Biochemical and Mechanistic Characterization of the Fungal Reverse N-1-Dimethylallyltryptophan Synthase DMATS1Ff

Article abstract of DOI:10.1021/acschembio.9b00828

Dimethylallyltryptophan synthases catalyze the regiospecific transfer of (oligo)prenylpyrophosphates to aromatic substrates like tryptophan derivatives. These reactions are key steps in many biosynthetic pathways of fungal and bacterial secondary metabolites. In vitro investigations on recombinant DMATS1_Ff from Fusarium fujikuroi identified the enzyme as the first selective reverse tryptophan-N-1 prenyltransferase of fungal origin. The enzyme was also able to catalyze the reverse N-geranylation of tryptophan. DMATS1_Ff was shown to be phylogenetically related to fungal tyrosine O-prenyltransferases and fungal 7-DMATS. Like these enzymes, DMATS1_Ff was able to convert tyrosine into its regularly O-prenylated derivative. Investigation of the binding sites of DMATS1_Ff by homology modeling and comparison to the crystal structure of 4-DMATS FgaPT2 showed an almost identical site for DMAPP binding but different residues for tryptophan coordination. Several putative active site residues were verified by site directed mutagenesis of DMATS1_Ff. Homology models of the phylogenetically related enzymes showed similar tryptophan binding residues, pointing to a unified substrate binding orientation of tryptophan and DMAPP, which is distinct from that in FgaPT2. Isotopic labeling experiments showed the reaction catalyzed by DMATS1_Ff to be nonstereospecific. Based on these data, a detailed mechanism for DMATS1_Ff catalysis is proposed.

Full text of DOI:10.1021/acschembio.9b00828

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Article
Biochemical and Mechanistic Characterization of the fungal
Ff
reverse N-1-Dimethylallyltryptophan Synthase DMATS1
Immo Burkhardt, Zhongfeng Ye, Slavica Janevska, Bettina Tudzynski, and Jeroen S. Dickschat
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00828 • Publication Date (Web): 22 Nov 2019
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Biochemical and Mechanistic Characterization of the fungal reverse N-
-Dimethylallyltryptophan Synthase DMATS1
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#
,‡
#,‡
§
§
#,
Immo Burkhardt, Zhongfeng Ye, Slavica Janevska, Bettina Tudzynski, and Jeroen S. Dickschat *
#
5
Kekulé Institut für Organische Chemie und Biochemie, Rheinische Friedrich Wilhelms-Universität Bonn, Gerhard-Domagk-Str. 1,
3121 Bonn, Germany
§
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48143 Münster,
Germany.
ABSTRACT: Dimethylallyltryptophan synthases catalyze the regiospecific transfer of (oligo)-prenylpyrophosphates to aromatic
substrates like tryptophan derivatives. These reactions are key steps in many biosynthetic pathways of fungal and bacterial secondary
metabolites. In vitro investigations on recombinant DMATS1Ff from Fusarium fujikuroi identified the enzyme as the first selective
reverse tryptophan-N-1 prenyltransferase of fungal origin. The enzyme was also able to catalyze the reverse N-geranylation of
tryptophan. DMATS1Ff was shown to be phylogenetically related to fungal tyrosine O-prenyltransferases and fungal 7-DMATS. Like
these enzymes, DMATS1Ff was able to convert tyrosine into its regularly O-prenylated derivative. Investigation of the binding sites
of DMATS1Ff by homology modeling and comparison to the crystal structure of 4-DMATS FgaPT2 showed an almost identical site
for DMAPP binding, but different residues for tryptophan coordination. Several putative active site residues were verified by site
directed mutagenesis of DMATS1Ff. Homology models of the phylogenetically related enzymes showed similar tryptophan binding
residues, pointing to a unified substrate binding orientation of tryptophan and DMAPP, which is distinct from that in FgaPT2. Isotopic
labeling experiments showed the reaction catalyzed by DMATS1Ff to be non-stereospecific. Based on these data, a detailed
mechanism for DMATS1Ff catalysis is proposed.
Prenylations are ubiquitous biochemical modifications that
significantly influence the overall polarity of biomolecules and
thus their activity and availability within a living cell. Examples
The general reaction mechanism of DMATS is thought to
follow an S 1 reaction with generation of an isoprenyl cation
N
and subsequent attack of the nucleophile, but although several
DMATS are characterized to date, the mechanistic details
including the origin of regio- and stereoselectivity as well as
substrate selectivity of the particular enzymes is still not
1
2
are post-translational prenylations of proteins and peptides as
well as prenylation steps in the biosynthesis of important
3
primary metabolites like ubiquinones. Prenylations also occur
1
0,15,16
in biosyntheses of microbial secondary metabolites, where they
produce intermediates that allow for structural diversification
towards a plethora of bioactive alkaloids or non-ribosomal
completely understood.
O
OH
O
O
OH
NH2
OH
NH2
4
,5
peptides.
These reactions are usually carried out by
4
5
NH2
6
3a
3
prenyltransferases belonging to the ABBA superfamily. The
common overall fold of this superfamily consists of five
5
6
1
3
2
PPO
N
4
7
a H 1
2
N
N
repeating  structural elements, which result in a barrel of
7
H
7,8
1
0 antiparallel -sheets that harbors the active site. These
1
2
3
4
enzymes catalyze the regioselective prenylation of aromatic
substrates in a Friedel-Crafts alkylation. Many of them utilize
Figure 1. Carbon numbering of the indole nucleus in tryptophan (1) and of
DMAPP (2). The structures 3 and 4 are examples for regular C-4- and
reverse N-1-prenylations.
9
tryptophan (Trp, 1) or tryptophan derivatives and dimethylallyl
pyrophosphate (DMAPP, 2) as substrates and thus make up the
1
0
group of dimethylallyltryptophan synthases (DMATS).
Despite the fact that no alkaloids or non-ribosomal peptides that
contain prenylated tryptophan are known from Fusarium spp.,
five putative DMATS-coding genes were identified in genome
sequenced members of the Fusarium fujikuroi species
Enzymes for the regioselective prenylation with DMAPP are
known for every available (non-bridgehead) position of the
1
1
electron rich aromatic indole system (Figure 1). Further,
prenylation of the indole ring can proceed by nucleophilic
1
7,18
complex.
Two of the putative enzymes, DMATS1 and
attack at C-1 or C-3 of DMAPP, resulting in “regular” or
DMATS3, are coded on the genome of the rice pathogen F.
fujikuroi IMI58289. Genomic analysis showed DMATS1 to
10
18
“
reverse” prenylation (Figure 1). Regularly prenylated 3 is the
first intermediate in the biosynthesis of ergot alkaloids in
Aspergillus spp. and Claviceps spp., while reversely N-1-
be part of a putative gene cluster, but only the in vivo
overexpression of dmats1 itself showed an impact on the
metabolome, i.e. the occurrence of 4 and derivatives in extracts
12
prenylated 4 is known as a building block for the NRPS derived
1
3
19
antibiotics ilamycin from Streptomyces atratus and the
from mutant cultures. Although the natural function of
1
4
cyclomarins from Salinispora arenicola. Although most
DMATS members are known from filamentous fungi, 4 is a
DMATS1 stayed unclear, these data suggested that it catalyzes
reverse N-1-prenylation on 1. Since this modification on free
tryptophan was unkown from fungal biochemistry before, we
1
1
building block only occurring in bacterial natural products.
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now report on the in vitro characterization of DMATS1 from F. structure of the product was unambiguously elucidated by 1-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fujikuroi (from now on called DMATS1Ff), including the
biochemical characterization, substrate scope screening, as well
as mechanistic investigations based on structural data from
homology modeling, site directed mutagenesis and isotopic
labeling experiments.
and 2-dimensional NMR spectroscopy as the novel compound
10, showing an unusual reverse N-geranylation of the indole
substructure (Scheme 1, Table S4). The geranylation of
tryptophan was only reported for two 6-DMATS from
Streptomyces violaceusniger and Streptomyces ambofaciens
2
8
that both produced 6-geranyl tryptophan. Fungal DMATS
usually do not accept GPP instead of DMAPP, but lately
RESULTS AND DISCUSSION
2
9
enzyme variants of the 4-DMATS FgaPT2 and several
Cloning, heterologous expression and in vitro activity of
DMATS1Ff. The gene coding for DMATS1Ff was obtained
3
0
diketopiperazine (DKP) converting DMATS obtained by site
directed mutagenesis were shown to produce geranylated
products. The only occurrence of reverse N-geranylation on the
indole nucleus was reported as one of four products from the
incubation of a F335G mutant of CdpC3PT with cyclo-Trp-
1
9
from the gDNA of F. fujikuroi IMI58289 and cloned into the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0
expression vector pYE-express. The expression construct was
transformed into Escherichia coli BL21 (DE3). Standard
expression conditions and purification via Ni-NTA affinity-
chromatography yielded soluble hexahis-DMATS1Ff (Figure
S1). In vitro incubations with L-tryptophan and DMAPP in Tris-
buffer at pH 7.5 at room temperature yielded one new
compound. A large-scale incubation, product purification by
RP18-column chromatography and NMR-analysis confirmed
that DMATS1Ff selectively catalyzes the production of 4 (Table
S1) that was identified by comparison of NMR-spectroscopic
3
0
Val. Thus, the selective enzymatic reverse N-geranylation on
an indole moiety as catalyzed by DMATS1Ff is unprecedented.
O
O
O
O
OH
NH2
OH
NH2
PPO
2
DMATS1Ff
N
2
1,22
N
H
data to those of a synthetic standard (Figure S2).
Biochemical characterization and substrate scope. The
optimum in vitro parameters were examined by comparison of
the substrate conversion within 5 min reaction time. The
temperature optimum was found at 35 °C (Figure S3). The pH
values were tested in a range from 6 to 9 and showed no clear
maximum, as the enzyme reached similar conversion rates at
pH values between 7.0 and 9.0 (Figure S4). DMATS1Ff showed
1
4
O
OH
NH2
OH
NH2
PPO
2
DMATS1Ff
N
N
H
ent-1
2
+
no dependency on divalent metal cations, but addition of Mg
ent-4
2
+
and Ca led to slightly enhanced reaction rates. This behavior
O
OH
NH2
1
2,23,24
was also reported for other DMATS.
Addition of different
OH
NH2
transition-metal cations inhibited the reactivity (Figure S5).
Since studies on other DMATS showed substrate
PPO
2
HO
HO
2
5,26,27
promiscuity,
the substrate scope of DMATS1Ff was
DMATS1Ff
N
N
surveyed by testing different trypthophan derivatives and
structurally related compounds (Figure S6). For substrates
missing either the free amino group or the free carboxylic acid
group no product formation was observed. Histidin and N-1-
methyl tryptophan as alternative amino acids were also not
converted. Selective conversion was shown for the alternative
substrates D-tryptophan (ent-1), 5-hydroxy-L-tryptophan (5)
and L-tyrosine (7) (Figures S7-10). The observed conversion of
ent-1 was low and the product was identified by HPLC
comparison to 4, showing the same retention time on a non-
chiral column (Figures S7 and S8). For substrates 5 and 7 a
better conversion was observed and large scale incubations
were conducted. The products were purified by RP18-column
chromatography and their structures were determined by one
and two-dimensional NMR analysis as 6 and 8, respectively
(Tables S2 and S3). While tryptophan derived substrates were
reversely prenylated at the N-1 position of the indole system,
tyrosine (7) was selectively converted into the regularly O-
prenylated compound 8.
H
5
7
6
O
O
PPO
2
OH
OH
NH2
NH2
DMATS1Ff
O
HO
8
O
O
OH
OH
H
PPO
2
NH2
NH2
9
DMATS1Ff
N
N
H
1
10
Scheme 1. In vitro reactions of DMATS1Ff with different substrates.
The kinetic parameters of DMATS1Ff for the different identified
substrates were determined to quantify the substrate selectivity
(Table 1, Figure S13). Tryptophan (1) showed a kcat value of
The prenyl donor DMAPP (2) was alternated to its isomer
isopentenyl pyrophosphate (IPP) and the higher homologs
geranyl pyrophosphate (GPP, 9), farnesyl pyrophosphate (FPP)
and geranylgeranyl pyrophosphate (GGPP) (Figure S11). While
IPP, FPP and GGPP did not show conversion, faint product
formation was observed when DMATS1Ff was incubated with
-1
m
1.27 s and a K value of 0.31 mM. The alternative prenyl
acceptor 5 was also converted well and showed a higher kcat
value than 1, probably due to its higher electron density in the
indole system, which promotes a faster nucleophilic attack. The
9
as a cosubstrate (Figure S12). A large scale incubation
K
m
value, however, was approximately 18 times higher than for
1, showing a much lower affinity towards DMATS1Ff. The
catalytic efficiency (kcat/K ) of tyrosine (7) conversion was 400
experiment with 1 and 9 was conducted and the product was
purified by RP18-column chromatography and HPLC. The
m
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are also able to convert tyrosine to 8.34 Thus, the named
times smaller than that of 1, and ent-1 was converted even
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
slower, the catalytic efficiency dropping by four orders of
magnitude compared to the reaction with 1. In a kinetic
comparison of DMAPP (2) and GPP (9), DMAPP shows a more
enzymes are related regarding their sequences and their
substrate and product scopes (Scheme 2A). All the product
structures can be explained by the same relative orientation of
the substrates to each other (Scheme 2B), which suggests a
common substrate binding mode for the named DMATS. The
other two tryptophan converting fungal enzymes FgaPT2 and
5-DMATSAc that show distinct regioselectivity are closer
related to the DKP converting DMATS (Figure 2).
than 800 times higher kcat value. The small apparent K
m
value of
5
µM observed in the conversion of 9 is accompanied by strong
i
substrate inhibition with a K value of 278 µM (Figures S13F).
At a concentration of 2 mM, the product formation was already
too slow to be measurable. These data show a clear preference
of 1 as the prenyl acceptor and of 2 as the prenyl donor by
DMATS1Ff among the tested substrates.
Table 1. Kinetic data for the conversion of different substrates
by DMATS1Ff.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comparison of DMATS1Ff with other characterized
DMATS.
A
phylogenetic comparison of characterized
-1 a
[mM] a
substrate
k
cat [s ]
K
m
kcat/K
m
DMATS-type prenyltransferases, which convert tyrosine (5),
tryptophan (1) or tryptophan-containing diketopiperazines
-1
-1 a
[
s *mM ]
(DKPs), showed a clear separation of fungal and bacterial
1b
1.27±0.08
0.31±0.02
5.61±0.54
3.40±1.88
7.94±3.35
0.34±0.08
4.07±0.40
enzymes (Figure 2). Consequently, the two characterized
bacterial DMATS CymD and IlaO that are responsible for
reverse N-prenylation of 1 in the biosynthesis of cyclomarin and
ilamycin do not show the highest sequence similarity to
DMATS1Ff. Instead, the two characterized fungal tyrosine O-
5b
2.26±0.13
0.40±0.05
7b
(5.6±1.0)·10-3
(1.0±0.6)·10-2
ent-1b
(3.8±1.1)·10-3
(4.8±2.4)·10-4
31
prenyltransferases SirD from Leptosphaeria maculans and
2c
0.69±0.04
1.99±0.45
32
TyrPT from Aspergillus niger form a branch with DMATS1Ff
.
These enzymes are also able to use 1 as a substrate to produce
9c,d
(8.4±1.1)·10-4 0.005±0.002 0.16±0.07
3
1,32
regularly 7-prenylated tryptophan (11).
SirD also produces
a: values and standard errors determined from the fit of the
Michaelis Menten equation to the data. b: determined with 2
mM of 2. c: determined with 1 mM of 1. d: 9 showed strong
small amounts of 4 as a second product, when incubated in vitro
3
3
with 1. The biochemically characterized 7-DMATS from
Aspergillus fumigatus and its homolog from Aspergillus
fischeri are located next to the branch that contains DMATS1Ff
and take tryptophan as the preferred substrate to produce 11, but
substrate inhibition above concentrations of 0.005 mM (K
i
=
0
.28±0.10 mM).
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Figure 2. Phylogenetic tree of characterized DMATS. Bacterial and fungal enzymes form two clades as indicated by yellow and gray background. DMATS1Ff
is marked in red, names of enzymes with available crystal structures are marked in blue. The scale shows changes per site, numbers at branches are bootstrap
values. For accession numbers and further information see Table S5.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
bound state, and the R105K variant exhibited only 9% of the
catalytic efficiency of the wild type. These data hint towards
A
O
7-DMATSAfu
OH
DMATS1Ff
distinct roles of the two conserved Arg residues. While R105
probably tightly interacts with the pyrophosphate group of 2
during catalysis, R251 could be more important for substrate
and product transport, which can also be promoted by the
charged Lys side chain.
NH2
O
main
product
main
product
8
main
product
O
O
Besides these conserved residues, K332 and S400 are unique to
DMATS1Ff and are both placed in proximity to the
pyrophosphate. The corresponding positions are occupied by
Q343 and Y409 in FgaPT2 (Figure S15). The K332Q and
S400Y variants showed 50% and 56% catalytic efficiency, and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
OH
only SirD
NH2
N
H
NH2
SirD,
TyrPT
N
1
1
4
B
4
was observed as the only product in both cases, suggesting a
non-critical role for both residues.
O
O
O
OH
NH2
OH
NH2
OH
The putative binding site for 1 was also investigated (Figures
3C and 3D). The indole system of 1 is oriented in parallel to the
prenyl unit of 2 and closes the hydrophobic cavity (Figure 3B).
Since the prenyl acceptors are more variable between different
DMATS, not many conserved residues are found in this part of
the active site. One exception is a conserved E90 in DMATS1Ff
NH2
N
H
N
H
HO
PPO
PPO
PPO
(
Figure 3C), which is capable of forming a hydrogen bond with
3
6
the indole nitrogen N-1 (Figure S15). Its exchange against Ala
H+
- OPP-
- H+
- OPP-
- H+
- OPP-
-
(E90A) led to a completely inactive enzyme, as has been
demonstrated for FgaPT2. Also as in FgaPT2, two unpolar
8
4
11
amino acids (F81 and M82 in DMATS1 ) point their amide
8
Ff
oxygens inside the active site, providing hydrogen bondings to
Scheme 2. A: Products of phylogenetically related DMATS when
incubated with either tyrosine or tryptophan. B: The same relative
orientation of the two respective substrates facilitate regular C-7- and
reverse N-1-prenylation of tryptophan as well as the O-prenylation of
tyrosine.
the -amino group of 1 (Figures 3 and S15). Via the adjacent
S80 these two residues are connected to a hydrogen bonding
network, the central point of which is the highly conserved
E109 in DMATS1 (Figure S16 and S19). The E109M variant
Ff
showed drastically reduced catalytic efficiency of less than 1%
of the wild type. Especially the K for 1 was strongly elevated,
m
pointing to an important role of E109 for binding of 1.
Active site analysis and mutagenesis of DMATS1Ff. To gain
insights into the orientation of the substrates, a homology model
of DMATS1Ff was generated using the SWISS model server
Differences in the tryptophan binding site of FgaPT2 compared
to DMATS1Ff are exhibited by the three polar residues K174,
Y191 and R244 that are positioned near 1 in the FgaPT2 active
site (Figure S15B and C). In FgaPT2, K174 is possibly involved
in the final deprotonation towards the product 3 at indole
8
and the ligand-bound crystal structure of FgaPT2 (PDB code:
3I4X) as the template (Figure S14). The homology model of
DMATS1Ff showed the same conserved active site residues
compared to the structures of FgaPT2 (Figure S15) or the DKP
3
5
prenyltransferase FtmPT1.
Docking of the substrates
37
position C-4, while Y191 and R244 interact with 1 via
tryptophan (1) and DMAPP (2) into the active site of the model
in a similar manner as observed in the FgaPT2 crystal structure
explain the formation of 4 (Figure 3). The pyrophosphate group
of DMAPP is surrounded by two rings of polar residues
8
hydrogen bonds to the carboxylic acid function. In the model
structure of DMATS1Ff the corresponding three positions are
occupied by F174, I191 and F238, which are all not suitable for
polar interactions with 1. However, Arg257 is located near F238
on the neighboring beta sheet and could interact with the
carboxylic acid moiety of 1 through hydrogen bonds (Figure
(Figures 3A and 3B). The first ring consists of K187, K253,
K332 and R105, the second ring is composed of Y189, Y255,
Y334 and Y404. Despite K332 all these residues are highly
3
C). While it was not possible to heterologously express
3
6
conserved in the enzymes included in Figure 2 (Figure S16).
recombinant DMATS1Ff-F174K, the variants I191Y, F238R
and R257L all showed below 2% residual catalytic efficiency
To check the importance of these residues for catalysis in
DMATS1Ff, a library of enzyme variants was constructed and
their kinetics were measured (Table 2, Figures S17 and S18).
Exchanges of K187, K253 and R105 against Met, and of Y189,
Y255 and Y404 against Phe resulted in a clearly diminished
of the wild type (Table 2), with significantly higher K
The strongest effect was observed for the R257L variant, by a
near 15-fold increase of the K . These data suggest that all three
m
values.
m
amino acid exchanges disfavor the binding of 1. Since R257 is
the only positively charged residue in the tryptophan binding
site of DMATS1Ff, it is probably responsible for interacting
with the tryptophan carboxylic acid moiety by formation of a
salt bridge and thus enables correct substrate orientation (Figure
m
catalytic efficiency (kcat/K ) in comparison to the wild type in
all cases (Table 2). R251 is located near the active site and part
of the highly conserved RXKXY motif that is found in all
DMATS discussed in this work (Figure S16). This residue was
exchanged by Met and Lys. While the R251M variant showed
21% catalytic efficiency of the wild type enzyme, the R251K
variant retained 58%. R105 is in close proximity to 2 in its
3
C and D). Elimination of this polar charge or introduction of
other polar groups could lead to wrong binding, lowering the
enzymatic affinity towards 1 and thus hamper catalysis.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Graphic representation of the DMATS1Ff homology model (blue) with docked substrates (green). A and B: Binding site for 2. C and D: Binding
site for 1.
Table 2. Kinetic data for the conversion of 1 and 2 by different variants of DMATS1Ff.
DMATS1Ff
variant
k
cat (s-1) a
K
m
(mM) a
k
cat/K
m
DMATS1Ff
variant
k
cat (s-1) a
K
m
(mM) a
k
cat/K
m
-1
-1
a
-1
-1 a
[s *mM ]
[s *mM ]
2.40±0.37
0.87±0.31
0.06±0.03
0.98±0.22
0.10±0.02
2.02±0.27
2.27±0.39
n. m.b
E90A
n. m.b
n. m.b
n. m.b
R251K
R251M
K253M
Y255F
R257L
K332Q
S400Y
Y404F
2.87±0.15
0.12±0.01
0.02±0.00
0.06±0.00
0.44±0.03
0.72±0.02
1.49±0.08
n. m.b
1.20±0.17
0.13±0.05
0.38±0.22
0.06±0.01
4.53±0.59
0.36±0.05
0.66±0.11
n. m.b
R105K
R105M
E109M
K187M
I191Y
Y189F
F238R
0.10±0.01
0.16±0.02
0.16±0.03
0.21±0.01
0.10±0.01
0.03±0.00
0.11±0.01
0.27±0.11
0.66±0.25
7.89±2.32
0.22±0.05
1.14±0.34
0.11±0.04
2.10±0.23
0.38±0.16
0.24±0.09
0.02 ±0.01
0.98±0.22
0.09±0.03
0.24±0.09
0.05±0.01
a: values and standard errors determined from the fit of the Michaelis Menten equation to the data. b: n. m. = not measurable.
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Since the exchange of non-conserved R257 resulted in a A recently published crystal structure of substrate bound CymD
4
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
severely diminished catalytic efficiency, the other fungal
DMATS were compared regarding the occurrence of an Arg in
the putative amino acid binding site. Such a residue could
indeed be observed for 7-DMATSAfu, 7-DMATSAfi, TyrPT and
SirD two positions downstream the RXKXY motif (Figure
S16). Structural models were created, showing that the
strongly suggests a direct nucleophilic attack also in that case.
Despite the similar reaction mechanisms, the active site
architectures of CymD and DMATS1Ff disclose strong
differences. While for DMATS1Ff binding of the substrates
results in a similar aromatic cavity as in FgaPT2 (Figure S15),
DMAPP and tryptophan are found with inverted relative
4
3
corresponding Arg residues for DMATS1Ff, 7-DMATSAfu
,
orientation in the CymD crystal structure. Further, CymD does
not seem to coordinate significantly to the polar residues of the
amino acid side chain. This is indicated on the one hand by the
substrate positioning in the substrate bound crystal and on the
other hand by the distinct substrate scope, as indole was
converted equally fast as 1 and turnover of ent-1 was only four
TyrPT and SirD are all located at the same position (Figure
S20). In contrast, none of the DKP-DMATS models exhibited
a corresponding Arg in the prenyl-acceptor binding site. Thus,
an Arg residue seems to be vital for amino acid-
prenyltransferases, while the uncharged, larger DKPs cannot
interact with such a residue, which is consequently not needed
for catalysis. This is further supported by mutagenesis studies
on FgaPT2, in which the enzyme showed better catalytic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
3
times slower, which is in contrast to the findings for
DMATS1Ff from this work. A mechanism, analogous to the
reverse prenylation of 1 can also be assumed for the regular
prenylation of 7 in DMATS1Ff (Scheme S1). The role of E90 is
not clear in this case, but it may be flexible enough to reach the
phenol OH of 7 for deprotonation (Figure S21).
3
8,39
40
parameters towards DKPs,
R244 was substituted.
and worse towards 1, after
Also tyrosine (7) was docked into the model structure of
DMATS1Ff (Figure S21). Tyrosine (7) can adopt a similar
orientation as 1, which would allow for the coordination of the
acid group to R257, and of the amino group towards F91 and
M92, while the aromatic ring lies in parallel to 2 and closes the
hydrophobic pocket. This binding mode is in line with the
proposed relative substrate orientations shown in Scheme 2 and
places the phenolic oxygen of 7 in closer proximity to C-1 rather
than C-3 of DMAPP (2), in contrast to the findings for substrate
R257
HN
H2N
NH2
Y189, Y255
O
K332
O
NH3
K187, K253
PPO
2
R105
Y334, Y404
N
H
1
. E90 is not positioned accordingly to interact with the
1
O
O
phenolic hydroxyl group in the docked structure but may be
flexible enough to reach a fitting orientation (Scheme S1). In
this manner, the reverse N-prenylation of 1 and the regular O-
prenylation of 7 by DMATSFf can be explained.
The reaction mechanism of DMATS1Ff. By combining the
results of the model structure analysis of the binding site, the
site directed mutagenesis experiments and the characterized
products, a detailed reaction mechanism of DMATS1Ff can be
proposed (Scheme 3). Since the DMAPP binding site is highly
conserved among DMATS, the pyrophosphate abstraction from
- OPP-
E90
R257
R257
HN
HN
H2N
O
H N
2
NH2
NH2
O
O
O
a)
NH3
12
NH3
a)
nucleophilic
attack of N-1
N
N
H
4
2
is likely the same as in all related enzymes. Thus, the polar
1
a)
residues around the bound pyrophosphate facilitate its
abstraction by tight coordination to produce the allyl cation 12.
Experimental evidence for this dissociative mechanism has
O
O
O
OH
E90
E90
4
1
42
been reported for FgaPT2 and CymD. The emerging prenyl
cation 12 is shielded by the hydrophobic cavity, which is closed
by the aromatic substrate (Figure 3B). Tryptophan (1) is held in
the right position by the polar interactions of R257 and the
amide carbonyl groups of F81 and M82, while E90 coordinates
to the amine hydrogen of the indole ring, thereby increasing the
nucleophilicity of N-1 (Figures 3C and 3D). The relative
positioning of the substrates 1 and 2 suggests a direct attack by
the indole nitrogen at C-3 of 2 in which E90 could serve as the
base to deprotonate N-1 in a concerted reaction (Scheme 3, path
a). For the bacterial enzyme CymD, which also selectively
produces 4, a reaction mechanism involving a nucleophilic
attack of the indole C-3 position at C-1 of DMAPP (2) and a
subsequent aza-Cope rearrangement was discussed (path b),
since the nitrogen of the aromatic indole system is a rather weak
b)
rotation of 12
aza-Cope
rearrangement
R257
R257
HN
HN
H2N
O
H2N
NH2
NH2
O
O
O
12
NH3 nucleophilic
attack of C-3
NH3
N
H
N
1
13
O
O
O
OH
E90
E90
4
2
nucleophile. However, our homology-based structural model
is in favor of a direct inverse N-1 prenylation. A rotation of
cation 12 of more than 90° would be necessary to enable the
reaction of path b, which makes this mechanism less likely than
the direct attack by the indole nitrogen.
Scheme 3. Proposed reaction mechanism of DMATS1Ff by direct reverse
N-1 prenylation (path a) and alternative mechanism by normal C-3
prenylation and aza-Cope rearrangement (path b).
Stereochemical course of the prenylation. The
stereoselectivity of the reaction was surveyed by isotopic
labelling experiments. Regioselectively labeled (4- C)IPP
1
3
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(
14)44 was transformed in situ into (4- C)-2 using the
13
the signals for the H-labeled hydrogen positions do not appear.
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
isopentenyl diphosphate isomerase from Serratia plymuthica
In this experiment a scrambling of the isotopic labeling was
4
5
(IDISp
)
and directly converted by DMATS1Ff in a one-pot
observed, again with a distribution of 4:1, leading to mainly Z-
2
13
reaction with 1 as a cosubstrate (Figure 4A). Since the methyl
groups of 4 are diastereotopic, they show two distinct signals in
labeled 4 from (R)-(1- H,1- C)-14 (Figure 5D) and mainly E-
2
13
labeled 4 from (S)-(1- H,1- C)-14 (Figure 5E). Since the
distribution of labeling from the geminal methyl groups and the
enantiotopic C-1 hydrogens of DMAPP occurs with the same
ratio, it is unlikely that a rotation of the prenyl cation in the
active site is responsible for the isotopic scrambling of the
methyl groups, because this would not influence the fate of the
C-1 hydrogen atoms in 4. Also, a bond rotation between C-1
and C-2 after formation of cation 12 is unlikely, because of the
1
3
the C-NMR spectrum of 4 (Figure 4B). The resulting spectrum
from the labeling experiment showed strongly enhanced signal
intensities for both methyl groups in 4 with a ratio of 4:1 (Figure
4
C and S22). To exclude an impact of IDISp on this outcome, a
control reaction was carried out, using the enzyme in a coupled
46
assay with FPP synthase from Streptomyces coelicolor and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
47
isodaucenol synthase from Streptomyces venezuelae for the
1
3
49
production of stereoselectively
C-labeled isodaucenol,
high rotational barrier in allyl cations. Instead, different
proving complete stereoselectivity of IDISp (Figure S23).
substrate binding modes for 2 in the active site of DMATS1Ff
provide a more likely explanation (Scheme 4). The main
2
isotopomers observed in both H-labeling experiments can be
explained by the substrate orientations as shown in Figure 3,
with the pyrophosphate leaving to the Re face at C-1 of
DMAPP, in conjunction with the indole-nitrogen attacking at
the Re face of C-3 (Scheme 4A). In this way, the 1-pro-R
hydrogen of 2 becomes H
Z
in 4 and the 1-pro-S hydrogen
becomes H . A second binding mode of 2 is sterically similarly
E
demanding but exhibits a stereochemically distinct orientation
in the active site (Scheme 4B).
Figure 4. A: Enzymatic conversion of (4- C)-14 into labeled 4. B: 13C-
1
3
1
3
NMR signals for the methyl groups of unlabeled 4. C: C-NMR signals for
1
3
the methyl groups of labeled 4 obtained from (4- C)-14.
These findings imply that the observed isotopic scrambling
occurs during catalysis by DMATS1Ff. The distribution of the
methyl labeling must be caused by switching of Re and Si attack
from 1 to 2 and thus by a change of the relative substrate
orientations. This could originate from rotation of the prenyl
cation 12 before the reaction with 1, or from different initial
substrate binding modes. To distinguish between these two
scenarios, the stereochemical course of the dephosphorylation
3
was analyzed. Since the sp center at C-1 in DMAPP (2) is
2
converted to a planar sp center during formation of cation 12
and is preserved in the final product, the prochiral CH
hydrogen atoms at this position are converted into E- and Z-
2
1
13
oriented hydrogens, which are distinguishable in H, C-HSQC
spectroscopy (Figures 5B and 5C). To track the hydrogen atoms
through the reaction, a new set of incubation experiments was
2
13
48
carried out using (R)- and (S)-(1- H,1- C)-14, which were
2
13
converted in situ into (R)- and (S)-(1- H,1- C)-2 by IDISp and
then directly by DMATS1Ff into labeled 4 (Figure 5A). The
resulting simultaneously C- and H-labeled positions are
suitable for sensitive analysis by HSQC spectroscopy, in which
2
Figure 5. A: Incubation experiments with in situ generated (R)-(1- H,1-
13
2
13C)-2 (up) and (S)-(1- H,1-13C)-2 (down). B: Indicated positions of the
2
Z E Z E
observed H and H atoms in 4. C: HSQC-signals for H and H . D: HSQC
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2
13
signals resulting from the incubation with (R)-(1- H,1- C)-2. E: HSQC
including an Arg that is placed distinctively (Figure 6). DKP-
2
13
signals resulting from the incubation with (S)-(1- H,1- C)-2.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
converting DMATS do not show such an active site Arg,
making this residue probably one of the factors responsible for
substrate selection.
O
P
O
A
O
O
O
O
B
O
O
P
O
O
P
R
P
R
O
R
O
O
A
FgaPT2,
5-DMATSAc
O
O
HS
1
B
O
HS
1
3
3
HR
N
H
N
H
HR
N
H
R
1
2
1
2
O
O
DMATS1Ff, SirD,
TyrPt,7-DMATSAfu
N
E90
H
E90
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. A: Enzymes catalyzing “downside” indole prenylation. B:
Enzymes catalyzing “upside” prenylation.
O
O
O
O
O
O
O
O
P
P
O
O
The combined data shed light on the reaction mechanisms of
DMATS1Ff and related amino acid prenyltransferases. In the
P
P
O
O
O
O
R
O
HS
1
R
HS
1
3
case of DMATS1 a direct attack of the indole nitrogen at C-3
Ff
HR
3
of 2 takes place, followed by deprotonation by E90 at the same
HR
N
H
N
nitrogen (Scheme 3) is the most likely scenario. A mechanism
including an aza-Cope rearrangement on the other hand seems
unlikely because of the relative orientations of the substrates,
which would require a major rotation of the prenyl cation. The
incubations with stereoselectively labeled 2 in this work
showed clear scrambling of the labeled positions by the reaction
of DMATS1Ff, which can be explained by relaxed binding
modes of 2 in the active site, but not by a rotation of the prenyl
cation 12. The occurrence of more than one reaction course
displays a certain dynamic freedom in the active site, subtle
changes of which could be responsible for the distinct
prenylation regiochemistry of different enzymes that start from
the same substrate binding modes. This might be caused by
different active site residues or by small changes in the overall
three-dimensional structure of the enzymes and will have to be
further investigated in future studies to gain a comprehensive
understanding of how DMATS enzymes accomplish their
remarkable regioselectivity.
1
1
12
12
H
O
O
O
E90
E90
O
O
O
O
O
O
O
P
O
O
P
O
R
R
P
O
P
O
O
O
N
HS
HR
N
4
HS
4
HR
O
OH
O
OH
E90
E90
Scheme 4. Proposed stereochemical reaction pathways that explain the
observations from the labeling experiments. A: Major reaction path. B:
Minor reaction path.
ASSOCIATED CONTENT
Starting from this alternative relative substrate orientation, the
pyrophosphate leaves to the Si face at C-1 of 12, with attack of
the indole nitrogen from the Si face of C-3. This reaction results
in the opposite outcome for the C-1 hydrogens of 2, i.e. the 1-
pro-R hydrogen ends up in the E position and the 1-pro-S
hydrogen in the Z position of the vinyl group in 4.
Supporting Information. Experimental procedures including
general and analytical methods, cloning, site-directed mutagenesis,
heterologous expression of proteins, in vitro incubation
experiments, structural elucidation of enzymatic products, kinetic
analyses, in silico analyses, NMR-spectra, supporting figures,
schemes and tables. This material is available free of charge via the
Internet at http://pubs.acs.org.
CONCLUSION
In summary, the first reverse prenylating N-1-
dimethylallyltryptophan synthase from a fungus, DMATS1Ff
AUTHOR INFORMATION
,
was characterized in vitro. The enzyme was shown to accept 1,
ent-1, and 5 as prenyl acceptors for selective reverse N-
prenylation and 7 as prenyl acceptor for selective regular O-
prenylation. DMATS1Ff was able to catalyze the unprecedented
reverse N-geranylation of 1. A homology model of DMATS1Ff
in comparison to the crystal structure of FgaPT2 allowed for
site directed mutagenesis of single active site residues in
DMATS1Ff. The strongest effect was seen for the R257L variant
Corresponding Author
* Email: dickschat@uni-bonn.de
Author Contributions
‡
These authors contributed equally to this work.
I.B., Z.Y. and S.J. performed the experimental work. J.S.D. and
B.T. designed the research. The manuscript was written by I.B.
and J.S.D. with proofreading by all other authors.
m
by a rise of the K . This finding is in line with an interaction
Funding Sources
This work was supported by the DFG with grant DI1536/9-1.
between R257 and the substrate 1 through a salt bridge. A
corresponding arginine residue is observed in all enzymes that
employ a putative similar binding mode for prenylation on the
downside” of 1, while FgaPT2 and the closely related 5-
DMATSAc that prenylate on the “upside” of the indole moiety
Notes
“
The authors declare no competing financial interest.
show different polar residues for the interaction with 1,
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ACKNOWLEDGMENT
ACS Chemical Biology
Regulation of Secondary Metabolism and Novel Metabolites. PLOS
Pathog. 9, e1003475.
18) Niehaus, E.-M., Münsterkötter, M., Proctor, R. H., Brown, D.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We thank A. J. Schneider for HPLC analyses and A. Babczyk for
the synthesis of 4 and experimental assistance.
(
W., Sharon, A., Idan, Y., Oren-Young, L., Sieber, C. M., Novák, O.,
Pencik, A., Tarkowská, D., Hromadová, K., Freeman, S., Maymon, M.,
Elazar, M., Youssef, S. A., El-Shabrawy, E. S. M., Shalaby, A. B. A.,
Houterman, P., Brock, N. L., Burkhardt, I., Tsavkelova, E. A.,
Dickschat, J. S., Galuszka, P., Güldener, U. and Tudzynski, B. (2016)
Comparative “Omics” of the Fusarium fujikuroi Species Complex
Highlights Differences in Genetic Potential and Metabolite Synthesis
Genome Biol. Evol. 8, 3574-3599.
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21) Luzung, M. R., Lewis, C. A. and Baran, P. S. (2009) Direct,
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regular tryptophan C4-prenyltransferase to a reverse tryptophan
containing cyclic dipeptide C3-prenyltransferase by sequential site-
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