Friedel-Crafts Alkylation of Acylphloroglucinols Catalyzed by a Fungal Indole Prenyltransferase

Article abstract of DOI:10.1021/np5009784

Naturally occurring prenylated acylphloroglucinol derivatives are plant metabolites with diverse biological and pharmacological activities. Prenylation of acylphloroglucinols plays an important role in the formation of these intriguing natural products and is catalyzed in plants by membrane-bound enzymes. In this study, we demonstrate the prenylation of such compounds by a soluble fungal prenyltransferase AnaPT involved in the biosynthesis of prenylated indole alkaloids. The observed activities of AnaPT toward these substrates are much higher than that of a microsomal fraction containing an overproduced prenyltransferase from the plant hop.

Full text of DOI:10.1021/np5009784

Note
pubs.acs.org/jnp
Friedel−Crafts Alkylation of Acylphloroglucinols Catalyzed by a
Fungal Indole Prenyltransferase
Kang Zhou, Lena Ludwig, and Shu-Ming Li*
Institut fur Pharmazeutische Biologie und Biotechnologie, Philipps-Universitat Marburg, Marburg 35037, Germany
̈
̈
S
* Supporting Information
ABSTRACT: Naturally occurring prenylated acylphloroglucinol derivatives
are plant metabolites with diverse biological and pharmacological activities.
Prenylation of acylphloroglucinols plays an important role in the formation of
these intriguing natural products and is catalyzed in plants by membrane-
bound enzymes. In this study, we demonstrate the prenylation of such
compounds by a soluble fungal prenyltransferase AnaPT involved in the
biosynthesis of prenylated indole alkaloids. The observed activities of AnaPT
toward these substrates are much higher than that of a microsomal fraction containing an overproduced prenyltransferase from
the plant hop.
olyprenylated acylphloroglucinols are found in a limited
number of plant families including Clusiaceae, Hyper-
phlorisovalerophenone (2a) in the presence of dimethylallyl
diphosphate (DMAPP) and also accepted phlorisobutyrophe-
none (1a) as prenylation substrate. Its catalytic activities for
both substrates were very low, being 11 pmol per mg of
microsomal fraction per minute for phlorisovalerophenone
(2a), and 29.5% of that for phlorisobutyrophenone (1a). Like
most membrane-bound proteins, HIPT-1 is more difficult to
overproduce and purify than soluble enzymes. These features
strongly prohibit its potential use as a biocatalyst for
chemoenzymatic synthesis for production of prenylated
acyphloroglucinols. Therefore, there is a need to find alternative
enzymes with better properties. In this study, we tested the
acceptance of such acylphloroglucinols by prenyltransferases of
the DMATS superfamily from fungi, which are soluble proteins
and can be easily overproduced in Escherichia coli with
significantly higher yields.10
P
icaceae, and Cannabaceae.1−5 Their fascinating chemical
structures and intriguing biological activities have attracted
increasing attention. Hyperforin, a polyprenylated acylphlor-
oglucinol (Scheme S1, Supporting Information), and the
naphthodianthrone hypericin are considered as active con-
stituents in extracts of Hypericum perforatum (St John’s wort,
Hypericaceae) used for treatment of depression in Europe and
the USA.2 Humulone (α-acid) and lupulone (β-acid, Scheme
S1, Supporting Information) are prenylated acylphloroglucinols
in hop cones, the female flowers of Humulus lupulus
(Cannabaceae), and are responsible for the bitter taste and
pharmacological effects.3 Hops are used primarily as a flavoring
and stabilizing agent in beer. In traditional medicines, hops are
also used as mild sedative drugs.3
Biogenetically, the acylphloroglucinol cores in hyperforin and
lupulone are formed by condensation of three malonyl-CoA
molecules with different start units, isobutyryl-CoA in the case
of the hyperforin precursor phlorisobutyrophenone (1a) and
isovaleryl-CoA in the case of the lupulone precursor
phlorisovalerophenone (2a). This key step is catalyzed by a
polyketide synthase (PKS) and followed by an alkylation on the
benzene ring catalyzed by prenyltransferases (Scheme S1,
Supporting Information).1,6,7 Several plants from the families
Clusiaceae and Hypericaceae also use benzoyl-CoA as start unit
and produce phlorbenzophenone (3a) and derivatives thereof
such as grandone (Scheme S1, Supporting Information).4,5,8,9
In sharp contrast to many new prenylated acylphloroglucinol
derivatives reported in 2014,1,5,8 little is known about the
enzymes related to the biosynthesis of these compounds. Until
now, only one recombinant enzyme involved in their
biosynthesis, the prenyltransferase HIPT-1 from Humulus
lupulus, was investigated biochemically.6 The membrane-
bound HIPT-1 was overproduced in baculovirus-infected insect
cells6 and the microsomal fraction of the protein extract was
used for enzyme assays. It catalyzed the prenylation of
Phlorisobutyrophenone (1a), phlorisovalerophenone (2a),
and phlorbenzophenone (3a) were synthesized according to
protocols described previously11−13 (Scheme 1) and incubated
with 13 purified soluble fungal prenyltransferases including four
tryptophan, seven cyclic dipeptide, and two tyrosine prenyl-
transferases. HPLC analysis of the incubation mixtures revealed
that AnaPT from Neosartorya fischeri, which catalyzes the C3-
Scheme 1. Synthesis of Acylphloroglucinols
Received: December 5, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Note
Figure 1. HPLC chromatograms and prenyl transfer reactions catalyzed by AnaPT. Different wavelengths were used for illustration of product
formation: 230 nm (4a), 254 nm ((R)-benzodiazepinedione and 5a), 266 nm (6a), 291 nm (1a, 2a, and 7a), and 306 nm (3a).
prenylation of (R)-benzodiazepinedione (Figure 1A) and is
involved in the biosynthesis of acetylaszonalenine,14 showed
higher activities toward these compounds than other tested
enzymes (Table S1, Supporting Information). Conversion
yields between 12% and 14% were achieved for these substrates
after incubation with 5 μg of AnaPT in 100 μL assay at 37 °C
for 16 h. No product formation was detected with heat-
inactivated AnaPT (data not shown). Using 50 μg of protein,
B
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Note
a
1
Table 1. H NMR Data (500 MHz) of the Enzyme Products 5b, 6b, 7b, and 7c in Methanol-d4
a
Chemical shifts (δ) are given in ppm and coupling constants (J) in Hz.
we found product formation to be nearly linear in the first 6 h
(Figure S1, Supporting Information). Conversion yields of
32.1%, 32.3%, and 28.2% were calculated for 1a, 2a, and 3a,
respectively, after incubation with 50 μg of protein at 37 °C for
6 h (Figure 1). Under this condition, 93% of (R)-
benzodiazepinedinone was converted to aszonalenin (Figure
1A). The tryptophan prenyltransferase 7-DMATS from
Aspergillus fumigatus15 showed slightly lower activities than
AnaPT, with product yields of about 10% in the assays of 1a
and 2a with 5 μg of protein (Table S1, Supporting
Information). Compound 1a was accepted by CdpC3PT
from Neosartorya fischeri16 with a conversion yield of 5.7%
(Table S1, Supporting Information).
These results encouraged us to test the acceptance of
phloroglucinol (4a) and its carboxylic acid 5a, as well as
orsellinic acid (6a) and 6-methylsalicylic acid (7a), as
substrates. The latter two compounds were identified as PKS
products in different microorganisms.17−19 As shown in Figure
1, all of these substances were accepted by AnaPT. Conversion
yields of 4.6−10.8% were observed using 50 μg of AnaPT after
incubation at 37 °C for 6 h. It is obvious that acylphlor-
oglucinols 1a−3a are better substrates for AnaPT than 4a−7a.
Interestingly, 6a with a methyl instead of a hydroxy group in 5a
was a better substrate for AnaPT (Figure 1F,G). Detailed
inspection of the HPLC chromatograms B−H in Figure 1
revealed the presence of one predominant product each in the
incubation mixtures of 1a−3a, 5a, and 6a. Compounds 4a and
7a were converted to at least two products.
To elucidate the structures, nine enzyme products, 1b, 2b,
3b, 4b, 4c, 5b, 6b, 7b, and 7c, were isolated from the
incubation mixtures of 1a−7a with AnaPT and DMAPP and
subjected to NMR and HR-EI-MS analysis (Table 1 and Tables
S2 and S3 and Figures S3−S14, Supporting Information). In
addition, 1b was also isolated from the incubation mixtures of
1a with CdpC3PT or 7-DMATS. The spectra of 1b from the
three different incubation mixtures are nearly identical, proving
the same product of different enzymes. With the exception of
4c, the M+ ions of the isolated products are 68 Da larger than
the respective substrates (Table S2, Supporting Information),
proving the monoprenylation of these compounds. The M+ ion
of 4c is 136 Da larger than that of 4a, proving the diprenylation
in its structure. This conclusion was confirmed by their
molecular formula deduced from HR-MS analysis (Table S2,
Supporting Information). The signals at δ3.16−3.29 (d, 2H
−CH2−), 5.02−5.32 (m, 1H, −CCH), 1.61−1.71 (s, 3H,
−CC−CH3), and 1.72−1.76 ppm (s, 3H, −CC−CH3) in
1
the H NMR spectra of the isolated products confirmed the
presence of regular dimethylallyl moieties in their structures.
The resonance of the methylene group in the range of 3.16−
3.29 ppm proved their attachment to aromatic carbon atoms.21
Comparison of the NMR data with those of previously reported
compounds led to identification of the structures of 1b,22 2b,22
3b,23 4b,24 and 4c,24 as shown in Figure 1. The structures of 5b,
6b, 7b, and 7c have not been reported prior to this study.
Only one singlet for an aromatic proton at δ5.73 ppm was
found in the 1H NMR spectrum of 5b, which proved
unequivocally the attachment of the prenyl moiety at C-3 of
the benzene ring (Table 1, Figure 1F). One singlet for an
In a previous study,20 we demonstrated that AnaPT also used
geranyl diphosphate (GPP) as prenyl donor. Therefore, 1a−7a
were incubated with 50 μg of AnaPT in the presence of GPP.
Indeed, product formation was observed in all incubation
mixtures, but with relatively lower activities than with DMAPP
for a given aromatic substrate. After incubation for 16 h,
conversion yields of 6−10% were obtained for 1a−3a and less
than 5% for other substrates (Figure S2, Supporting
Information).
1
aromatic proton (δ6.13 ppm) was also observed in the H
NMR spectrum of 6b, confirming the prenylation at C-3 or C-5
of the benzene ring. Coupling between CH3 at C-6 and H-5
was observed in the 1H NMR spectrum of 6a (data not shown).
No coupling between the signal for this methyl group at δ2.50
1
ppm with the singlet at δ6.13 ppm was observed in the H
NMR spectrum of 6b, indicating a prenylation at C-5. The
NOESY correlation of the signal at δ2.50 ppm with that of H-
C
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Note
1′of the prenyl moiety at δ3.28 ppm proved unequivocally the
prenylation at C-5, as shown in Figure 1. The 1H NMR
spectrum of 7b showed two doublets at δ6.97 (1H, d, J = 8.4
Hz, ArH-3) and δ6.57 ppm (1H, d, J = 8.4 Hz, ArH-4), and the
NOESY correlation of the signal at δ2.46 ppm for CH3 at C-6
with that of H-1′of the prenyl moiety at 3.24 (d, J = 7.8 Hz)
confirmed the prenyl moiety at C-5 of the benzene ring. The
spectrum of 7c also showed signals of two aromatic protons at
δ6.91 (1H, d, J = 7.5 Hz, ArH-3) and δ6.50 (1H, d, J = 7.5 Hz,
ArH-4). NOESY correlation was observed for the signal of CH3
at C-6 with that of H-5, suggesting the prenylation at C-3 in 7c
(Figure 1H).
that of (R)-benzodiazepinedione determined in this study.
Undoubtedly, catalytic efficiency of AnaPT toward acylphlor-
oglucinols should be improved in the future by suitable
approaches such as mutagenesis experiments. However, it
should be mentioned that the turnover number of AnaPT
toward its natural substrate is much higher (up to 10-fold) than
most prenyltransferases of the DMATS superfamily to their
natural substrates. The determined Vmax values for 1a−3a are in
the range of 3.7−5.1 nmol mg protein−1 min−1. Therefore, it
can be concluded that AnaPT is already an interesting
candidate as a biocatalyst for prenylation of phloroglucinol
analogues, especially of acylphloroglucinols.
In summary, our data provide evidence that the soluble
fungal indole prenyltransferase AnaPT catalyzed the same
prenylation reaction of acylphloroglucinols as the membrane-
bound prenyltransferases involved in the biosynthesis of the
prenylated acylphloroglucinols in plants like HIPT-1,6 but with
much higher conversion yields than HIPT-1. To the best of our
knowledge, this is the first report on the prenylation of
acylphloroglucinols by microbial enzymes. Furthermore,
AnaPT also prenylated hydroxylated benzoic acids such as
orsellinic acid (6a) and 6-methylsalicylic acid (7a), which are
typical PKS products of microorganisms.17−19 Therefore,
prenylated and hydroxylated benzoic acids could be produced
by introducing anaPT into the producers of 6a and 7a or by
coexpression of the responsible PKS genes17−19 with anaPT in
suitable hosts. Thus, this work extends significantly the
substrate and catalytic promiscuity of the prenyltransferases
of the DMATS superfamily as well as their potential
applications.
To get more insights into the catalytic efficiency of the
tetrameric AnaPT14,25 toward acylphloroglucinols and hydrox-
ybenzoic acids, kinetic parameters including Michaelis−Menten
constants (KM) and turnover numbers (kcat) were determined
at pH 7.5 for (R)-benzodiazepinedinone, 1a−7a, and DMAPP
by Hanes−Woolf, Eadie−Hofstee, and Lineweaver−Burk plots
and were compared with each other (Figures S15−24,
Supporting Information). As given in Table 2, AnaPT displayed
EXPERIMENTAL SECTION
■
General Experimental Procedures. Phloroglucinol (4a) was
obtained from Acros Organics, 2,4,6-trihydroxybenzoic acid (5a) and
orsellinic acid (6a) from Alfa Aesar, and 6-methylsalicylic acid (7a)
from Chempur. NMR spectra were recorded on a JEOL ECA-500
spectrometer, processed with MestReNova 5.2.2. Chemical shifts were
referenced to the signal of acetone-d6 at 2.05 ppm or methanol-d4 at
3.31 ppm. The enzyme products were also analyzed by electron impact
mass spectrometry (EI-MS) on an Auto SPEC (Micromass Co. UK
Ltd.).
Synthesis of DMAPP, GPP, and Acylphloroglucinols 1a, 2a,
and 3a. The triammonium salts of dimethylallyl diphosphate
(DMAPP) and geranyl diphosphate (GPP) were synthesized
according to the method described for geranyl diphosphate by
Woodside et al.26 Compounds 1a, 2a, and 3a in yields of 70.8%,
67.1%, and 65.8% were prepared by Friedel−Crafts acylations of
phloroglucinol with isobutyryl chloride,11 methylbutanal chloride,13
and benzoyl chloride12 in the presence of AlCl3, respectively (Scheme
1).
Overproduction and Purification of the Recombinant AnaPT
and Enzyme Assay. Protein overproduction and purification were
carried out as described previously.14 The enzyme assays (100 μL)
contained 1a−7a (1 mM), CaCl2 (5 mM), DMAPP/GPP (2 mM),
glycerol (1.0−6% v/v), dimethyl sulfoxide (DMSO, 5% v/v), 50 mM
Tris-HCl (pH 7.5), and purified recombinant protein (5−50 μg). The
reaction mixtures were incubated at 37 °C for different times and
terminated by addition of 100 μL of methanol. The protein was
removed by centrifugation at 13000 rpm for 20 min. Assays for
isolation of the enzyme products were carried out in large scale (10−
15 mL) containing aromatic substrates (1 mM), DMAPP (2 mM),
CaCl2 (5 mM), glycerol (1.0−9.9% v/v), DMSO (5% v/v), 50 mM
Tris-HCl (pH 7.5), and 5 mg of recombinant protein per 10 mL assay.
After incubation for 16 h at 37 °C, the reaction mixtures of 1a−3a
were extracted 3−4 times with a double volume of ethyl acetate. The
organic phases were combined and evaporated. The residues were
dissolved in methanol (0.5−1.0 mL) and purified by HPLC. The
reaction mixtures of 4a−7a were terminated by addition of a double
volume of methanol. Protein was removed by centrifugation at 6000
rpm for 30 min. The supernatants were collected, concentrated, and
dried on a rotary evaporator. The residues were dissolved in methanol
(0.5−1.0 mL) and after centrifugation, the supernatants were purified
on HPLC. Assays for determination of kinetic parameters (100 μL)
contained CaCl2 (5 mM), glycerol (1.0−9.9% v/v), DMSO (5% v/v),
50 mM Tris-HCl (pH 7.5), DMAPP (2 mM), 50 μg of AnaPT, and
(R)-benzodiazepinedinone or 1a−7a at final concentrations of up to
20.0 mM. For determination of the kinetic parameters of DMAPP, 1a
or 2a at a final concentration of 1 mM and DMAPP of up to 5.0 mM
were used. The reaction mixtures were incubated for 60 min ((R)-
benzodiazepinedione), 120 min (1a, 2a, 3a, 5a, DMAPP, and 6a), or
240 min (4a and 7a). Product formation was found to be linear within
such incubation times (Figure S1, Supporting Information). The
reactions were then terminated with 100 μL of methanol. Protein was
removed by centrifugation at 13000 rpm for 20 min.
Table 2. Kinetic Parameters of AnaPT toward Selected
Substrates
Vmax (nmol
KM
mg protein−1
kcat
kcat/KM
substrate
[mM]
min−1
526
)
[s−1
1.72
]
[s−1 M−1
]
(R)-
benzodiazepinedione
0.22
7818
1a
0.22
0.33
0.24
1.64
0.87
0.42
0.52
0.21
0.38
3.71
5.07
4.54
0.71
1.21
3.54
1.11
2.56
3.29
0.012
54.5
51.5
62.5
1.4
2a
0.017
3a
0.015
4a
0.0023
0.0040
0.012
5a
4.8
6a
28.6
7.1
7a
0.0037
0.0084
0.011
DMAPP with 1a
DMAPP with 2a
40.0
28.9
similar affinity to 1a−3a as to its natural aromatic substrate (R)-
benzodiazepinedinone,14 while 4a−7a showed lower affinity to
AnaPT. With 1a as aromatic substrate, a slightly higher KM
value was determined for DMAPP than in the presence of (R)-
benzodiazepinedione,14 while a significantly higher KM value
was obtained in the presence of 2a. As expected, the turnover
numbers of AnaPT for 1a−3a are very low, only 0.7−1.0% of
Analysis of Enzyme Products by HPLC and Structure
Elucidation by NMR and MS Analysis. An Agilent HPLC series
D
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Note
1200 was used for analysis and isolation of the enzyme products. A
Multospher 120 RP-18 column (250 mm × 4 mm, 5 μm C+S
Chromatographie Service, Langerwehe, Germany) was applied for
analysis at a flow rate of 1 mL/min, and a Multospher 120 RP18
column (250 mm × 10 mm, 5 μm) was used for isolation at a flow rate
of 2.5 mL/min. Water containing 0.5% trifluoroacetic acid (solvent A)
and acetonitrile containing 0.5% trifluoroacetic acid (solvent B), were
used as solvents. A linear gradient of 2−100% (v/v) solvent B in 30
min was used for analysis of the enzymatic products. The column was
then washed with 100% solvent B for 5 min and equilibrated with 2%
solvent B for another 5 min. Detection was carried out on a
photodiode array detector. Solvents for isolation of the enzyme
products are water (solvent C) and acetonitrile (solvent D) without
acid. The enzyme products were isolated with a linear gradient of 10−
100% D in C in 25 min. After each run, the column was equilibrated
with 10% solvent D for 5 min. Analysis of product from (R)-
benzodiazepinedione by HPLC was carried out as described
previously.14
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ASSOCIATED CONTENT
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* Supporting Information
Examples of polyprenylated acylphloroglucinols and their
biosynthetic origins, results of different enzyme assays as tables
or HPLC chromatograms, MS and 1H NMR data, NMR
spectra, and, kinetic parameters. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: shuming.li@staff.uni-marburg.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported in part by a grant from the
Deutsche Forschungsgemeinschaft (Li844/4-1 to S.-M. L.) We
thank Nina Zitzer and Stefan Newel for taking MS and NMR
spectra, respectively. Kang Zhou is a recipient of a scholarship
from China scholarship council.
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