Tyrosine O -prenyltransferase SirD catalyzes S -, C -, and N -prenylations on tyrosine and tryptophan derivatives

Article abstract of DOI:10.1021/cb400691z

The tyrosine O-prenyltransferase SirD in Leptosphaeria maculans catalyzes normal prenylation of the hydroxyl group in tyrosine as the first committed step in the biosynthesis of the phytotoxin sirodesmin PL. SirD also catalyzes normal N-prenylation of 4-aminophenylalanine and normal C-prenylation at C7 of tryptophan. In this study, we found that 4-mercaptophenylalanine and several derivatives of tryptophan are also substrates for prenylation by dimethylallyl diphosphate. Incubation of SirD with 4-mercaptophenylalanine gave normal S-prenylated mercaptophenylalanine. We found that incubation of the enzyme with tryptophan gave reverse prenylation at N1 in addition to the previously reported normal prenylation at C7. 4-Methyltryptophan also gave normal prenylation at C7 and reverse prenylation at N1, whereas 4-methoxytryptophan gave normal and reverse prenylation at C7, and 7-methyltryptophan gave normal prenylation at C6 and reverse prenylation at N1. The ability of SirD to prenylate at three different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is similar to behavior seen for dimethylallyltryptophan synthase. The multiple products produced by SirD suggests it and dimethylallyltryptophan synthase use a dissociative electrophilic mechanism for alkylation of amino acid substrates.

Full text of DOI:10.1021/cb400691z

Articles
pubs.acs.org/acschemicalbiology
Tyrosine O‑Prenyltransferase SirD Catalyzes S‑, C‑, and
N‑Prenylations on Tyrosine and Tryptophan Derivatives
Jeffrey D. Rudolf† 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 tyrosine O-prenyltransferase SirD in Leptos-
phaeria maculans catalyzes normal prenylation of the hydroxyl
group in tyrosine as the first committed step in the biosynthesis of
the phytotoxin sirodesmin PL. SirD also catalyzes normal N-
prenylation of 4-aminophenylalanine and normal C-prenylation at
C7 of tryptophan. In this study, we found that 4-mercaptophe-
nylalanine and several derivatives of tryptophan are also substrates
for prenylation by dimethylallyl diphosphate. Incubation of SirD
with 4-mercaptophenylalanine gave normal S-prenylated mercap-
tophenylalanine. We found that incubation of the enzyme with
tryptophan gave reverse prenylation at N1 in addition to the
previously reported normal prenylation at C7. 4-Methyltryptophan also gave normal prenylation at C7 and reverse prenylation at
N1, whereas 4-methoxytryptophan gave normal and reverse prenylation at C7, and 7-methyltryptophan gave normal prenylation
at C6 and reverse prenylation at N1. The ability of SirD to prenylate at three different sites on the indole nucleus, with normal
and reverse prenylation at one of the sites, is similar to behavior seen for dimethylallyltryptophan synthase. The multiple
products produced by SirD suggests it and dimethylallyltryptophan synthase use a dissociative electrophilic mechanism for
alkylation of amino acid substrates.
hytopathogenic fungi produce toxic secondary metabolites
that provide selective advantages against other micro-
P
organisms. Epipolythiodioxopiperazines (ETPs) and prenylated
indole alkaloids constitute two classes of chemically diverse
biologically active natural products.1−4 Biosynthetically, the
carbon skeletons of many of these compounds are constructed
from prenyl diphosphates and tyrosine, tryptophan, or
derivatives of tryptophan. Well-known examples of ETPs
include gliotoxin and sirodesmin PL; examples of prenylated
indole alkaloids include the ergot alkaloids, the asterriquinones,
and the nodulisporic acids (Figure 1).
A family of enzymes has been identified that catalyzes
alkylation of the indole ring in tryptophan by an isoprenyl
diphosphate, typically dimethylallyl diphosphate (DMAPP).5−8
The isoprenyl moiety can be attached at C1′ (“normal”
prenylation) or C3′ (“reverse” prenylation), further increasing
structural diversity. These dimethylallyltryptophan synthases
(DMATS) catalyze alkylation of the indole ring in tryptophan
or tryptophan-containing dipeptides at positions N1, C2, C3,
C4, C5, C6, or C7 to give a variety of naturally occurring
prenylated indoles.9−16 Both normal and reverse prenylation is
seen at N19,17 and at C2.10,18 Reverse prenylation is seen at C3,
giving products with an α (AnaPT)11 or a β (CdpC3PT)12
configuration. Recently, FtmPT1 (a normal C2 prenyltransfer-
ase) was reported to also catalyze normal prenylation at C3 of
tryptophan-containing cyclic dipeptides.19 Only normal pre-
nylation is found at C4,13,18 C5,14 and C6.15 7-DMATS
catalyzes normal prenylation at C7 of L-tryptophan,16 whereas
MpnD catalyzes reverse prenylation at C7 of an indolactam.20
Figure 1. Indole ring numbering, possible prenylation orientations,
and representative structures of ETPs and prenylated indole alkaloids.
4-DMATS catalyzes the normal prenylation of tryptophan at
C4 as the first committed step in ergot alkaloid biosynthesis21
and has been extensively studied from Claviceps purpurea13,22,23
and Aspergillus fumigatus.18,24−27 4-DMATS (FgaPT2) from A.
fumigatus consists of a 10-stranded antiparallel β-barrel
surrounded by α-helices.26 This “ABBA” prenyltransferase
Received: September 8, 2013
Accepted: October 1, 2013
Published: October 1, 2013
© 2013 American Chemical Society
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(PT) fold has been reported for five other enzymes. FtmPT128
and CdpNPT29 catalyze normal C2 and reverse C3 alkylation
of indole, respectively, whereas NphB (formerly Orf2),30
CloQ,31 and EpzP,32 alkylate naphthalene, benzene, and
phenazine rings, respectively.
Table 1. Kinetic Parameters for SirD with Tyrosine,
Tryptophan, and Their Analogues
kcat/Km
(kcat/Km)Rel
Km (mM)
kcat (s‑1)
(s‑1 M‑1)
(%)
Tyr
0.30 0.04
1.1 0.2
1.3 0.1
4.3 × 103
100
The mechanism proposed for prenylation by DMATSs is a
dissociative electrophilic alkylation of the indole ring by the
dimethylallyl cation generated by heterolytic cleavage of the C−
O bond in DMAPP, followed by loss of a proton to give the
prenylated product.22,33 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 give dimethylallyltryptophan (DMAT).
Relaxed selectivity for the aromatic substrate is reported for
several of the DMATSs. 7-DMATS prenylated both enan-
tiomers of tryptophan, tryptophan-containing linear and cyclic
dipeptides, and a variety of tryptophan derivatives, although no
activity was observed when 7-DMATS was incubated with
tyrosine.16,34 CTrpPT from Aspergillus oryzae DSM1147 was
shown to simultaneously prenylate two different sites on the
indole ring of tryptophan-containing cyclic dipeptides with
normal prenylation at C7 and reverse prenylation at N1 of the
indole nucleus.17 We recently reported that 4-DMATS
prenylates several 4-substituted tryptophans to give products
with a normal prenylation at N1, C3, C5, or C7 or a reverse
prenylation at C3.35
An aromatic amino acid prenyltransferase from the
pathogenic fungus Leptosphaeria maculans, dimethylallyltyro-
sine synthase (SirD), was recently reported.36 The enzyme
likely has an ABBA PT fold and catalyzes normal O-prenylation
of the hydroxyl group in tyrosine as the first pathway specific
step in the biosynthesis of sirodesmin PL.37 SirD also catalyzes
normal N-prenylation of the aromatic amino group in 4-
aminophenylalanine and normal C-prenylation of C7 of the
indole ring in L-tryptophan.37,38 We now report a much broader
substrate selectivity for SirD, which includes normal prenylation
of the sulfhydryl group in 4-mercaptophenylalanine and normal
prenylation of C6 and C7 and reverse prenylation of N1 and
C7 of the indole ring in tryptophan derivatives. These results
are consistent with a dissociative electrophilic alkylation similar
to that reported for 4-DMATS.
4-SH-
Phe
(2.7 0.2) × 10−2
25
0.58
Trp
0.37 0.06
5.1 2.0
(9.5 0.2) × 10−2
(1.3 0.3) × 10−2
2.6 × 102
5.8
4-OCH3-
Trp
2.5
0.06
7-CH3-
Trp
2.0 0.4
(1.3 0.1) × 10−2
6.5
0.15
determined from a nonlinear fit of initial velocities versus
concentration using GraFit 5.0.11. The values for tyrosine, Km
= 0.30 mM and kcat = 1.3 s−1, compared well with the
previously reported values.37,38 Km = 1.1 mM and kcat = 2.7 ×
10−2 s−1 for 4-mercaptophenylalanine. The catalytic efficiency
(kcat/Km) for alkylation of 1 was <1% of the normal reaction.
The ∼4-fold increase in Km and ∼50-fold decrease in kcat of 4-
mercaptophenylalanine indicates that the replacement of the
side chain oxygen with sulfur decreases substrate binding and
reduces the rate of reaction. Since one would have expected
that a sulfur nucleophile would facilitate capture of the putative
dimethylallyl cation relative to oxygen, the decrease in kcat
might result from a conformation for 1 and DMAPP in the
active site that reduces the rate of C−O bond cleavage in
DMAPP.
Km and kcat for tryptophan are comparable with the
previously reported values.37 Km’s are somewhat higher and
kcat’s substantially lower for the 7-methyl and 4-methoxy
analogues, respectively, with catalytic efficiencies (kcat/Km) for
the 7-methyl- and 4-methoxytryptophan analogues that were
<1% of the normal reaction. Maximal velocity was not reached
at concentrations before 4-methyltryptophan precipitated (10
mM) under our assay conditions, and values for the kinetic
constants were not determined.
Product Studies. 4-Mercaptophenylalanine. Incubation
of 1 and DMAPP with SirD gave a single product (6) (Figure
2a). The UV spectrum of 6 (10.3 min) had a peak at 257.0 nm.
Monoprenylation was confirmed by HRMS where the sodium
adduct of dimethylallyl mercaptophenylalanine gave a peak at
m/z of 288.1041 Da. The structure of 6 was established using a
RESULTS AND DISCUSSION
■
1
Synthesis of Tyrosine and Tryptophan Analogues.
The symmetric disulfide of 4-mercapto-L-phenylalanine (1) was
prepared by the palladium-catalyzed cross-coupling of N-t-Boc-
4-iodo-L-phenylalanine and tert-butylthiol,39 followed by
simultaneous removal of the Boc and S-tert-butyl protecting
groups with concentrated HCl.40 Deprotection under oxidative
conditions gave the disulfide, which was purified and stored.
Immediately before an incubation, the disulfide was treated
with β-mercaptoethanol to give 1. 4-Vinyl-L-phenylalanine (2)
was synthesized according to the procedure of Yao and co-
workers,41 except for changes in the protecting groups. Fmoc
was used instead of Cbz, and a methyl ester instead of a benzyl
ester in order to avoid deprotection by catalytic hydrogenation.
Enantiomerically pure 4-methyl-L-tryptophan (3), 4-methoxy-L-
tryptophan (4), and 7-methyl-L-tryptophan (5) were synthe-
sized using the PLP-dependent tryptophan synthase β
subunit,42 as previously described.35
combination of H and 2D NMR spectroscopy (Table 2).
Normal prenylation was confirmed by the presence of signals
for the methylene protons at C1′ (3.55 ppm, d, J = 7.8 Hz), the
proton at C2′ (5.24 ppm, t, J = 7.2 Hz), and the methyl groups
at C3′ (1.66 and 1.58 ppm). An AB quartet for the aromatic
protons at 7.14 and 7.19 ppm, each integrating to two protons,
indicated that the benzene ring was para-substituted. Thus,
prenylation occurred at the sulfur atom. The HMBC spectrum
revealed correlations between the C1′ dimethylallyl methylene
protons and C2′ (119.2 ppm), C3′ (135.5 ppm), and the para
carbon C4 (133.1 ppm). These data demonstrate that SirD
catalyzes a normal S-prenylation of 4-mercaptophenylalanine.
4-Vinylphenylalanine. Incubation of up to 10 mM 2 and
DMAPP with SirD yielded no identifiable products (data not
shown). We surmise that either the vinyl moiety is too large to
be accommodated in the active site or 2 is not properly
positioned for prenylation.
Kinetic Studies. Michaelis−Menten kinetic parameters for
tyrosine, tryptophan, and their substituted analogues are listed
in Table 1. Rates (kcat) and Michaelis constants (Km) were
Tryptophan. Kremer and Li reported that SirD catalyzes the
normal prenylation of tryptophan at C7.37 In our hands,
incubation of L-tryptophan and DMAPP with SirD gave two
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Figure 2. HPLC chromatograms of preparative enzymatic assays for isolation and purification. Each aromatic amino acid was incubated with
DMAPP and SirD. (a) 4-Mercaptophenylalanine, (b) tryptophan, (c) 4-methyltryptophan, (d) 4-methoxytryptophan, and (e) 7-methyltryptophan.
products (Figure 2b). The UV spectra of compounds 7 (12.7
min, 7%) and 8 (15.3 min, 93%) had peaks at 222.7, 284.3 nm
and 221.5, 277.2, 297.9 nm, respectively, suggesting the indole
chromophores of compounds 7 and 8 were intact. HMRS
analysis revealed that the molecular ions of both compounds
were 68 Da higher than tryptophan, or its sodium adduct,
indicating the addition of one isoprene unit.
Compound 7 has a dimethylallyl moiety attached to the
indole ring through C3′ as evidenced by signals for the three
vinyl protons at 6.03 ppm (dd, J = 10.8, 17.4 Hz), 5.11 ppm (d,
J = 10.8 Hz), and 5.02 ppm (d, J = 17.4 Hz) and the methyl
groups at 1.62 ppm. Although the NMR sample was too dilute
to collect relevant TOCSY, HMQC, or HMBC data, the
ROESY spectrum showed a correlation between the proton at
C2 (7.33 ppm, s) and the methyl groups of the prenyl moiety.
Aromatic protons at C2, C4 (7.60 ppm), C5 (7.05 ppm), C6
(7.09 ppm), and C7 (7.54 ppm) suggested that the
dimethylallyl moiety was attached at the indole nitrogen.
Collectively, these data indicate that 7 is the product of reverse
prenylation of tryptophan at N1. Thus, SirD catalyzes normal
prenylation at C7 and reverse prenylation at N1 of the indole
1H NMR analysis (Table 2) of compound 8 revealed a
spectrum very similar to the data reported for 7-dimethylallyl-
tryptophan.16 The HMBC spectrum showed a correlation
between the methylene protons at C1′ (3.47 ppm, d, J = 7.2
Hz) and C7 of the indole ring (128.1 ppm). Together, these
data establish that 8 is normal 7-dimethylallyltryptophan.
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a
1
Table 2. H and 13C NMR Data for Products 6−14
a
δH and δC in ppm. J in Hz. n.f. = not found. exc. = exchanged.
nucleus of tryptophan, similar to the cyclo-L-Trp-L-Trp
dimethylallyltransferase from A. oryzae DSM1147.17
4-Methyltryptophan. Zou and co-workers38 reported
activity for prenylation of 4-methyl-DL-tryptophan at C7 by
SirD at a rate ∼8% of L-tyrosine. Similar to our results for
tryptophan, we found that incubation of 3 and DMAPP with
SirD gave two products (Figure 2c). HMRS and UV analysis of
compounds 9 (18.8 min, 79%) and 10 (24.6 min, 21%)
revealed that they are monoprenylated derivatives of 4-
methyltryptophan.
Compound 10 was identified as 7-dimethylallyl-4-methyl-
tryptophan by NMR analysis. H signals were seen for the
1
methylene protons at C1′ (3.47−3.51 ppm, m), the proton at
C2′ (5.33 ppm, t, J = 6.0 Hz), and the methyl groups at 1.61
and 1.63 ppm of the dimethylallyl group (Table 2). Signals for
protons attached to C2 (7.14 ppm, s), C5 (6.75 ppm, d, J = 7.2
Hz), and C6 (6.86 ppm, d, J = 7.2 Hz), along with HMBC
correlations between the methylene protons at C1′ and C7 in
the indole ring (125.6 ppm), confirmed prenylation at C7.
We suspect that Li and co-workers were unable to see the N-
prenylated products due to their use of HPLC solvents
containing 0.5% trifluoroacetic acid (TFA).37,38 The product
from A. fumigatus CdpNPT was originally assigned incorrectly
due to disassociation and rearrangement of the dimethylallyl
moiety at N1 of the indole ring upon exposure to trichloro-
acetic acid.43,44 We found that products seen by HPLC analysis
of samples from incubation of tryptophan or 3, DMAPP, and
SirD disappeared when the elution solvent contained 0.5% TFA
(data not shown).
1
The H NMR spectrum of 9 showed three vinyl signals at
6.01 ppm (dd, J = 10.8, 18.0 Hz), 5.09 ppm (d, J = 10.8 Hz),
and 5.00 ppm (d, J = 17.4 Hz) for the protons at C2′ and C1′,
respectively, and methyl groups at 1.61 and 1.60 ppm, all
characteristic of a reverse prenylated structure. The ROESY
spectrum displayed a correlation between the methyl groups of
the prenyl moiety and the proton at C2 (7.30 ppm). The
presence of signals at C2, C4 (6.79 ppm), C5 (6.96 ppm), and
C6 (7.38 ppm) suggested that prenylation occurred at N1.
Thus, compound 9 is reverse N1-(dimethylallyl)-4-methyl-
tryptophan.
4-Methoxytryptophan. Incubation of 4 and DMAPP with
SirD gave compounds 11 (16.1 min, 8%) and 12 (20.8 min,
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Figure 3. Products from incubation of tyrosine or tryptophan analogues and DMAPP with SirD. Structures for compounds with asterisks were
determined previously.37,38
92%) (Figure 2d). HRMS analysis indicated that the molecular
ions were 68 Da higher than the sodium adduct of 4, as
expected for monoprenylation. UV spectra for 11 and 12
suggested that the indole nuclei were intact.
NMR analysis (Table 2) confirmed that compound 13 is
reverse N1-(dimethylallyl)-7-methyltryptophan. The orienta-
tion of the dimethylallyl unit is clear from the 1H NMR
spectrum of 13, where signals were seen for the three vinyl
protons at 6.30 ppm (dd, J = 10.8, 17.4 Hz), 4.94 ppm (d, J =
10.8 Hz), and 4.49 ppm (d, J = 17.4 Hz) and the methyl groups
at 1.64 ppm (s). The TOCSY spectrum showed correlations
between the dimethylallyl methyl groups and the C2 indole
proton at 7.43 ppm. Signals for protons at C2, C4 (7.46 ppm),
C5 (7.00 ppm), and C6 (6.96 ppm) confirm prenylation of the
indole nitrogen.
The dimethylallyl moiety in compound 11 was attached in a
reverse orientation as evidenced by distinctive signals for the
vinyl protons at 6.00 ppm (dd, J = 10.8, 17.4 Hz), 5.03 ppm
(dd, J = 10.8 Hz), and 4.98 ppm (dd, J = 17.4 Hz) and two
methyl groups at 1.35 ppm. The HMBC spectrum showed
correlations between the methyl peaks of the prenyl moiety and
C7 (127.8 ppm). The ROESY spectrum showed correlations
between the methyl peaks of the prenyl moiety and the
aromatic proton at C6 (7.06 ppm) and between the methoxy
substituent at C4 (57.8 ppm) and the proton at C5 (6.55 ppm).
Together, these data confirm that 11 is formed by reverse
prenylation at C7 of 4-methoxytryptophan.
Compound 12 contained a dimethylallyl moiety attached in a
normal orientation as indicated by signals for the methylene
protons at C1′ (3.35−3.37 ppm, m), the proton at C2′ (5.30
ppm, t, J = 6.6 Hz), and the methyl groups at 1.60 and 1.59
ppm. Signals for the proton at C2 (7.00 ppm, s) and two other
aromatic protons (6.84 ppm, d, J = 7.8 Hz and 6.47 ppm, d, J =
7.8 Hz) showed that prenylation had occurred at either C5 or
C7. The ROESY spectrum showed a weak correlation between
the methylene protons of the prenyl moiety and the aromatic
proton at C6 (6.84 ppm). The HMBC spectrum showed
correlations between the methylene protons of the prenyl
moiety and C7 (121.5 ppm). Collectively, these data indicate
that 12 is formed by normal prenylation at C7 position of 4-
methoxytryptophan.
The 1H NMR spectrum of 14 had peaks for the dimethylallyl
group at C1′ (3.46 ppm, d, J = 7.2 Hz) and C2′ (5.32 ppm, d, J
= 7.2 Hz) and the methyl groups at C3′ (1.77 and 1.69 ppm)
indicative of normal prenylation. Resonances for the proton at
C2 (7.23 ppm, s) and aromatic protons at 7.48 ppm (d, J = 7.8
Hz) and 7.03 ppm (d, J = 7.8 Hz) indicated that prenylation
occurred at C4 or C6 in the indole ring. The HMBC spectrum
showed a correlation between the methyl group at C7 (2.42
ppm) and C3′ (135.8 ppm). Although we were not able to
unambiguously distinguish between prenylation at C4 and C6,
1
a comparison of our H NMR spectrum with those published
for 4-dimethylallyl-7-methyltryptophan22 and 7-dimethylallyl-6-
methyltryptophan15 suggests 14 is prenylated at C6. The
reported resonances for C5 (6.99 ppm) and C6 (6.89 ppm) in
4-dimethylallyl-7-methyltryptophan are only 0.1 ppm apart,
while those for C4 (7.41 ppm) and C5 (6.86 ppm) in 7-
dimethylallyl-6-methyltryptophan are separated by 0.55 ppm.
The separation in 14 is 0.45 ppm. Collectively, these data
suggest that SirD catalyzes normal prenylation at C6 in 7-
methyltryptophan.
7-Methyltryptophan. Zou and co-workers reported that 7-
methyl-DL-tryptophan was not a substrate of SirD (relative yield
<0.03%).38 On the basis of our results with 3 and 4, we
anticipated that 5 might be a substrate for regioselective
prenylation at N1. Interestingly, incubation of 5 and DMAPP
with SirD gave two products, 13 (16.3 min, 13%) and 14 (19.6
min, 87%) (Figure 2e). HRMS and UV spectra indicated that
the products were monoprenylated with intact indole nuclei.
Promiscuity and Mechanism. SirD catalyzes normal
prenylation of the hydroxyl group in tyrosine as the first
committed step in the biosynthesis of the phytotoxin
sirodesmin PL. The protein is a presumed member of the
ABBA-fold family of aromatic prenyltransferases, whose other
members catalyze alkylation of benzene,31 naphthalene,30 and
phenazine32 rings and the indole moiety in tryptophan.26,28,29
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The most studied member of the family, 4-dimethylallyltrypto-
phan synthase (4-DMATS), gives a single product when
incubated with its normal substrates. However, alternate
substrates for tryptophan with methyl, methoxy, or amino
substituents at C4 give different mixtures of products, which
collectively are formed by normal alkylation at N1, C3, C5, and
C7 and reverse alkylation at C3.35 This behavior can be
explained by a dissociative electrophilic alkylation of tryptophan
by the dimethylallyl cation generated by cleavage of the C−O
bond in DMAPP, where blocking the preferred alkylation at C4
results in electrophilic attack of other nucleophilic sites in the
indole moiety. The regiopromiscuity seen for 4-DMATS
provides a logical path for evolution of other dimethylallyl-
tryptophan synthases that are regioselective for alkylation of the
indole nucleus at positions other than C4.
As part of a study to explore structure and function in the
ABBA aromatic prenyltransferases, we examined the ability of
SirD to catalyze alkylation of a variety of tyrosine and
tryptophan analogues. In addition to its normal role in the
synthesis of dimethylallyltyrosine, SirD accepts 4-amino-
phenylalanine,38 4-mercaptophenylalanine, tryptophan,37 4-
methyltryptophan,38 4-methoxytryptophan, and 7-methyltryp-
tophan as alternate substrates to generate the products shown
in Figure 3. Given the diverse group of aromatic amino acid
derivatives that are alkylated by SirD, we were surprised that 4-
vinylphenylalanine was not a substrate, even at high
concentrations, where alkylation of the vinyl group would be
mechanistically similar to alkylation of the vinyl group in
protoheme IX by the farnesyltransferase encoded by cyoE in E.
coli.45 We surmise that the bound conformations of 4-
vinylphenylalanine and DMAPP are incompatible with catalysis.
A comparison of the regiochemistry for alkylation of the
indole moiety in tryptophan by SirD and 4-DMATS (see
Figure 4) emphasizes the regiopromiscuity of both enzymes. All
alkylate tryptophan at all of the nucleophilic sites in the indole
ring.
Finally, the electronic properties of the substituents on the
indole ring appear to influence the regiochemistry of the
alkylation. Incubation of 4-methyltryptophan (σ+methyl = 0.31)46
and DMAPP with SirD gives normal prenylation at C7 and
reverse prenylation at N1. In contrast, 4-methoxytryptophan
(σ+
= 0.78)46 gives normal and reverse prenylation
methoxy
exclusively at C7. This observation is consistent with an
enhancement in the nucleophilicity of C7 relative to N1 by the
strongly electron donating methoxy group.
Conclusion. SirD catalyzes aromatic S-prenylation of 4-
mercaptophenylalanine and N- and C-prenylations of trypto-
phan and tryptophan analogues at three different sites on the
indole ring (N1, C6, C7): normal prenylation at C6, normal
and reverse prenylation at C7, and reverse prenylation at N1.
Our results provide further evidence for a dissociative
electrophilic alkylation by ABBA aromatic prenyltransferases,
where substrate orientation within the active site and
substituent electronic effects determines the position and type
of prenylation.
METHODS
■
Synthesis of Amino Acid Analogues. Procedures for syntheses
of 4-mercapto-L-phenylalanine (1), 4-vinyl-L-phenylalanine (2), 4-
methyl-L-tryptophan (3), 4-methoxy-L-tryptophan (4), and 7-methyl-
L-tryptophan (5) are described in the Supporting Information.
Production and Purification of SirD. L. maculans sirD (NCBI
GenBank AY553235.1) was optimized for heterologous expression in
E. coli and synthesized by Genscript (Supplementary Figure S2).
Modified L. maculans sirD in pUC57 was cloned into pET21a(+)
following standard restriction digestion protocols (see Supporting
Information) to give pLmSirD. pLmSirD was transformed into E. coli
BL21 (DE3) according to the manufacturer’s instructions. E. coli strain
pLmSirD-BL21 (DE3) was incubated in 1 L of LB medium containing
50 μg mL−1 ampicillin at 37 °C with shaking at 225 rpm until an
OD600 of 0.6 was reached. The culture was induced with IPTG to a
final concentration of 0.6 mM. After 5 h, cells were harvested by
centrifugation (4,000 × g, 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−1 of lysozyme, and 10 μg mL−1
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 (10,000 × g, 20 min, 4 °C). SirD 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% v/v
glycerol. The protein was stored at −80 °C until use.
Figure 4. Regiochemistry for alkylation of the indole nucleus by SirD
and 4-DMATS.
Kinetic Studies. All kinetic assays were performed in a total
volume of 100 μL at 30 °C for 10 min unless otherwise noted and
quenched by heating at 100 °C for 30 s. After centrifugation, a 10 μL
portion of the reaction mixture was spotted and developed by RP-
TLC. For tyrosine assays, RP-C18 plates were developed with 25:75
H2O/methanol. For all other substrates, RP-C8 plates were developed
with 4:6 25 mM NH4HCO3/methanol. The developed TLC plates
were imaged on a storage phosphor screen (Molecular Dynamics) and
scanned by a Typhoon 8600 Variable Mode Imager (GE Healthcare).
The data were visualized and processed with ImageQuant 5.2.
Background radioactivity was determined from incubations without
the aromatic substrate. Each kinetic assay was performed in triplicate
or quadruplicate. [1-14C]DMAPP and [14C(U)]-L-tyrosine were
purchased from American Radiolabeled Chemicals, Inc.
of the SirD products are formed by alkylation of sites on the
“southern” (N1, C6, C7) edge of the indole nucleus, as are two
of the 4-DMATS products (N1, C7). Four additional 4-
DMATS products are from alkylation of the “northern” (C3,
C4, C5) edge. Both enzymes catalyze normal and reverse
alkylations. The catalytic efficiencies of the alternative
substrates for both enzymes are remarkably high, with values
of kcat/Km ∼10−3 relative to those of the normal substrates for
the least efficient analogues. Thus, the ABBA aromatic
prenyltransferases appear to have remarkably plastic active
sites. Proteins with this fold have evolved to give a family of
biosynthetic enzymes that collectively have the ability to
For L-tyrosine, the assay was conducted in 20 mM Tris-HCl buffer,
pH 8.0, containing 80 nM SirD, 1 mM DMAPP, and [14C(U)]-L-
tyrosine concentrations of 0.04, 0.05, 0.07, 0.08, 0.1, 0.15, 0.2, 0.25,
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Articles
0.3, 0.35, and 0.4 mM (0.25−25 μCi μmol−1) with 10 min incubations.
For 4-mercaptophenylalanine, the assay was conducted in 100 mM
Tris-HCl buffer, pH 8.0, containing 10 mM βME, 2% v/v glycerol, 5
μM SirD, 1 mM [1-14C]DMAPP (1 μCi μmol−1), and 4-mercapto-L-
phenylalanine concentrations of 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, and 10
mM with 30 min incubations. For tryptophan, the assay was
conducted in 100 mM Tris-HCl buffer, pH 8.0, containing 2% v/v
glycerol, 5 μM SirD, 1 mM [1-14C]DMAPP (1 μCi μmol−1), and L-
tryptophan concentrations of 0.1, 0.2, 0.3, 0.6, and 1 mM with 20 min
incubations. For 4-methoxy and 7-methyltryptophan, concentrations
were 0.3, 0.6, 1, 2, 3, 4, 6, and 10 mM with 30 min incubations.
Product Studies. Incubations with 4-mercaptophenylalanine were
conducted in 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM
MgCl2, 20 mM βME, 5 mM DMAPP, 10 mM compound 1, and 14.5
μM SirD in a total volume of 5 mL. Glycerol present in the SirD
dialysis buffer was removed by repeated dialysis/centrifugation using
an Amicon Ultra 10 kDa molecular weight cutoff (MWCO) filter. The
reaction mixture was incubated at 30 °C for 21 h. SirD 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 MV C18 5 μm column at a flow rate of 1
mL min−1 with isocratic elution using acetonitrile/H2O (25:75).
Products were detected with a Waters 996 photodiode array detector
with simultaneous monitoring at 254 nm.
ACKNOWLEDGMENTS
■
We thank R. Sterner for supplying the tmTrpB vector. We
thank J. Muller and J. Skalicky for performing mass spectros-
copy and NMR assistance, respectively. This work was
supported by NIH grant GM 21328.
REFERENCES
■
(1) Gardiner, D. M., Waring, P., and Howlett, B. J. (2005) The
epipolythiodioxopiperazine (ETP) class of fungal toxins: Distribution,
mode of action, functions and biosynthesis. Microbiology (Reading, U.
K.) 151, 1021−1032.
(2) Li, S.-M. (2010) Prenylated indole derivatives from fungi:
Structure diversity, biological activities, biosynthesis and chemo-
enzymic synthesis. Nat. Prod. Rep. 27, 57−78.
(3) Hulvova, H., Galuszka, P., Frebortova, J., and Frebort, I. (2012)
Parasitic fungus Claviceps as a source for biotechnological production
of ergot alkaloids. Biotechnol. Adv. 31, 79−89.
(4) Wallwey, C., and Li, S.-M. (2011) Ergot alkaloids: structure
diversity, biosynthetic gene clusters and functional proof of
biosynthetic genes. Nat. Prod. Rep. 28, 496−510.
(5) Heide, L. (2009) Prenyl transfer to aromatic substrates: genetics
and enzymology. Curr. Opin. Chem. Biol. 13, 171−179.
Incubations with tryptophan and tryptophan derivatives were
identical to those above except as follows: 10 mM MgCl2, 2.92 mM
DMAPP, 20 mM tryptophan analogue, 5 μM SirD in a total volume of
5 mL. The reaction mixtures were incubated for 21−42 h, and the
products were detected at 225 nm. Product ratios were calculated by
integrating the area under each product peak visualized at 220 nm (4-
methoxy) or 225 nm (tryptophan/4-methyl/7-methyl).
(6) Tello, M., Kuzuyama, T., Heide, L., Noel, J. P., and Richard, S. B.
(2008) The ABBA family of aromatic prenyltransferases: broadening
natural product diversity. Cell. Mol. Life Sci. 65, 1459−1463.
(7) Saleh, O., Haagen, Y., Seeger, K., and Heide, L. (2009) Prenyl
transfer to aromatic substrates in the biosynthesis of aminocoumarins,
meroterpenoids and phenazines: The ABBA prenyltransferase family.
Phytochemistry (Elsevier) 70, 1728−1738.
Determination of Structures. Purified products were lyophilized,
dissolved in D2O (D, 99.9%), lyophilized again, dissolved in DMSO-d6
(D, 99.96%) or D2O (D, 99.96%) (Cambridge Isotope Laboratories,
Inc.), and placed in a Norell 3-mm NMR tube (Sigma). NMR spectra
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.
(8) Li, S.-M. (2009) Evolution of aromatic prenyltransferases in the
biosynthesis of indole derivatives. Phytochemistry 70, 1746−1757.
(9) Grundmann, A., Kuznetsova, T., Afiyatullov, S. S., and Li, S.-M.
(2008) FtmPT2, an N-prenyltransferase from Aspergillus fumigatus,
catalyses the last step in the biosynthesis of fumitremorgin B.
ChemBioChem 9, 2059−2063.
(10) Grundmann, A., and Li, S.-M. (2005) Overproduction,
purification and characterization of FtmPT1, a brevianamide F
prenyltransferase from Aspergillus fumigatus. Microbiology (Reading,
U. K.) 151, 2199−2207.
(11) Yin, W.-B., Grundmann, A., Cheng, J., and Li, S.-M. (2009)
Acetylaszonalenin biosynthesis in Neosartorya fischeri: Identification
of the biosynthetic gene cluster by genomic mining and functional
proof of the genes by biochemical investigation. J. Biol. Chem. 284,
100−109.
(12) Yin, W.-B., Yu, X., Xie, X.-L., and Li, S.-M. (2010) Preparation
of pyrrolo[2,3-b]indoles carrying a β-configured reverse C3-
dimethylallyl moiety by using a recombinant prenyltransferase
CdpC3PT. Org. Biomol. Chem. 8, 2430−2438.
(13) Gebler, J. C., and Poulter, C. D. (1992) Purification and
characterization of dimethylallyl tryptophan synthase from Claviceps
purpurea. Arch. Biochem. Biophys. 296, 308−313.
(14) Yu, X., Liu, Y., Xie, X., Zheng, X.-D., and Li, S.-M. (2012)
Biochemical characterization of indole prenyltransferases: Filling the
last gap of prenylation positions by a 5-dimethylallyltryptophan
synthase from Aspergillus clavatus. J. Biol. Chem. 287, 1371−1380.
(15) Takahashi, S., Takagi, H., Toyoda, A., Uramoto, M., Nogawa, T.,
Ueki, M., Sakaki, Y., and Osada, H. (2010) Biochemical character-
ization of a novel indole prenyltransferase from Streptomyces sp. SN-
593. J. Bacteriol. 192, 2839−2851.
(16) Kremer, A., Westrich, L., and Li, S.-M. (2007) A 7-
dimethylallyltryptophan synthase from Aspergillus fumigatus: over-
production, purification and biochemical characterization. Microbiology
(Reading, U. K.) 153, 3409−3416.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional methods; bacterial strains, plasmids and culture
conditions; overproduction and purification of tryptophan
synthase; synthesis of DMAPP and amino acid analogues;
synthetic schemes for preparation of amino acid analogues
(Schemes S1−S3); NMR spectra of aromatic substrate
analogues and precursors (Figures S3−S18); NMR spectra of
prenylated tyrosine and tryptophan products (Figures S19−
S71); structures of products with pertinent 2D NMR
correlations (Figure S72); HRMS data for products (Table
S1); Michaelis−Menten analysis of kinetic data (Figures S73).
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: poulter@chem.utah.edu.
Present Address
†Department of Chemistry, The Scripps Research Institute,
Scripps Florida, Jupiter, Florida 33458.
(17) Zou, H.-X., Xie, X.-L., Linne, U., Zheng, X.-D., and Li, S.-M.
(2010) Simultaneous C7- and N1-prenylation of cyclo-L-Trp-L-Trp
catalyzed by a prenyltransferase from Aspergillus oryzae. Org. Biomol.
Chem. 8, 3037−3044.
Notes
The authors declare no competing financial interest.
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Articles
(18) Unsoeld, I. A., and Li, S.-M. (2005) Overproduction,
purification and characterization of FgaPT2, a dimethylallyltryptophan
synthase from Aspergillus fumigatus. Microbiology (Reading, U. K.) 151,
1499−1505.
of the plant pathogenic fungus Leptosphaeria maculans. Mol. Microbiol.
53, 1307−1318.
(37) Kremer, A., and Li, S.-M. (2010) A tyrosine O-prenyltransferase
catalyses the first pathway-specific step in the biosynthesis of
sirodesmin PL. Microbiology (Reading, U. K.) 156, 278−286.
(38) Zou, H.-X., Xie, X., Zheng, X.-D., and Li, S.-M. (2011) The
tyrosine O-prenyltransferase SirD catalyzes O-, N-, and C-
prenylations. Appl. Microbiol. Biotechnol. 89, 1443−1451.
(39) Rajagopalan, S., Radke, G., Evans, M., and Tomich, J. M. (1996)
Synthesis of N-t-Boc-4-S-t-butyl-L-thiophenylalanine via palladium
catalyzed cross-coupling reaction of N-t-Boc-4-iodo-L-phenylalanine
with t-butylthiol or sodium t-butylthiolate. Synth. Commun. 26, 1431−
1440.
(19) Wollinsky, B., Ludwig, L., Xie, X., and Li, S. M. (2012) Breaking
the regioselectivity of indole prenyltransferases: identification of. Org.
Biomol. Chem. 10, 9262−9270.
(20) Ma, J., Zuo, D., Song, Y., Wang, B., Huang, H., Yao, Y., Li, W.,
Zhang, S., Zhang, C., and Ju, J. (2012) Characterization of a single
gene cluster responsible for methylpendolmycin and pendolmycin
biosynthesis in the deep sea bacterium Marinactinospora thermotoler-
ans. ChemBioChem 13, 547−552.
(21) Lee, S.-L., Floss, H. G., and Heinstein, P. (1976) Purification
and properties of dimethylallylpyrophosphate:tryptophan dimethylallyl
transferase, the first enzyme of ergot alkaloid biosynthesis in Claviceps.
sp. SD 58. Arch. Biochem. Biophys. 177, 84−94.
(22) Gebler, J. C., Woodside, A. B., and Poulter, C. D. (1992)
Dimethylallyltryptophan synthase. An enzyme-catalyzed electrophilic
aromatic substitution. J. Am. Chem. Soc. 114, 7354−7360.
(23) Tsai, H.-F., Wang, H., Gebler, J. C., Poulter, C. D., and Schardl,
C. L. (1995) The Claviceps purpurea gene encoding dimethylallyl-
tryptophan synthase, the committed step for ergot alkaloid biosyn-
thesis. Biochem. Biophys. Res. Commun. 216, 119−125.
(24) Steffan, N., Unsoeld, I. A., and Li, S.-M. (2007) Chemo-
enzymatic synthesis of prenylated indole derivatives by using a 4-
dimethylallyltryptophan synthase from Aspergillus fumigatus. Chem-
BioChem 8, 1298−1307.
(25) Luk, L. Y. P., and Tanner, M. E. (2009) Mechanism of
dimethylallyltryptophan synthase: Evidence for a dimethylallyl cation
intermediate in an aromatic prenyltransferase reaction. J. Am. Chem.
Soc. 131, 13932−13933.
(26) Metzger, U., Schall, C., Zocher, G., Unsoeld, I., Stec, E., Li, S.-
M., Heide, L., and Stehle, T. (2009) The structure of dimethylallyl
tryptophan synthase reveals a common architecture of aromatic
prenyltransferases in fungi and bacteria. Proc. Natl. Acad. Sci. U. S. A.
106, 14309−14314.
(40) Clavier, S., Rist, O., Hansen, S., Gerlach, L.-O., Hoegberg, T.,
and Bergman, J. (2003) Preparation and evaluation of sulfur-
containing metal chelators. Org. Biomol. Chem. 1, 4248−4253.
(41) Yao, Z.-J., Gao, Y., and Burke, T. R. (1999) Preparation of L-Nα-
Fmoc-4-[di-(tert-butyl)-phosphonomethyl]phenylalanine from L-tyro-
sine. Tetrahedron: Asymmetry 10, 3727−3734.
(42) Hettwer, S., and Sterner, R. (2002) A novel tryptophan synthase
β-subunit from the hyperthermophile Thermotoga maritima. J. Biol.
Chem. 277, 8194−8201.
(43) Yin, W.-B., Ruan, H.-L., Westrich, L., Grundmann, A., and Li, S.-
M. (2007) CdpNPT, an N-prenyltransferase from Aspergillus
fumigatus: overproduction, purification and biochemical character-
ization. ChemBioChem 8, 1154−1161.
(44) Ruan, H.-L., Yin, W.-B., Wu, J.-Z., and Li, S.-M. (2008)
Reinvestigation of a cyclic dipeptide N-prenyltransferase reveals
rearrangement of N-1 prenylated indole derivatives. ChemBioChem 9,
1044−1047.
(45) Saiki, K., Mogi, T., Ogura, K., and Anraku, Y. (1993) In vitro
heme O synthesis by the cyoE gene product from Escherichia coli. J.
Biol. Chem. 268, 26041−26044.
(46) Brown, H. C., and Okamoto, Y. (1958) Derective effects in
aromatic substitution. XXX. Electrophilic substituent constants. J. Am.
Chem. Soc. 80, 4979−4987.
(27) Luk, L. Y. P., Qian, Q., and Tanner, M. E. (2011) A Cope
rearrangement in the reaction catalyzed by dimethylallyltryptophan
synthase? J. Am. Chem. Soc. 133, 12342−12345.
(28) Jost, M., Zocher, G., Tarcz, S., Matuschek, M., Xie, X., Li, S.-M.,
and Stehle, T. (2010) Structure−function analysis of an enzymatic
prenyl transfer reaction identifies a reaction chamber with modifiable
specificity. J. Am. Chem. Soc. 132, 17849−17858.
(29) Schuller, J. M., Zocher, G., Liebhold, M., Xie, X., Stahl, M., Li,
S.-M., and Stehle, T. (2012) Structure and catalytic mechanism of a
cyclic dipeptide prenyltransferase with broad substrate promiscuity. J.
Mol. Biol. 422, 87−99.
(30) Kuzuyama, T., Noel, J. P., and Richard, S. B. (2005) Structural
basis for the promiscuous biosynthetic prenylation of aromatic natural
products. Nature (London, U. K.) 435, 983−987.
(31) Metzger, U., Keller, S., Stevenson, C. E. M., Heide, L., and
Lawson, D. M. (2010) Structure and mechanism of the magnesium-
independent aromatic prenyltransferase CloQ from the clorobiocin
biosynthetic pathway. J. Mol. Biol. 404, 611−626.
(32) Zocher, G., Saleh, O., Heim Joel, B., Herbst Dominik, A., Heide,
L., and Stehle, T. (2012) Structure-based engineering increased the
catalytic turnover rate of a novel phenazine prenyltransferase. PLoS
One 7, e48427.
(33) Qian, Q., Schultz, A. W., Moore, B. S., and Tanner, M. E. (2012)
Mechanistic studies on CymD: A tryptophan reverse N-prenyltransfer-
ase. Biochemistry 51, 7733−7739.
(34) Kremer, A., and Li, S.-M. (2008) Potential of a 7-
dimethylallyltryptophan synthase as a tool for production of
prenylated indole derivatives. Appl. Microbiol. Biotechnol. 79, 951−961.
(35) Rudolf, J. D., Wang, H., and Poulter, C. D. (2013) Multisite
prenylation of 4-substituted tryptophans by dimethylallyltryptophan
synthase. J. Am. Chem. Soc. 135, 1895−1902.
(36) Gardiner, D. M., Cozijnsen, A. J., Wilson, L. M., Pedras, M. S.
C., and Howlett, B. J. (2004) The sirodesmin biosynthetic gene cluster
2714
dx.doi.org/10.1021/cb400691z | ACS Chem. Biol. 2013, 8, 2707−2714