Tryptophan prenyltransferases showing higher catalytic activities for Friedel-Crafts alkylation of o- and m-tyrosines than tyrosine prenyltransferases

Article abstract of DOI:10.1039/c5ob01040c

Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATS_Sv and 7-DMATS catalyse regiospecific C-prenylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation of the phenolic hydroxyl group. In this s

Full text of DOI:10.1039/c5ob01040c

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Tryptophan prenyltransferases showing higher
catalytic activities for Friedel–Crafts alkylation
of o- and m-tyrosines than tyrosine
prenyltransferases†
Cite this: DOI: 10.1039/c5ob01040c
Aili Fan,a Xiulan Xieb and Shu-Ming Li*a
Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS catalyse regiospecific C-pre-
nylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation
of the phenolic hydroxyl group. In this study, we report the Friedel–Crafts alkylation of L-o-tyrosine by
these enzymes. Surprisingly, no conversion was detected with SirD and three tryptophan prenyltrans-
ferases showed significantly higher activity than another tyrosine prenyltransferase TyrPT. C5-prenylated
L-o-tyrosine was identified as a unique product of these enzymes. Using L-m-tyrosine as the prenylation
substrate, product formation was only observed with the tryptophan prenyltransferases FgaPT2 and
7-DMATS. C4- and C6-prenylated derivatives were identified in the reaction mixture of FgaPT2. These
results provided additional evidence for the similarities and differences between these two subgroups
within the DMATS superfamily in their catalytic behaviours.
Received 22nd May 2015,
Accepted 3rd June 2015
DOI: 10.1039/c5ob01040c
www.rsc.org/obc
transferases, prenyl diphosphate synthases and aromatic pre-
nyltransferases.4,7 In the last few years, significant progress
Introduction
Prenylated secondary metabolites are widely distributed in has been achieved for aromatic prenyltransferases, especially
nature. The biological and pharmacological activities of preny- for those from the dimethylallyl tryptophan synthase (DMATS)
lated products are usually distinct from their non-prenylated superfamily.6,8,9 So far, more than 40 such prenyltransferases
precursors and therefore contribute largely to drug discovery have been identified and characterised biochemically from
and development programmes.1–3 Prenyltransferases are microorganisms, especially from fungi and bacteria.6,8,9 The
involved in the biosynthesis of these natural products and cata- majority of the DMATS superfamily uses indole derivatives
lyse the regiospecific, in most cases Friedel–Crafts alkylations including tryptophan and tryptophan-containing cyclic
by transferring prenyl moieties from different prenyl donors to dipeptides as substrates. In the presence of dimethylallyl
various acceptors. The prenyl moieties with different carbon diphosphate (DMAPP), for example, FgaPT2,10 5-DMATS,11
chain lengths (C5, C10, C15 or C20 units) can be attached in 6-DMATSSv and 7-DMATS12 from this family catalyse regio-
8
the reverse or regular pattern and further modified by cycliza- specific prenylations of L-tryptophan (1a) at C-4, C-5, C-6 and
tion, oxidation and more. Therefore, prenyltransferases play an C-7 of the indole ring, respectively (Fig. 1). FgaPT2 and
important role in the structural diversity of such products.4–6 7-DMATS from Aspergillus fumigatus are involved in the bio-
These features have drawn considerable attention from synthesis of fumigaclavine C13 and astechrome,14 respectively.
scientists in different disciplines to prenyltransferases and 6-DMATSSv is likely to be involved in the biosynthesis of 6-di-
prenylated derivatives. Based on their primary sequences, bio- methylallylindole-carbaldehyde in Streptomyces violaceusniger.8
chemical properties and structures, prenyltransferases can be A few members of the DMATS superfamily are responsible for
divided into different subgroups including protein prenyl- the prenylation of non-indole substances. For example, SirD
from Leptosphaeria maculans catalyses an O-prenylation of tyro-
sine (2a) (Fig. 1),15 the first specific step in the biosynthesis of
sirodesmin PL.16 Last year, a new tyrosine O-prenyltransferase
TyrPT (Fig. 1) was identified in Aspergillus niger, which was
demonstrated to have similar functions to SirD.17 Further
study showed that SirD and TyrPT also accepted L-tryptophan
(1a) as a substrate in vitro and catalyse the same C7-prenylation
as 7-DMATS (Fig. 1).15,17 Correspondingly, 7-DMATS catalyses
aInstitut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität
Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany.
E-mail: shuming.li@staff.uni-marburg.de
bFachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032
Marburg, Germany
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob01040c
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Fig. 1 Selected examples of prenyl transfer reactions catalysed by tryptophan and tyrosine prenyltransferases.
the same O-prenylation of tyrosine as SirD and TyrPT (Fig. 1). 2a, 5 mM CaCl2 and 1 mM DMAPP in 100 µL reaction mix-
Very recently, we demonstrated the C3-prenylation of L-tyrosine tures. With the exception of 2a with 5-DMATS and 6-DMATSSv,
(2a) by the tryptophan C4-prenyltransferase FgaPT2 (Fig. 1).18 HPLC analysis of the reaction mixtures (Fig. 2A–L) revealed the
These results demonstrated the close relationship between formation of one product each, confirming the results pub-
tryptophan C-prenyltransferases and tyrosine O-prenyltrans- lished previously.17,18,22 Product yields of these reactions are
ferases. The different regioselectivities of the mentioned summarized in Table 1. It is obvious that tryptophan prenyl-
enzymes with 2a encouraged us to test the acceptance of the transferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS
2a isomers, L-o-tyrosine (3a) and L-m-tyrosine (4a), by trypto- accepted 1a much better than 2a. Over 50% conversion yields
phan and tyrosine prenyltransferases. 3a and 4a are important were observed with 1a as the substrate by tryptophan prenyl-
amino acid analogues found in metabolic pathways of human transferases after incubation at 37 °C for 1.5 h (Table 1;
beings and have shown potential for treatments of different Fig. 2A–D). With 2a as the substrate, product formation was
diseases.19–21 Prenylations of these two compounds have not detected only in the reaction mixtures of FgaPT2 and 7-DMATS
yet been reported and their prenylated derivatives could be for tryptophan prenyltransferases, with product yields of 6.2
interesting candidates for further biological and pharmaco- 1.0 and 13.8 1.3%, respectively (Table 1). As expected, 2a was
logical investigations.
much better accepted by tyrosine than by tryptophan prenyl-
transferases. Almost total conversion of 2a was observed for
TyrPT and SirD (Table 1; Fig. 2K and L). Product yields of 1a
with TyrPT and SirD were found to be 5.2 0.40 and 5.9
1.0% (Table 1, Fig. 2), respectively.
Results and discussion
Comparison of the enzyme activities of tryptophan and
tyrosine prenyltransferases towards tryptophan (1a) and
tyrosine (2a)
Acceptance of L-o-tyrosine (3a) by all the tested tryptophan
prenyltransferases, but not by the tyrosine prenyltransferase
SirD
For better comparison of their catalytic activities, we carried
out incubation of 1a and 2a with FgaPT2, 5-DMATS,
6-DMATSSv, 7-DMATS, SirD and TyrPT under the same con- In the presence of 1 mM DMAPP, L-o-tyrosine (3a) was incu-
ditions, i.e. 2 µg enzyme for 1a and 20 µg for 2a, 1 mM 1a or bated with the six prenyltransferases (20 µg in 100 µL assay)
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Fig. 2 HPLC analysis of the reaction mixtures of 1a–4a with tryptophan and tyrosine prenyltransferases and the enzyme reactions of 3a and 4a. The
assays contained 1 mM aromatic substrate, 5 mM CaCl2, 1 mM DMAPP, and 2 (for 1a) or 20 (for 2a–4a) µg purified recombinant protein and were
incubated at 37 °C for 1.5 h. Results are illustrated for absorption at 277 nm.
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Table 1 Prenylation positions and product yields [%] of enzyme reactions
1a
2a
3a
4a
Prenylated
positiona
Product yield
[%]
Prenylated
positiona
Product
yield [%]
Prenylated
positiona
Product
yield [%]
Prenylated
positiona
Product
yield [%]
FgaPT2
5-DMATS
6-DMATSSv C-6
7-DMATS
TyrPT
C-4
C-5
51.2 4.6
62.1 2.8
63.6 2.9
52.1 1.9
5.2 0.40
5.9 1.0
C-3
—
—
O-4
O-4
O-4
6.2 1.0
≤0.5
C-5
C-5
C-5
C-5
C-5
C-5
73.6 3.6
10.9 2.5
29.9 2.5
40.2 3.8
10.8 0.7
≤0.5
C-4; C-6
33.4 1.4
≤0.5
—
—
—
—
—
≤0.5
≤0.5
C-7
C-7
C-7
13.8 1.3
100
100
4.8 1.6
≤0.5
≤0.5
SirD
a For structures see Fig. 1; “—” not determined.
Table 2 1H-NMR and 13C-NMR data of the enzyme products
(500 MHz)
mentioned in Fig. 2 at 37 °C for 1.5 h. Subsequent HPLC analy-
sis revealed product formation in five reaction mixtures
(Fig. 2M–Q). Surprisingly, 3a was accepted by all the tested
tryptophan prenyltransferases, but not by the tyrosine prenyl-
transferase SirD (Fig. 2R), although TyrPT and SirD shared
nearly the same behaviour towards the diverse substrates
tested before.17 Furthermore, the tryptophan prenyltrans-
ferases FgaPT2, 6-DMATSSv and 7-DMATS with product yields
of 73.6 3.6, 29.9 2.5 and 40.2 3.8%, respectively, showed
significantly higher activities than TyrPT with a product yield
of 10.8 0.7%, which is comparable to that of 5-DMATS. The
product peaks detected in these assays display the same reten-
tion time, indicating the formation of an identical enzyme
product by different prenyltransferases.
Pos δH, multi, J
δH, multi, J
δC
δH, multi, J
1
2
3
4
—
—
—
134.9
116.4
155.4
114.1
—
6.69, s
—
6.69, d, 2.5
—
6.76, d, 8.0
6.91, dd, 8.0, 2.0 6.64, dd, 8.0,
2.5
—
5
6
7
—
7.00, d, 8.0
—
3.42, dd, 15.0,
4.5
130.6
131.2
34.4
7.00, d, 7.5
6.67, d, 7.5
3.19, dd, 14.0,
3.0
6.99, d, 2.0
Approx. 3.30a
C5-prenylated o-tyrosine (3b) as the unique product of tyrosine
and tryptophan prenyltransferases
2.95, dd, 14.3,
8.5
3.85, dd, 8.5, 4.0 3.72, dd, 10.5,
4.5
2.80, dd, 15.0,
10.5
2.86, dd, 14.0,
9.3
3.69, m
8
55.5
To elucidate their structures, the enzyme products of 3a with
FgaPT2, 5-DMATS, 6-DMATSSv, 7-DMATS and TyrPT were iso-
lated on HPLC from 5–10 ml of incubation mixtures and sub-
jected to NMR (Table 2, Fig. S1 in the ESI†) and HR-MS
analyses (Experimental section). HR-MS data confirmed the
monoprenylation of the isolated products by detection of
molecular masses, which are 68 Da larger than that of 3a.
1H-NMR data proved that FgaPT2, 5-DMATS, 6-DMATSSv,
7-DMATS and TyrPT converted indeed 3a to the same product
9
1′
2′
—
—
172.6
30.0
123.4
—
3.21, d, 7.0
5.25, tsept, 7.5,
1.5
3.33, d, 7.0
5.19, br t, 7.0
3.24, d, 7.5
5.28, br t, 7.5
3′
4′
5′
—
—
131.3
16.6
24.4
—
1.70, d, 1.0
1.69, br s
MeOH-d4
1.73, s
1.72, s
MeOH-d4
1.70, d, 1.0
1.69, s
MeOH-d4 MeOH-d4
a Signals overlapping with those of solvent.
1
3b. Inspection of the H-NMR spectra (Fig. S1 in the ESI†) of
3b revealed the presence of signals for a regular prenyl moiety
at δH 3.21 (d, H-1′), 5.25 (tsept, H-2′), 1.70 (d, H-4′) and
1.69 ppm (br s, H-5′). Furthermore, signals for three aromatic
protons indicated that the prenylation took place on the aro-
matic ring, rather than at the hydroxyl group. The signals of
the aromatic protons at δH 6.76 (d, 8.0 Hz), 6.91 (dd, 8.0,
2.0 Hz) and 6.99 (d, 2.0 Hz) of 3b proved the prenylation at C-4
or C-5 of 3a (Table 2). For the C5-prenylated 3a, it is expected
that the signal of H-3 should be found in the high-field with a
coupling constant of approximately 8.0 Hz and the signal of
H-6 in the low-field with a coupling constant of 2.0 Hz. The
an O-prenylated derivative was identified in the reaction mix-
tures of L-tyrosine (2a) with 7-DMATS and TyrPT.17,22 However,
the C3-prenylated derivative was the enzyme product of
FgaPT2 with 2a (Fig. 1).18
Acceptance of L-m-tyrosine (4a) by two tryptophan
prenyltransferases and identification of C4- and C6-prenylated
derivatives as products of the FgaPT2 reaction
obtained data corresponded very well to this prediction and After the identification of the C5-prenylated 3a from reaction
therefore confirmed 3b to be the C5-prenylated product mixtures of tyrosine and tryptophan prenyltransferases, we
(Fig. 2Y). These results sound somewhat surprising, because tested the behaviour of these enzymes towards L-m-tyrosine
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(4a) under the same conditions as for 3a. As shown in Fig. 2S– interaction with the enzyme active sites. Furthermore, these
X, product formation was only observed in the reaction mix- results reveal that tryptophan and tyrosine prenyltransferases
tures of FgaPT2 and 7-DMATS, with product yields of 33.4
possess very likely similar active sites and strengthen their
1.4 and 4.8 1.6% (Table 1), respectively. Unexpectedly, 4a was close relationship in the evolution.
not accepted by the two tyrosine prenyltransferases SirD and
TyrPT (Fig. 2W and X). Another surprise is the much better
acceptance of 4a by FgaPT2 than by 7-DMATS (Fig. 2S and V),
which differs clearly from the preference of 2a towards these
two enzymes (Fig. 2G and J). It seems that the positions of the
hydroxyl groups at the benzene ring of phenylalanine have
critical influence on their acceptance by tryptophan and tyro-
sine prenyltransferases.
Kinetic study of the enzymatic Friedel–Crafts reactions
To get insights into the catalytic efficiency, kinetic parameters
were determined for FgaPT2, 5-DMATS, 6-DMATSSv, 7-DMATS
and TyrPT towards 3a and FgaPT2 towards 4a (Table 3;
Fig. S6–S11 in the ESI†). As expected for unnatural substrates,
KM values between 0.17 0.0076 and 0.58 0.081 mM were
determined for 3a, much higher than for their natural
substrates.8,10–12,17 The KM value of FgaPT2 towards 4a was
Inspection of the HPLC chromatograms in Fig. 2S and V
revealed the presence of two product peaks 4b and 4c with a
ratio of 1 : 2 in the reaction mixture of FgaPT2 at 10.3 and
11.4 min and only one in that of 7-DMATS at 11.4 min. The UV
maxima of 4b at 220 and 283 nm differed slightly from those
of 4c at 220 and 278 nm. For structure elucidation, 4b and 4c
were isolated on HPLC from the incubation mixture of 4a with
FgaPT2 and subjected to NMR (Table 2; Fig. S2–S5 in the ESI†)
and HR-MS analyses (Experimental section). HR-MS data con-
firmed the monoprenylation of the isolated products. Inspec-
tion of the 1H-NMR spectra (Table 2) of 4b and 4c revealed the
presence of signals for a regular prenyl moiety each at δH 3.33
or 3.24 (d, H-1′), 5.19 or 5.28 (br t H-2′), 1.73 or 1.70 (s H-4′)
and 1.72 or 1.69 ppm (s, H-5′). The signals of the aromatic
protons at 6.69 (d, 2.5 Hz, H-2), 6.64 (dd, 8.0, 2.5 Hz, H-4), and
7.00 (d, 8.0 Hz, H-5) of 4b and 6.69 (s, H-2), 7.00 (d, 7.5 Hz,
H-5), and 6.67 (d, 7.5 Hz, H-6) of 4c indicated the prenylation
at C-4 in one case and C-6 in another case. To determine the
structures of 4b and 4c, we recorded HSQC and HMBC spectra
for 4b (Fig. 3, Fig. S3 and S4 in the ESI†). The important corre-
lation between H-1′ and C-1 prove unequivocally that 4b is the
C6-prenylated derivative, i.e. prenylation at the para-position to
the phenolic hydroxyl group (Fig. 2Z). Consequently, 4c is the
C4-prenylated product (Fig. 2Z). Due to the low quality, the
structure of the enzyme product of 4a with 7-DMATS could not
be elucidated in this study. However, from its same retention
time and UV absorption maxima with those of 4c, it could be
speculated that this substance has the same structure as 4c.
Our results mentioned above suggest that the acceptance of
a substrate by tryptophan or tyrosine prenyltransferases would
not strictly depend on the chemical category of the aromatic
nucleus, but strongly rely on the position of the functional
groups on the aromatic nucleus, which perform the direct
found to be 0.90
0.013 mM, higher than that with 3a
(Table 3). In agreement with the product yields given in
Table 1, FgaPT2 showed the highest turnover number and cata-
lytic efficiency toward 3a, followed by 7-DMATS and
6-DMATSSv. TyrPT and 5-DMATS demonstrated comparable
kinetic parameters for their reactions with 3a (Table 3).
Table 3 Kinetic parameters of the tested enzymes for 3a and 4a
KM (mM)
L-o-Tyrosine (3a)
kcat (s−1
)
kcat/KM (s−1 M−1
)
FgaPT2
0.47 0.0024
0.49 0.0031
0.58 0.081
0.17 0.0076
0.58 0.014
0.29 0.029
600 58
27 0.40
59 3.6
270 8.9
24 1.7
5-DMATS
6-DMATSSv
7-DMATS
TyrPT
0.013 0.00011
0.034 0.00038
0.045 0.036
0.014 0.00066
L-m-Tyrosine (4a)
FgaPT2 0.90 0.013
0.0065 0.00044
6.8 0.093
Proposed reaction mechanisms of Friedel–Crafts alkylation
of o- and m-tyrosine
Previous studies on structures of several prenyltransferases
and mutagenesis experiments have shown that the prenyl
transfer reactions catalysed by the prenyltransferases of the
DMATS superfamily are initiated by the formation of a di-
methylallyl carbocation.23–26 Nucleophilic attack of this ion by
an electron-rich aromatic ring leads to the formation of non-
aromatic intermediates, which will be rearomatized by elimin-
ation of a proton and result in the formation of the prenylated
derivatives. A similar mechanism could also be proposed for
reactions observed in this study. As shown in Fig. 4, attack of
the dimethylallyl carbocation by C-5 of 3a would result in
the formation of the intermediate I, which would undergo
proton elimination to form the final product 3b. For the
FgaPT2 reaction with 4a, attack from two positions, C-6 and
C-4, would be possible. It can be expected that two different
intermediates II and III will be formed via routes A and B,
respectively, and then converted to 4b and 4c after proton
elimination (Fig. 4).
Fig. 3 Important HMBC correlations of 4b.
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Enzyme assays for determination of the kinetic parameters
Assays for determination of the kinetic parameters contained
CaCl2 (5 mM), glycerol (1.0–9.9% v/v), DMSO (0–5.0% v/v),
50 mM Tris HCl (pH 7.5), DMAPP (1 mM) and an aromatic
substrate at final concentrations up to 2.0 mM. The reactions
were then terminated with 100 µl MeOH. Protein was removed
by centrifugation at 17 000 rpm for 20 min.
HPLC analysis and isolation of enzyme products for structure
elucidation
The enzyme products were routinely analysed by HPLC on an
Agilent series 1200 by using a Multospher 120 RP-18 column
(250 × 4 mm, 5 µm C + S Chromatographie Service, Langer-
wehe, Germany) at a flow rate of 1 ml min−1. Water (solvent A)
and methanol (solvent B) were used as solvents for analysis
and isolation of the enzyme products. A linear gradient of
30–100% (v/v) solvent B in 20 min was used for analysis of the
enzymatic products. The column was then washed with 100%
solvent B for 5 min and equilibrated with 30% solvent B for
another 5 min. Detection was carried out on a photodiode
array detector.
For isolation of the enzyme products, the same HPLC
equipment with a Multospher 120 RP-18 column (250 ×
10 mm, 5 μm, C + S Chromatographie Service, Langerwehe,
Germany) was used. A linear gradient was run with 50–100%
(v/v) of methanol (solvent B) in water (solvent A) in 50–80 min
and a flow rate at 2.5 ml min−1. The column was then washed
with 100% (v/v) solvent B for 10 min and equilibrated with
50% (v/v) solvent B for 10 min.
Fig. 4 Proposed reaction mechanisms for the formation of 3b from 3a
as well as 4b and 4c from 4a.
Experimental section
Chemicals
DMAPP was synthesized according to the method described
for geranyl diphosphate (GPP) reported previously.27 L-o-Tyro-
sine and L-m-tyrosine used for the enzyme assays were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA) and Alfa Aesar (Karlsruhe, Germany), respectively.
Overexpression and purification of recombinant proteins
Protein overproduction and purification were carried out
for FgaPT2,10 5-DMATS,11 6-DMATSSv,8 7-DMATS,12 SirD15 and
TyrPT17 as described previously.
HR-ESI-MS and NMR analysis of the enzyme products
The obtained products were analysed by NMR (in CD3OD) and
MS analyses. NMR spectra were recorded at room temperature
on a Bruker Avance 600 MHz or a JEOL ECA-500 spectrometer.
The heteronuclear single quantum correlation (HSQC) and
heteronuclear multiple bond correlation (HMBC) spectra were
recorded with standard methods28 and processed with MestRe-
Nova.5.2.2. The positive HR-ESI-MS was carried out on an
AutoSpec instrument (Micromass Co. UK Ltd) with the follow-
ing results:
Assays for determination of enzyme activities
Reaction mixtures (100 µl) for determination of the enzyme
activities contained an aromatic substrate (1 mM), CaCl2
(5 mM), DMAPP (1 mM), glycerol (1.0–6.0% v/v), dimethyl sulf-
oxide (DMSO, 0–5.0% v/v), 50 mM Tris-HCl (pH 7.5) and a pur-
ified recombinant protein (2 or 20 µg). The reaction mixtures
were incubated at 37 °C for 1.5 h and then terminated by
the addition of 100 µl MeOH. Protein was removed by centri-
fugation at 17 000 rpm for 20 min.
3b: m/z 249.1374 [M]+ (C14H19NO3, cal.: 249.1365); 4b: m/z
249.1373 [M]+ (C14H19NO3, cal.: 249.1365); 4c: m/z 249.1348
[M]+ (C14H19NO3, cal.: 249.1365).
Enzyme assays for product isolation and structure elucidation
Assays for isolation of the enzyme products were carried out in
large scales (5–10 ml) containing an aromatic substrate
Conclusions
(1 mM), DMAPP (1.5 mM), CaCl2 (5 mM), glycerol (1.0–9.9% Prenyltransferases of the DMATS superfamily are known for
v/v), DMSO (0–5.0% v/v), 50 mM Tris-HCl (pH 7.5) and a their broad aromatic substrates.6 As shown in Fig. 2, tyrosine
recombinant protein (0.2–0.6 mg per ml assay). After incu- prenyltransferases TyrPT and SirD also accepted tryptophan
bation for 16 h at 37 °C, the reaction mixtures were terminated as the substrate, but with lower activity than with tyrosine.
by the addition of 5–10 ml methanol. After removal of the pre- Correspondingly, tryptophan prenyltransferases FgaPT2 and
cipitated protein by centrifugation at 6000 rpm for 30 min, the 7-DMATS also prenylated tyrosine, but with lower activity than
reaction mixtures were concentrated in a rotating vacuum for tryptophan. In this study, we demonstrated surprisingly
evaporator at 35 °C to a final volume of 1 ml before injection that L-o-tyrosine (3a) was accepted by all of the tested trypto-
into HPLC.
phan prenyltransferases for prenylations at C-4, C-5, C-6 and
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C-7 of the indole ring, but not by the tyrosine prenyltransferase 10 I. A. Unsöld and S.-M. Li, Microbiology, 2005, 151, 1499–
SirD. The unique product C5-prenylated o-tyrosine was identi- 1505.
fied in all the reaction mixtures of FgaPT2, 5-DMATS, 11 X. Yu, Y. Liu, X. Xie, X.-D. Zheng and S.-M. Li, J. Biol.
6-DMATSSv, 7-DMATS and TyrPT, although these enzymes Chem., 2012, 287, 1371–1380.
utilized different natural substrates and catalysed diverse 12 A. Kremer, L. Westrich and S.-M. Li, Microbiology, 2007,
regioselective reactions. 153, 3409–3416.
L-m-Tyrosine (4a) was only converted by two tryptophan prenyl- 13 S.-M. Li and I. A. Unsöld, Planta Med., 2006, 72, 1117–
transferases, but by none of the tyrosine prenyltransferases. 1120.
Structure elucidation of the enzyme products of 4a with 14 W.-B. Yin, J. A. Baccile, J. W. Bok, Y. Chen, N. P. Keller and
FgaPT2 revealed a low regioselectivity. C4- and C6-prenylated
m-tyrosines with a ratio of approximately 2 : 1 were identified
as the reaction products.
F. C. Schroeder, J. Am. Chem. Soc., 2013, 135, 2064–
2067.
15 A. Kremer and S.-M. Li, Microbiology, 2010, 156, 278–286.
Comparison studies in the future on the structure level of 16 D. M. Gardiner, A. J. Cozijnsen, L. M. Wilson, M. S. Pedras
these two groups of prenyltransferases could provide detailed and B. J. Howlett, Mol. Microbiol., 2004, 53, 1307–1318.
insights into the difference and similarity of their substrate 17 A. Fan, H. Chen, R. Wu, H. Xu and S.-M. Li, Appl. Microbiol.
and catalytic promiscuities. Meanwhile, it could even provide Biotechnol., 2014, 98, 10119–10129.
suggestions for protein engineering to create new biocatalysts 18 A. Fan, G. Zocher, E. Stec, T. Stehle and S.-M. Li, J. Biol.
in order to meet a given target, such as broader substrate
Chem., 2015, 290, 1364–1373.
spectra and/or different regioselectivity.29,30
19 G. A. Molnar, V. Nemes, Z. Biro, A. Ludany, Z. Wagner and
I. Wittmann, Free Radical Res., 2005, 39, 1359–1366.
20 G. A. Molnar, Z. Wagner, L. Marko, S. T. Ko, M. Mohas,
B. Kocsis, Z. Matus, L. Wagner, M. Tamasko, I. Mazak,
B. Laczy, J. Nagy and I. Wittmann, Kidney Int., 2005, 68,
2281–2287.
21 C. E. Humphrey, M. Furegati, K. Laumen, L. La Vecchia,
T. Leutert, J. C. D. Müller-Hartwieg and M. Vögtle, Org.
Process Res. Dev., 2007, 11, 1069–1075.
Acknowledgements
This work was supported in part by a grant from DFG (Li844/
4-1 to S.-M. Li). Aili Fan is a recipient of a scholarship from
CSC. We thank Lena Ludwig for synthesis of DMAPP, Nina
Zitzer and Stefan Newel, all from Philipps-Universität
Marburg, for recording MS and NMR spectra, respectively.
22 A. Fan and S.-M. Li, Tetrahedron Lett., 2014, 55, 5199–5202.
23 L. Y. P. Luk and M. E. Tanner, J. Am. Chem. Soc., 2009, 131,
13932–13933.
24 M. Jost, G. Zocher, S. Tarcz, M. Matuschek, X. Xie, S.-M. Li
and T. Stehle, J. Am. Chem. Soc., 2010, 132, 17849–17858.
25 U. Metzger, C. Schall, G. Zocher, I. Unsöld, E. Stec, S.-M. Li,
L. Heide and T. Stehle, Proc. Natl. Acad. Sci. U. S. A., 2009,
106, 14309–14314.
26 E. Stec, N. Steffan, A. Kremer, H. Zou, X. Zheng and
S.-M. Li, ChemBioChem, 2008, 9, 2055–2058.
27 A. B. Woodside, Z. Huang and C. D. Poulter, Org. Synth.,
1988, 66, 211–215.
28 S. Berger and S. Braun, 200 and More NMR Experiments. A
Practical Course, Wiley-VCH, Weinheim, Germany, 2004.
29 U. T. Bornscheuer and R. J. Kazlauskas, Angew. Chem., Int.
Ed., 2004, 43, 6032–6040.
30 M. Hohne and U. T. Bornscheuer, Angew. Chem., Int. Ed.,
2014, 53, 1200–1202.
References
1 S.-M. Li, Nat. Prod. Rep., 2010, 27, 57–78.
2 P. Ruiz-Sanchis, S. A. Savina, F. Albericio and M. Alvarez,
Chemistry, 2011, 17, 1388–1408.
3 C. Wallwey and S.-M. Li, Nat. Prod. Rep., 2011, 28, 496–510.
4 L. Heide, Curr. Opin. Chem. Biol., 2009, 13, 171–179.
5 K. Yazaki, K. Sasaki and Y. Tsurumaru, Phytochemistry,
2009, 70, 1739–1745.
6 X. Yu and S.-M. Li, Methods Enzymol., 2012, 516, 259–278.
7 S.-M. Li, Appl. Microbiol. Biotechnol., 2009, 84, 631–639.
8 J. Winkelblech and S.-M. Li, ChemBioChem, 2014, 15, 1030–
1039.
9 K. Miyamoto, F. Ishikawa, S. Nakamura, Y. Hayashi,
I. Nakanishi and H. Kakeya, Bioorg. Med. Chem., 2014, 22,
2517–2528.
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