Multisite prenylation of 4-substituted tryptophans by dimethylallyltryptophan synthase

Article abstract of DOI:10.1021/ja310734n

The aromatic prenyltransferase dimethylallyltryptophan synthase in Claviceps purpurea catalyzes the normal prenylation of tryptophan at C4 of the indole nucleus in the first committed step of ergot alkaloid biosynthesis. 4-Methyltryptophan is a competitive inhibitor of the enzyme that has been used in kinetic studies. Upon investigation of background activity during incubations of 4-methyltryptophan with dimethylallyl diphosphate, we found that the analogue was an alternate substrate, which gave four products. The structures of three of these compounds were established by ¹H NMR and 2D NMR studies and revealed that dimethylallyltryptophan synthase catalyzed both normal and reverse prenylation at C3 of the indole ring and normal prenylation of N1. Similarly, 4-methoxytryptophan was an alternate substrate, giving normal prenylation at C5 as the major product. 4-Aminotryptophan, another alternate substrate, gave normal prenylation at C5 and C7. The ability of dimethylallyltryptophan synthase to prenylate at five different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is consistent with a dissociative electrophilic alkylation of the indole ring, where orientation of the substrates within the active site and substituent electronic effects determine the position and type of prenylation. These results suggest a common mechanism for prenylation of tryptophan by all of the members of the structurally related dimethylallyltryptophan synthase family.

Full text of DOI:10.1021/ja310734n

Article
pubs.acs.org/JACS
Multisite Prenylation of 4‑Substituted Tryptophans by
Dimethylallyltryptophan Synthase
Jeffrey D. Rudolf,† Hong Wang,‡ and C. Dale Poulter*
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112, United States
S
* Supporting Information
ABSTRACT: The aromatic prenyltransferase dimethylallyl-
tryptophan synthase in Claviceps purpurea catalyzes the normal
prenylation of tryptophan at C4 of the indole nucleus in the
first committed step of ergot alkaloid biosynthesis. 4-
Methyltryptophan is a competitive inhibitor of the enzyme
that has been used in kinetic studies. Upon investigation of
background activity during incubations of 4-methyltryptophan
with dimethylallyl diphosphate, we found that the analogue
was an alternate substrate, which gave four products. The
structures of three of these compounds were established by 1H NMR and 2D NMR studies and revealed that
dimethylallyltryptophan synthase catalyzed both normal and reverse prenylation at C3 of the indole ring and normal
prenylation of N1. Similarly, 4-methoxytryptophan was an alternate substrate, giving normal prenylation at C5 as the major
product. 4-Aminotryptophan, another alternate substrate, gave normal prenylation at C5 and C7. The ability of
dimethylallyltryptophan synthase to prenylate at five different sites on the indole nucleus, with normal and reverse prenylation
at one of the sites, is consistent with a dissociative electrophilic alkylation of the indole ring, where orientation of the substrates
within the active site and substituent electronic effects determine the position and type of prenylation. These results suggest a
common mechanism for prenylation of tryptophan by all of the members of the structurally related dimethylallyltryptophan
synthase family.
dipeptides at positions N1, C2, C3, C4, C5, C6, or C7 by
dimethylallyl diphosphate (DMAPP) to give the different
naturally occurring carbon skeletons.4−11 The dimethylallyl
moiety can be attached at C1′ (“normal” prenylation) or C3′
(“reverse” prenylation), further increasing structural diversity.
For wild-type DMATS, both normal and reverse prenylation
are seen at N14,12 and at C2.5,13 Reverse prenylation at C3, in
either the α- or β-configuration, is catalyzed by the DMATS
AnaPT6 and CdpC3PT,7 respectively. Very recently, FtmPT1
(a normal C2 prenyltransferase) was shown to catalyze normal
prenylation at C3 of tryptophan-containing cyclic dipeptides.14
Only normal prenylation is seen at C4,8,15 C5,9 and C6.10 7-
DMATS catalyzes normal prenylation at C7 of L-tryptophan,11
and MpnD catalyzes reverse prenylation at C7 of an
indolactam.16
INTRODUCTION
■
Prenylated indole alkaloids constitute a large family of
chemically diverse biologically active natural products.1−3
Biosynthetically, the carbon skeletons of these compounds
are constructed from prenyl diphosphates and tryptophan or
derivatives of tryptophan. Well-known examples include the
ergot alkaloids, the brevianamides, epiamauromine, and
verruculogen (Figure 1). Collectively a family of enzymes, the
dimethylallyltryptophan synthases (DMATS) catalyze alkyla-
tion of the indole ring in tryptophan or tryptophan-containing
4-DMATS catalyzes the normal prenylation of tryptophan at
C4 by DMAPP as the first committed step in ergot alkaloid
biosynthesis.17 The enzyme was the first indole prenyltransfer-
ase to be purified, characterized, and later available from
expression clones and has been studied from Claviceps
purpurea8,18,19 and Aspergillus fumigatus.15,20−22 4-DMATS
(FgaPT2) from A. fumigatus consists of a 10-stranded
antiparallel β-barrel surrounded by α-helices.22 This “ABBA
PT” fold has been reported for five other prenyltransferases:
Figure 1. Indole ring numbering, possible prenylation orientations,
and representative structures of prenylated indole alkaloids.
Received: October 31, 2012
Published: January 9, 2013
© 2013 American Chemical Society
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4:1); 1H NMR (300 MHz, D2O/NaOD) δ 2.19 (s, 3H), 2.44 (dd, J =
8.1, 14.4 Hz, 1H), 2.83 (dd, J = 5.4, 14.4 Hz, 1H), 2.96 (dd, J = 5.7, 7.8
Hz, 1H), 6.38 (d, J = 7.5 Hz, 1H), 6.61 (t, J = 7.8 Hz, 1H), 6.69 (s,
1H), 6.86 (d, J = 8.1 Hz, 1H); UV λmax 219.1, 270.0, 278.4 (shoulder),
289.1 nm (shoulder); MS-ESI m/z 219.2 [M + H]+; HRMS-ESI TOF
m/z [M + H]+ calcd for C12H15N2O2, 219.1134; found, 219.1136.
4-Methoxy-L-tryptophan. 4-Methoxyindole (160 mg, 1.09
mmol) was incubated with 8 mg of tryptophan synthase for 24 h as
described above. Column chromatography yielded 120 mg (47%) of a
light-yellow solid; Rf 0.44 (ACN:H2O 4:1); 1H NMR (500 MHz,
D2O) δ 3.23 (dd, J = 8.5, 14.5 Hz 1H), 3.67 (dd, J = 4.5, 14.5 Hz, 1H),
4.01 (s, 3H), 4.14 (dd, J = 4.5, 8.5 Hz, 1H), 6.70 (d, J = 7.0 Hz, 1H),
7.17 (d, J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.22 (t, J = 7.5 Hz); 13C NMR
(125 MHz, D2O) δ 28.0, 55.5, 56.5, 100.1, 105.7, 107.7, 116.6, 123.3,
124.4, 138.3, 153.8, 174.8; UV λmax 217.9, 265.3, 279.6, 289.1 nm; MS-
ESI m/z 257.1 [M + Na]+, 273.1 [M + K]+; HRMS-ESI TOF m/z [M
+ H]+ calcd for C12H14N2O3Na, 257.0902; found, 257.0903.
4-Amino-L-tryptophan. 4-Aminoindole (160 mg, 1.21 mmol) was
incubated with 2 mg tryptophan synthase for 16 h as described above.
Column chromatography yielded 220 mg (83%) as a gray solid; Rf
0.34 (ACN:H2O 4:1); 1H NMR (500 MHz, D2O) δ 3.38 (dd, J = 8.0,
16.0 Hz, 1H), 3.62 (dd, J = 5.0, 16.0 Hz, 1H), 4.02 (dd, J = 5.0, 8.0 Hz,
1H), 6.57 (t, J = 4.5 Hz, 1H), 7.08−7.10 (m, 2H), 7.20 (s, 1H); UV
λmax 221.5, 267.7 (shoulder), 272.4, 283.1 nm (shoulder); MS-ESI m/z
220.1 [M + Na]+, 242.2 [M + Na]+, 258.1 [M + K]+; HRMS-ESI TOF
m/z [M + H]+ calcd for C11H14N3O2, 220.1086; found, 220.1078.
Production and Purification of 4-DMATS. A production strain
for soluble 4-DMATS was constructed by cotransforming E. coli BL21
Star (DE3) with pHDMAT and pGroESL36 according to the
manufacturer’s instructions. E. coli BL21 Star (DE3)/pHDMAT/
pGroESL was incubated in 1 L of LB medium containing 35 μg/mL
kanamycin and 34 μg/mL chloramphenicol at 37 °C with shaking at
225 rpm until an OD600 of 0.6 was reached. The culture was then
cooled to 20 °C, and protein expression was induced with IPTG to a
final concentration of 0.4 mM. After 18 h, cells were harvested by
centrifugation (4000g, 20 min, 4 °C). Pelleted cells were resuspended
in lysis buffer (50 mM NaH2PO4, pH 8.0, containing 300 mM NaCl,
10 mM imidazole, 1 mg/mL of lysozyme, and 10 μg/mL of RNase A).
After incubation at 0 °C for 30 min, the cell suspension was sonicated
for 6 × 10 s at 4 °C. The lysate was clarified by centrifugation (10
000g, 20 min, 4 °C). 4-DMATS was purified by nickel affinity
chromatography using a gradient elution 0−100% of lysis buffer/
elution buffer (50 mM NaH2PO4, pH 8.0, containing 300 mM NaCl,
and 500 mM imidazole). The purified protein was dialyzed against 2 ×
4 L of dialysis buffer (20 mM Tris-HCl, pH 8.0, containing 1 mM
βME, and then against 4 L of dialysis buffer containing 20% glycerol.
The protein was stored at −80 °C until use.
FtmPT123 and CdpNPT,24 normal C2 and reverse C3 indole
prenyltransferases, respectively; NphB (formerly Orf2),25
CloQ,26 and EpzP,27 nonindole aromatic prenyltransferases.
BLAST alignments with 4-DMATS from C. purpurea and other
DMATS report the following sequence identities: 56% with
FgaPT2 from A. fumigatus, 35% with FtmPT1 from A.
fumigatus, 35% with CdpC3PT from Neosartorya fischeri, 31%
with CdpNPT from A. fumigatus, 47% with 5-DMATS from A.
clavatus, 23% with IptA from Streptomyces sp. SN-593, and 28%
with 7-DMATS from A. fumigatus.28 A recent phylogenetic
analysis illustrates the relationships between aromatic and
indole prenyltransferases.10
The mechanism proposed for prenylation by DMATS is a
dissociative electrophilic alkylation of the indole ring by
DMAPP, where cleavage of the carbon−oxygen bond in
DMAPP gives a dimethylallyl cation−PPi ion pair, with
subsequent alkylation of the indole moiety, followed by loss
of a proton to give the prenylated product.19,29 In the case of 4-
DMATS, regiospecific alkylation at C4 of the indole ring by
C1′ of the allylic cation generates an arenium intermediate,
which rearomatizes by deprotonation at C4 to produce
dimethylallyltryptophan (DMAT). The crystal structure of
FgaPT2 from A. fumigatus in complex with tryptophan and
dimethylallyl S-thiolodiphosphate, a nonhydrolyzable DMAPP
analogue,30 revealed two active site amino acids thought to be
important for catalysis.22 A hydrogen bond between Glu89 and
the indole N−H likely increases the electron density in the
indole ring. Lys174 is situated near C4 and is a likely candidate
for removing the C4 proton during rearomatization. Recently,
Luk et al. reported that the K174A mutant of FgaPT2 gave a
C3 reverse-prenylated hexahydropyrroloindole as the major
product in addition to a small amount of 4-DMAT.31 The
authors suggest the initial alkylation is a reverse prenylation at
C3 of the indole ring, followed by a Cope rearrangement, a
mechanism originally proposed by Wenkert and Sliwa,32 to give
the normal C4 prenylated arenium intermediate followed by
deprotonation facilitated by Lys174 to produce 4-DMAT.
Although there is no direct experimental evidence for the Cope
rearrangement in a 3-substituted indole, Schwarzer et al.
reported a related homo-Cope rearrangement at room
temperature driven by relief of cyclopropyl ring strain.33
4-Methyltryptophan has been used as a competitive dead-end
analogue of tryptophan in kinetic studies of 4-DMATS.34 The
substitution of the hydrogen atom at the C4 by a methyl group
should block alkylation at that position. During inhibition
studies with 4-DMATS from C. purpurea, we utilized 4-methyl-
D,L-tryptophan as a competitive substrate inhibitor and found a
consistently high background of activity. We subsequently
discovered that 4-methyltryptophan is an alternate substrate for
4-DMATS, and we now report studies with the analogue that
provide insights about the mechanism of the reaction.
Kinetic Studies. All kinetic assays were performed in 50 mM Tris-
HCl buffer, pH 8.0, containing 4 mM MgCl2 and 50 μM 14C-DMAPP
(10 μCi/μmol), in a total volume of 100 μL. Each reaction was
incubated at 30 °C and quenched by heating at 100 °C for 30 s. After
centrifugation, 10 μL of reaction mixture were spotted and developed
by RP-C8 TLC using a solvent system of 4:6 25 mM NH4HCO3/
methanol. The developed TLC plates were then imaged on a storage
phosphor screen (Molecular Dynamics) and scanned by a Typhoon
8600 Variable Mode Imager (GE Healthcare), and the data were
visualized and processed with ImageQuant 5.2. Background radio-
activity was determined from incubations without the tryptophan
substrate. Each kinetic assay was performed in duplicate or triplicate.
For L-tryptophan, the assay contained 50 nM 4-DMATS and L-
tryptophan concentrations of 3, 10, 20, 30, and 60 μM, with a 5 min
incubation. For the tryptophan analogues, the assay contained 500 nM
4-DMATS with 20 min incubations. For 4-methyltryptophan, final
concentrations were 30, 100, 300, and 600 μM; for 4-methoxy-
tryptophan, 30, 100, 300, 600, 1000, and 3000 μM; and for 4-
aminotryptophan, 3, 10, 20, 30, 60, and 100 μM.
EXPERIMENTAL SECTION
■
4-Methyl-L-tryptophan. In a nitrogen atmosphere, 4-methyl-
indole (160 mg, 1.22 mmol) and L-serine (250 mg, 2.38 mmol) were
incubated at 80 °C with 7.5 mg of recombinant tryptophan synthase
from the hyperthermophilic bacterium Thermotoga maritime35 in 25
mL of buffer TS (100 mM K2HPO4, pH 7.5. containing 180 mM KCl,
120 μM PLP, and 2% DMSO) for 89 h. After incubation, the reaction
mixture was cooled to rt, and the enzyme was removed by filtration
(Millipore Centricon, 10 000 molecular weight cutoff (MWCO)). The
filtrate was concentrated in vacuo, the resulting residue was purified by
silica gel flash chromatography using a gradient of 0−20% H2O in
ACN to give 175 mg (66%) of an off-white solid; Rf 0.65 (ACN/H2O
Product Studies. Incubations were in 50 mM Tris-HCl buffer, pH
8.0, containing 10 mM MgCl2, 8.8 mM DMAPP, 20 mM tryptophan
analogue, and 15 μM 4-DMATS in a total volume of 5 mL. Glycerol
present in the 4-DMATS dialysis buffer was removed by repeated
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dialysis/centrifugation using an Amicon Ultra 10 kDa MWCO filter.
The reaction mixture was incubated at 30 °C for 18−42 h. 4-DMATS
was removed by MWCO filtration, samples were concentrated by
lyophilization, and the products were purified by HPLC on a Waters
2690 Separation Module using a Microsorb MVTM C18 5 μm column
at a flow rate of 1 mL/min with isocratic elution using ACN:H2O (25/
75). Products were detected with a Waters 996 photodiode array
detector with simultaneous monitoring at 206, 225, and 254 nm.
Product ratios were calculated by integrating the area under each
product peak visualized at 214 nm (4-methyl/4-methoxy) or 225 nm
(4-amino).
239.2, 289.1, 293.8 nm, respectively, suggesting that the indole
chromophore (λmax ≈ 278 nm) had been disrupted to give an
indoline-like structure.40 Compounds 3 (24.6 min, 7%) and 4
(26.9 min, 44%) had peaks at 223.8, 272.4 nm and 225.0, 276.0,
290.3 nm, respectively, suggesting that they contained intact
indole chromophores. Chromatography of the reaction mixture
from 4-methyl-L-tryptophan on a C18 RP-HPLC column with
an acidic solvent system (1% TFA) resulted in complete loss of
1, while the ratios for compounds 2−4 did not change (data
not shown). HRMS analysis of the four products gave
molecular ions that were 68 Da higher than the corresponding
peaks for 4-methyl-L-tryptophan or its sodium adduct,
indicating the addition of one isoprene unit.
The structures of the products were determined from their
1H and 13C NMR spectra (Table 2). The dimethylallyl moiety
in compound 1 was attached in a reverse orientation as
evidenced by the distinctive signals for the three vinyl protons
at 5.85 ppm (dd, J = 10.8, 17.4 Hz), 4.95 ppm (dd, J = 0.6, 10.2
Hz), and 4.94 ppm (dd, J = 0.6, 17.4 Hz), and two methyl
groups at 0.93 and 0.85 ppm. The HMBC spectrum showed
correlations between the methyl peaks of the prenyl moiety and
C3′ (44.7 ppm) of the isoprene unit and C3 (68.1 ppm) in the
indoline moiety. Aromatic protons at C5 (6.61 ppm), C6 (7.00
ppm), and C7 (6.48 ppm) support prenylation at C3 of the
indole ring. In addition, the peak for the proton at C2 (5.06
ppm) is near reported values for C2 diamine protons in tricyclic
C3-prenylated tryptophan derivatives.24,31 Collectively, these
data confirm that 1 is formed by a reverse prenylation at the C3
position of 4-methyltryptophan, followed by cyclization to give
a hexahydropyrroloindole (HHPI) structure.
Structure of 4-Substituted Dimethylallyltryptophan Prod-
ucts. After purification, each product was lyophilized, dissolved in
D2O (D, 99.9%), lyophilized again, dissolved in D2O (D, 99.96%)
(Cambridge Isotope Laboratories, Inc.; Andover, MA), and placed in a
Norell 3 mm NMR tube (Sigma). NMR experiments were recorded
on an INOVA 600 NMR spectrometer equipped with a HCN
cryogenic probe. Unless otherwise noted, the following NMR
experiments were performed: 1D 1H, COSY 1H−1H, TOCSY
1H−1H, ROESY 1H−1H, HSQC or HMQC 1H−13C, and HMBC
1H−13C. All spectra were processed with MestReNova 7.1.
RESULTS
■
Kinetic Studies. Michaelis−Menten kinetic parameters for
tryptophan and its 4-substituted analogues are listed in Table 1.
Table 1. Kinetic Parameters for 4-DMATS with Tryptophan
and 4-Substituted Analogues
a
kcat/KM
relative activity
(%)
KM (μM)
20
kcat (s−1
60
)
(s−1 M−1
)
TRP
5
6
3.1 × 106
8.5 × 103
5.1 × 103
100
0.28
0.17
4-CH3-TRP
89 16
0.75 0.04
1.3 0.1
4-OCH3-
TRP
250 43
Compound 2 also contains an HHPI moiety formed by
normal prenylation at C3 of 4-methyltryptophan, as confirmed
by the presence of proton signals for methylene protons at C1′
[2.34 (dd, J = 8.4, 15.0 Hz) and 2.55 ppm (dd, J = 6.6, 15.0
Hz)], the proton at C2′ (4.77 ppm, app t, J ≈ 7.2 Hz), and the
methyl groups at 1.47 and 1.46 ppm. Cross peaks were also
seen between a proton at C1′ (2.34 ppm) and C3 (61.1 ppm)
and between the proton at C2 (4.84 ppm) and C1′ (37.4 ppm)
in the HMBC spectrum. In addition, signals were seen for the
aromatic protons at C5 (6.57 ppm), C6 (6.94 ppm), and C7
(6.45 ppm). Thus, 4-DMATS catalyzes both the normal and
reverse prenylation at the C3 of 4-methyltryptophan.
4-NH2-
TRP
17
3
0.42 0.02
2.5 × 104
0.81
a
Relative activity is defined as the percent kcat/KM compared to TRP
Rates (kcat) and Michaelis constants (KM) were determined
from a nonlinear regression fit of initial velocities versus
concentration by GraFit 5.0.11. KM = 20 μM for tryptophan,
which compared favorably with the previously reported value.19
KM = 17 μM for 4-aminotryptophan was comparable to the
normal substrate, while those for the 4-methyl- and 4-methoxy
analogues, 89 and 250 μM, respectively, were substantially
higher. Values of kcat for the tryptophan analogues ranged
between 0.42 and 1.3 s−1, substantially smaller than kcat = 60 s−1
for tryptophan. The catalytic efficiencies (kcat/KM) for the
analogues were <1% of the normal reaction.
Product Studies. In preliminary work with C. purpurea 4-
DMATS (dmaW), we used racemic 4-methyltryptophan as a
substrate. However, in a study of the A. fumigatus enzyme
(FgaPT2) D-tryptophan was also a substrate, although at a rate
of only 1.8% that of the L-enantiomer.15 Thus, we decided to
use enantiomerically pure 4-methyl-L-tryptophan in our study.
The substrate was synthesized from 4-methylindole and L-
serine upon incubation with the PLP-dependent tryptophan
synthase β subunit.35 The same procedure was used to convert
4-methoxyindole and 4-aminoindole to 4-methoxy-L-trypto-
phan and 4-amino-L-tryptophan, respectively. All of the NMR
data matched previously reported data.37−39
The 1H spectrum of 4 had peaks for the protons at C1′ (4.60
ppm, d, J = 6.6 Hz) and C2′ (5.24 ppm, t, J = 6.6 Hz) and the
methyl groups at C3′ (1.70 and 1.60 ppm) for normal
prenylation. Signals for aromatic protons at C5 (6.80 ppm), C6
(7.03 ppm), and C7 (7.19 ppm) indicated that prenylation had
occurred in the pyrrole ring. The chemical shifts of C1′ (46.3
ppm) and the directly attached protons (4.60 ppm), along with
cross peaks between the C1′ protons and C2 (130.6 ppm) in
the HMBC spectrum, indicate that normal prenylation
occurred at the N1 position on the indole ring. The UV
spectrum and the resonance at 7.07 ppm for the proton at C2
indicated that the indole ring was intact. A negative ion fast
atom bombardment mass spectroscopy spectrum of fully
exchanged product D in deuterated glycerol and D2O verified
indole ring nitrogen substitution (Figure S43). Unfortunately,
we were unable to obtain sufficient quantities of 3 to establish
its structure, although as mentioned earlier, the indole ring
appeared to be intact. Co-injection experiments (Figure S44)
with 3 and two products we had previously isolated and
characterized, normal 7-dimethylallyl-4-methyl-L-tryptophan
4-Methyltryptophan. Incubation of 4-methyl-L-tryptophan
and DMAPP with 4-DMATS gave four products (see Figure
2a). The UV spectra of compounds 1 (11.4 min, 44%) and 2
(15.1 min, 5%) had peaks at 209.7, 242.8, 293.8 nm and 206.2,
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Figure 2. HPLC chromatograms of preparative enzymatic assays for isolation and purification. (a) Incubation of 4-CH3-Trp and DMAPP with 4-
DMATS, (b) incubation of 4-OCH3-Trp and DMAPP with 4-DMATS, and (c) incubation of 4-NH2-Trp and DMAPP with 4-DMATS.
and reverse N-dimethylallyl-4-methyl-L-tryptophan (unpub-
lished results) excluded these structures as candidates for 3.
4-Methoxytryptophan. As described for 4-methyltrypto-
phan, incubation of 4-methoxy-L-tryptophan with 4-DMATS
gave products 5 (11.8 min, 5%), 6 (13.3 min, 7%), 7 (17.5 min,
71%), and 8 (21.4 min, 17%) (see Figure 2b). HRMS analysis
indicated that the molecular ions of the compounds were 68 Da
higher than 4-methoxytryptophan and its sodium adduct, as
expected for monoprenylation. UV spectra for 5 and 6
suggested that the indole moiety had been disrupted, while
those for 7 and 8 indicated that the indole nucleus was intact.
The 1H spectrum of 7 had peaks for the protons at C1′ (3.35
ppm, d, J = 7.2 Hz) and C2′ (5.25 ppm, t, J = 7.2 Hz) and the
methyl groups at C3′ (1.67 and 1.60 ppm) for normal
prenylation. Signals for the proton at C2 (7.10, s) and two
other aromatic protons [(7.15 ppm, d, J = 8.4 Hz) and (6.98
ppm, d, J = 8.4 Hz)] indicated that prenylation had occurred at
either the 5 or 7 position of the indole ring. Although we were
unable to unambiguously determine the prenylation site from
this data, we previously isolated and characterized normal 7-
dimethylallyl-4-methoxy-L-tryptophan (unpublished data). A
comparison of UV spectra and NMR chemical shifts for both
compounds confirm that they are distinct products, implying 7
is prenylated at C5. Thus, 4-DMATS catalyzes a normal
prenylation at C5 of 4-methoxytryptophan.
molecular ions of the compounds were 68 Da higher than 4-
aminotryptophan as expected for monoprenylation. The UV
spectra for 9 and 10 suggested that the indole nucleus was
intact.
The dimethylallyl moiety in compound 9 was attached in a
normal orientation as evidenced by the distinctive signals for
the methylene protons at C1′ (3.24 ppm, d, J = 7.2), the proton
at C2′ (5.19 ppm, t, J = 7.2 Hz), and the methyl groups at 1.66
and 1.61 ppm. Signals for the proton at C2 (7.02, s) and two
other aromatic protons [(6.89 ppm, d, J = 8.4 Hz) and (6.87
ppm, d, J = 8.4 Hz)] indicated that prenylation had occurred at
either the 5 or 7 position of the indole ring. The ROESY
spectrum showed correlations between the methylene protons
of the prenyl moiety and the aromatic proton at C6 (6.87
ppm). The HMBC spectrum showed correlations between the
methylene protons of the prenyl moiety and C3a (120.0 ppm),
C4 (138.4 ppm), and C6 (127.0 ppm). Collectively, these data
confirm that 9 is formed by a normal prenylation at the C5
position of 4-aminotryptophan.
Compound 10 also contains normal prenylation as
confirmed by the presence of proton signals for methylene
protons at C1′ (3.35, d, J = 7.8), the proton at C2′ (5.31 ppm,
t, J = 7.8), and the methyl groups at 1.62 and 1.61 ppm. Signals
for the proton at C2 (7.05, s) and two other aromatic protons
[(6.75 ppm, d, J = 7.2 Hz) and (6.36 ppm, d, J = 7.2 Hz)]
indicated that prenylation had occurred at either the 5 or 7
position of the indole ring. The COSY spectrum showed
correlations between the methylene protons of the prenyl
moiety and the aromatic proton at C6 (6.75 ppm). The HMBC
4-Aminotryptophan. As described for 4-methyl and 4-
methoxytryptophan, incubation of 4-amino-L-tryptophan with
4-DMATS gave products 9 (10.2 min, 82%) and 10 (14.2 min,
18%) (see Figure 2c). HRMS analysis indicated that the
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a
1
Table 2. H and 13C NMR Data of Isolated Enzymatic Products Including Structures
a
δH and δC in ppm;J in Hz; n.f. = not found; and exc. = exchanged.
spectrum showed correlations between the methylene protons
of the prenyl moiety and C7 (120.3 ppm) and C7a (138.9
ppm) as well as a correlation between the aromatic proton at
C6 and C1′ (31.2 ppm). Collectively, these data confirm that
10 is formed by a normal prenylation at the C7 position of 4-
aminotryptophan. Thus, 4-DMATS catalyzes normal prenyla-
tion at C5 and C7 of 4-aminotryptophan.
DISCUSSION
■
The 4-DMATS subgroup is an important member of the
dimethylallyltryptophan synthase superfamily. These enzymes,
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including 4-DMATS from C. purpurea8 and FgaPT2 from A.
fumigatus,15 catalyze normal prenylation at C4 in the indole ring
of L-tryptophan as the first pathway specific step in the
biosynthesis of ergot alkaloids.41 Although the natural substrate
is an unsubstituted tryptophan, both 4-DMATS and FgaPT2
catalyze C4 prenylation of tryptophan analogues with
substituents on the indole ring as well as the amino acid
chain.19,20 The methyl group at C4 in 4-methyltryptophan
blocks prenylation at that site, and the compound is an
inhibitor of the normal reaction.34 During studies in our
laboratory with 4-DMATS using 4-methyltryptophan as a
competitive inhibitor against tryptophan, we detected a higher
than expected background in our assays and discovered what
appeared to be prenylation of 4-methyltryptophan.
Incubation of 4-methyltryptophan with 4-DMATS gave four
prenylation products, two monoprenylated indoline derivatives
and two monoprenylated indoles. The major product (1) was a
hexahydropyrroloindole reverse prenylated at C3, accompanied
by a smaller amount of the normal prenylated isomer (2), both
of which eluted before the putative indoles. The more abundant
indole isomer (4) was normally prenylated at N1. Unfortu-
nately, we were unable to obtain a sufficient quantity of 3, the
less abundant isomer for NMR spectroscopy. Co-injection of 3
with reverse N-dimethylallyl-4-methyl-L-tryptophan and normal
7-dimethylallyl-4-methyl-L-tryptophan excluded both of these
structures.
both normal and reverse prenylation are seen at C3 for 4-
methyltryptophan. Alkylation of the indole ring at sites
separated by several angstroms and by normal and reverse
alkylation of C3 indicates that the enzyme can bind DMAPP in
different conformations. The high regioselectivity for C4
alkylation with the normal substrates suggests that binding
and catalysis of a single conformation have been optimized for
synthesis of 4-dimethylallyltryptophan, but when alkylation at
C4 is blocked, alkylation from other conformations becomes
competitive.
All of the substitutents introduced at C4 are electron-
donating relative to hydrogen. For the methyl substituent (σ+ =
0.31),42 alkylation is mostly at C3, giving normal and reverse
prenylated indolines, along with a smaller amount of the N1
alkylated indole. The methoxy group (σ+ = 0.78) directs
alkylation predominantly to C5 giving only trace amounts of
putative indolines. The amino group (σ+ = 1.3) directs
alkylation exclusively to C5 and C7. Thus, when a relatively
weak electron-donating substituent blocks C4, the dimethylallyl
cation alkylates C3 to generate normal and reverse prenylated
iminium intermediates, which cyclize to the corresponding
indolines. The more powerful electron-donating methoxy and
amino groups direct alkylation to the ortho (C5) and para (C7)
carbons, positions further activated by N1. Interestingly, the
amino group in 4-aminotryptophan is not alkylated.
Incubation with 4-methoxytryptophan also gave four
products. The elution and UV profiles for compounds 5−8
were similar to those of the prenylated 4-methyltryptophan
derivatives. Normally prenylated 5-dimethylallyl-4-methoxy-
tryptophan, whose elution profile was similar to 3 was the
major product and the only compound to be obtained in
sufficient quantity to be fully characterized. Given the similar
HPLC retention times and UV profiles for the four products
from 4-methyl- and 4-methoxytryptophan, it is likely that 5 and
6 are indolines normal and reverse prenylated at C3 and that 8
is a normal prenylated indole, most likely at N1 or C7. 4-
DMATS also catalyzes the normal prenylation of 4-amino-
tryptophan at the C5 and C7 positions of the indole nucleus.
Thus, 4-DMATS catalyzes normal or reverse prenylation of
tryptophan and C4-substituted analogues at five different sites
on the indole ring, N1, C3, C4, C5, and C7, depending on the
substituent (Figures 3 and 4).
Our results provide strong evidence for a dissociative
electrophilic alkylation of the indole nucleus by C1′ and in
one instance C3′ of the dimethylallyl cation generated by
heterolysis of DMAPP. 4-Dimethylallyltryptophan is the only
product of the normal reaction catalyzed by 4-DMATS. When
alkylation at C4 is blocked, the enzyme no longer catalyzes a
regioselective reaction but instead produces a variety of
products from alkylation at the nucleophilic positions (N1,
C3, C5, and C7) on the indole ring by the electrophilic
dimethylallyl cation. In addition, we saw only a 3-fold variation
in kcat for the methyl, methoxy, and amino tryptophans, which
is most readily explained by formation of the dimethylallyl
cation as the rate-limiting step, followed by alkylation of the
indole nucleus. These results are consistent with previous
studies with 7-methyl and 7-methoxy derivatives of tryptophan.
In this case, tryptophan and the 7-substituted analogues are
alkylated at C4 with only a 2-fold variation in kcat.19
Recently, Luk and co-workers reported that the K174A
mutant of A. fumigatus 4-DMATS catalyzes reverse prenylation
of C3.31 A crystal structure of the enzyme and kinetic studies
indicate that K174 is an important base, which facilitates
removal of the C4 hydrogen during the electrophilic aromatic
substitution reaction. They suggested that the reaction
catalyzed by the wild-type enzyme proceeds in two steps:
reverse alkylation at C3 followed by a Cope rearrangement and
deprotonation to form the normal C4 prenylation product, as
originally proposed by Wenkert and Sliwa.32 The multiplicity of
products we observe when C4 is blocked, including normal and
reverse alkylation at C3, do not support the Cope mechanism.
While a Cope rearrangement is consistent with reverse
alkylation at C3 and normal alkylation at N1, it does not
account for the concurrent normal prenylation at both C5 and
C7. In addition, the presence of normal alkylation at C3 and
the absence of a reverse N1 prenylation product suggest
rearrangement from C3 to N1 is unlikely.
Figure 3. Sites for alkylation of 4-methyltryptophan by 4-DMATS.
The indole ring in tryptophan is susceptible to electrophilic
alkylation at N1; at C2, C5, and C7 which are activated by
conjugation with N1 through the benzene ring; and at C4 and
C6, which are activated by conjugation with N1 through the
C2-C3 double bond and the benzene ring. When C4 is blocked,
4-DMATS catalyzes alkylation at all of the activated positions,
except weakly activated C6, depending on the electron-
donating properties of the blocking substituent. In addition,
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Figure 4. Characterized and proposed (bracketed) products from incubation of tryptophan analogues and DMAPP with 4-DMATS.
‡Division of Endocrinology, Metabolism and Diabetes,
University of Colorado Denver Anschutz Medical Campus,
Aurora, Colorado 80045.
CONCLUSION
■
In conclusion, our work with 4-substituted analogues of
tryptophan demonstrates that 4-DMATS has the innate ability
to alkylate the indole ring at five different positions and by
normal and reverse addition. The other reaction channels,
which were revealed by blocking the normal alkylation at C4,
represent five of the seven sites for alkylation of the indole
nucleus by the DMATS superfamily and provide a logical
rationale for evolution of members of the superfamily from a
common ancestor.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Reinhard Sterner and Jonathan Johnston for
supplying the tmTrpB and pHDMAT vectors, respectively. We
thank Dr. James Muller for performing mass spectroscopy and
Dr. Jack Skalicky for assistance with cryoprobe NMR. This
work was supported by NIH grant GM 21328.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental; preparation of L-tryptophan analogues
using tryptophan synthase (Scheme S1); NMR spectra of 4-
substituted analogues (Figures S1−S4); NMR data tables
(Tables S1−S6) and NMR spectra of prenylated products
(Figures S5−S40); prenylation product structures with
pertinent 2D NMR correlations (Figure S41); HRMS data of
prenylated products (Table S7); negative ion FABMS of
compound 4 (Figures S42−S43); HPLC chromatograms of
coinjection assay (Figure S44); radioTLCs and corresponding
Michaelis−Menten curves of enzyme kinetics (Figures S45−
S48). This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
poulter@chem.utah.edu
■
Present Addresses
†Department of Chemistry, The Scripps Research Institute,
Scripps Florida, Jupiter, Florida 33458.
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