160/
620-1 to S.-M. L.). K.Z. is a recipient of a scholarship from the
China Scholarship Council (201308440282).
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In conclusion, we have provided in this study the first
example of gem-diprenylation of APs by a member of the
DMATS superfamily, proving their unprecedented application
potential. AtaPT could be an interesting candidate for
production of polyprenylated APs like β-bitter acids by
synthetic biology.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b03594.
Experimental procedures, detailed NMR data, HR-ESI-
MS data, determination of kinetic parameters, as well as
NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: shuming.li@staff.uni-marburg.de.
ORCID
Shu-Ming Li: 0000-0003-4583-2655
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the following people from the University Marburg for
their help: Lena Ludwig and Edyta Stec for synthesis of prenyl
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DOI: 10.1021/acs.orglett.6b03594
Org. Lett. XXXX, XXX, XXX−XXX