Site-directed mutagenesis switching a dimethylallyl tryptophan synthase to a specific tyrosine C3-prenylating enzyme

Article abstract of DOI:10.1074/jbc.M114.623413

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C⁴- and C⁷-prenylation of the indole ring, respectively. 7-DMATS was found to accept L-tyrosine as substra

Full text of DOI:10.1074/jbc.M114.623413

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 3, pp. 1364–1373, January 16, 2015
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Site-directed Mutagenesis Switching a Dimethylallyl
Tryptophan Synthase to a Specific Tyrosine C3-Prenylating
Enzyme*
Received for publication, November 4, 2014, and in revised form, November 26, 2014 Published, JBC Papers in Press, December 4, 2014, DOI 10.1074/jbc.M114.623413
Aili Fan (范爱丽)‡1, Georg Zocher§, Edyta Stec‡, Thilo Stehle§, and Shu-Ming Li (李书明)‡2
From the ‡Institut fu¨r Pharmazeutische Biologie und Biotechnologie, Philipps-Universita¨t Marburg, 35037 Marburg and the
§Interfakulta¨res Institut fu¨r Biochemie, Eberhard Karls Universita¨t Tu¨bingen, 72076 Tu¨bingen, Germany
Background: Dimethylallyl tryptophan synthase FgaPT2 catalyzes in nature the C4-prenylation of indole ring.
Results: FgaPT2 also catalyzes in vitro a regular C3-prenylation of L-tyrosine; its mutant FgaPT2_K174F showed a much higher
catalytic activity toward L-tyrosine than L-tryptophan.
Conclusion: Single mutation on the key amino acid switches the tryptophan C4-prenyltransferase to a tyrosine C3-prenylating
enzyme.
Significance: The first L-tyrosine C3-prenylating enzyme was created by molecular modeling-guided mutagenesis.
The tryptophan prenyltransferases FgaPT2 and 7-DMATS
(7-dimethylallyl tryptophan synthase) from Aspergillus fumiga-
tus catalyze C4- and C7-prenylation of the indole ring, respec-
tively. 7-DMATS was found to accept L-tyrosine as substrate as
well and converted it to an O-prenylated derivative. An accept-
ance of L-tyrosine by FgaPT2 was also observed in this study.
Interestingly, isolation and structure elucidation revealed the
identification of a C3-prenylated L-tyrosine as enzyme product.
Molecular modeling and site-directed mutagenesis led to cre-
ation of a mutant FgaPT2_K174F, which showed much higher
specificity toward L-tyrosine than L-tryptophan. Its catalytic
efficiency toward L-tyrosine was found to be 4.9-fold in compar-
ison with that of non-mutated FgaPT2, whereas the activity
toward L-tryptophan was less than 0.4% of that of the wild-type.
To the best of our knowledge, this is the first report on an enzy-
matic C-prenylation of L-tyrosine as free amino acid and altering
the substrate preference of a prenyltransferase by mutagenesis.
According to their sequences, biochemical properties, and
structures, prenyltransferases can be divided into several sub-
groups (1, 9, 10). One of the most investigated subgroups is the
dimethylallyl tryptophan synthase (DMATS) superfamily from
microorganisms. Until now, more than 40 such enzymes have
been identified and characterized biochemically (8, 11–14).
The majority of the DMATS superfamily is involved in the bio-
synthesis of prenylated indole alkaloids and takes indole deriv-
atives including tryptophan and tryptophan-containing cyclic
dipeptides as substrates (3, 8). Usually, they showed significant
substrate tolerance toward different aromatic substrates, but
catalyzed regiospecific Friedel-Crafts alkylations of the indole
ring. These features make the DMATS prenyltransferases use-
ful tools as biocatalysts for production of prenylated products
(8). For example, FgaPT2 catalyzes the first pathway-specific
step in the biosynthesis of the ergot alkaloid fumigaclavine C in
Aspergillus fumigatus, i.e. a regular prenylation of L-tryptophan
at position C-4 of the indole moiety in the presence of DMAPP
(Fig. 1) (15, 16). It was later demonstrated that FgaPT2 also
accepted DMAPP analogues as alkyl donors (17, 18) and
accepted simple tryptophan derivatives (19), tryptophan-con-
taining cyclic dipeptides (19), or even hydroxynaphthalenes
(20) as acceptors. The crystal structure of FgaPT2 was deter-
mined in 2009 and used as basis for understanding the preny-
lation mechanism (21, 22). Another example of this superfam-
ily, 7-DMATS, catalyzes a C7-prenylation of L-tryptophan on
the indole ring (23) and is involved in the biosynthesis of
astechrome in A. fumigatus (24). FgaPT2 and 7-DMATS share
a sequence identity of 31% on the amino acid level.
Prenyltransferases catalyze the transfer reactions of prenyl
moieties from different prenyl donors, e.g. dimethylallyl
diphosphate (DMAPP),3 to various aliphatic or aromatic accep-
tors and play an important role as modification enzymes for
creating structural diversity of numerous natural products (1).
Prenylated derivatives often show biological and pharmacolog-
ical activities clearly distinct from their non-prenylated precur-
sors (1–5), which make these enzymes valuable biocatalysts in
the structural modification of small molecules. These features
have drawn noteworthy attention of scientists from different
disciplines (6–8).
A few members of the DMATS superfamily catalyze preny-
lations of non-indole derivatives, and in some cases, O-preny-
* This work was supported in part by Deutsche Forschungsgemeinschaft lations (25, 26). One example of such enzymes, SirD from Lep-
Grants Li844/4-1 (to S.-M. L.) and SFB766 (to G. Z. and T. S.).
tosphaeria maculans, is involved in the biosynthesis of
sirodesmin PL and responsible for the O-prenylation of the
phenolic hydroxyl group in L-tyrosine (26, 27). SirD shares a
sequence identity of 34% on the amino acid level with
7-DMATS and also accepts L-tryptophan and some of its deriv-
atives as aromatic substrates. It catalyzes mainly a C7-prenyla-
1 Recipient of a scholarship from the China Scholarship Council.
2 To whom correspondence should be addressed. Tel.: 49-6421-2822461; Fax:
49-6421-2825365; E-mail: shuming.li@staff.uni-marburg.de.
3 The abbreviations used are: DMAPP, dimethylallyl diphosphate; DMATS,
dimethylallyl tryptophan synthase; DMSPP, dimethylallyl S-thiolodiphos-
phate; HSQC, heteronuclear single quantum correlation; HMBC, heteronu-
clear multiple bond correlation.
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FIGURE 1. Prenyl transfer reaction catalyzed by FgaPT2 in the biosynthesis of fumigaclavine C.
tion of the indole ring (26, 28, 29). C7-Prenylation of L-trypto-
phan by tyrosine prenyltransferases was also demonstrated
recently with TyrPT from Aspergillus niger (14). Encouraged by
the results obtained for SirD and TyrPT with L-tryptophan as
substrate, we investigated the behavior of 7-DMATS from
A. fumigatus toward L-tyrosine in the presence of DMAPP.
7-DMATS was also able to take L-tyrosine and two derivatives
thereof as substrates and catalyzed the same O- or N-prenyla-
tion as the two tyrosine O-prenyltransferases SirD and TyrPT
(14, 28–30). These results demonstrated the complementary
substrate and catalytic promiscuity of tryptophan and tyrosine
prenyltransferases and meanwhile raised the question about
the acceptance of L-tyrosine and derivatives by other trypto-
phan prenyltransferases, such as FgaPT2, 5-DMATS, and
6-DMATSSa, which catalyze the L-tryptophan prenylation at
C-4, C-5, and C-6, respectively (11, 15, 31). To gain more
insights into the behavior of these enzymes toward tyrosine and
derivatives, FgaPT2, 5-DMATS, and 6-DMATSSa were over-
produced and purified as described previously (11, 19, 31) and
incubated with L-tyrosine at 37 °C for 16 h. Incubations with
L-tryptophan were used as positive controls. The incubation
mixtures were then analyzed on HPLC.
TABLE 1
Mutated derivatives of FgaPT2 and respective primers used for site-
directed mutagenesis
Annealing temperatures for PCR varied from 55 to 65 °C, and elongation time was
7.5 min; bold letters in primers indicated mutation in comparison with the original
sequence of FgaPT2.
EXPERIMENTAL PROCEDURES
used for plasmids pALF23 to pALF28. pES26 was used as tem-
Chemicals—DMAPP was synthesized according to the plate for construction of the double mutant K174F_R244E in
method described for geranyl diphosphate reported previously pES34 with the primers R244E 2fw and R244E 2rev (Table 1).
(32). Substrates used for the enzyme assays were purchased at The obtained plasmids were subjected to sequencing to con-
the highest available purity.
firm the desired mutations in the respective constructs.
Bacterial Strains, Plasmids, and Culture Conditions—Esche-
Overproduction and Purification of the Recombinant Pro-
richia coli XL1 Blue MRFЈ (Stratagene, Heidelberg, Germany) teins—FgaPT2 and its mutated derivatives were overproduced
and E. coli BL21 (DE3)pLysS (Invitrogen, Karlsruhe, Germany) and purified as described previously (19) and analyzed on SDS-
were used for cloning and expression experiments, respectively. PAGE (Fig. 2). Protein yields between 0.6 and 4.9 mg/liter of
pIU18 was used as construct for FgaPT2 overproduction as culture were obtained in this study.
described previously (19) and as DNA template for site-di-
Enzyme Assays with Recombinant Purified Proteins—To
rected mutagenesis experiments. E. coli cells harboring plas- determine the enzyme activity, the incubation mixtures con-
mids were grown in liquid Lysogeny Broth (LB) or Terrific tained 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, 1 mM aromatic
Broth (TB) medium and on solid LB medium with 1.5% (w/v) substrate, 1 or 2 mM DMAPP, 0.15–5% (v/v) glycerol, 0–5%
agar at 37 °C. 25 ␮g mlϪ1 of kanamycin were used for selection (v/v) dimethyl sulfoxide (DMSO), and 0.2–0.4 mg mlϪ1 of puri-
of recombinant E. coli strains.
fied recombinant protein in a total volume of 100 ␮l. After
Site-directed Mutagenesis—One-step site-directed mutagen- incubation at 37 °C for 6 or 16 h, the reactions were terminated
esis protocols were used to generate the mutated derivatives of by the addition of 100 ␮l of methanol. Protein was removed by
FgaPT2 listed in Table 1. The QuikChange site-directed centrifugation at 13,000 rpm for 20 min, and the supernatant
mutagenesis kit (Stratagene, Heidelberg, Germany) was used was analyzed on HPLC. Data given in this study were calculated
for construction of plasmids pES23 to pES26 and pES34, the from two or three independent measurements.
Expand Long Template PCR dNTP pack (Roche Diagnostics,
To determine the kinetic parameters of aromatic substrates,
Mannheim, Germany) was used for plasmids pALF13, pALF15, DMAPP at 1 mM and aromatic substrates with concentrations
pALF16, pALF18, and pALF22, and the Expand Long Template of up to 2 mM were used. Variable protein concentrations and
PCR system (Roche Diagnostic, Mannheim, Germany) was incubation times were used for different enzymes.
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TABLE 2
NMR data of the enzyme products in D2O (500 MHz)
Chemical shifts (␦) are given in ppm, and coupling constants (J) are in Hz.
a Signals overlapping with those of methanol.
methanol (solvent B) in water (solvent A) in 50–80 min was car-
ried out with a flow rate at 2.5 ml minϪ1. The column was then
washed with 100% (v/v) solvent B for 10 min and equilibrated with
50% (v/v) solvent B for 10 min.
NMR and Mass Spectrometric Analyses—For structural elu-
1
cidation, the isolated enzyme products were subjected to H
NMR, HSQC, HMBC, and MS analyses. High resolution electron
impact mass spectrometry data were obtained on a Micromass
Auto Spec spectrometer (Waters, Milford, MA). The enzyme
products were: 1b, m/z 249.1361 (C14H19NO3, calculated,
249.1365); 3b, m/z 248.1524 (C14H20N2O2, calculated, 248.1525).
For NMR analysis, the enzyme products were dissolved in
CD3OD or D2O. Spectra were recorded at room temperature
with an ECX-500 spectrometer (JEOL, Tokyo, Japan) equipped
with a broadband probe with z-gradient. Chemical shifts were
referenced to the solvent signal at 3.30 ppm for CD3OD or 4.79
ppm for D2O. All spectra were processed with MestReNova
5.2.2 (Mestrelab Research, Santiago de Compostela, Spain).
NMR data of the isolated products are given in Table 2.
Molecular Docking Calculation—The structure of FgaPT2
(3I4X) in complex with tryptophan was used as a template for
the docking calculation using AUTODOCK4 (33). The trypto-
phan and surrounding water molecules were removed, and the pre-
nylation substrate, dimethylallyl S-thiolodiphosphate (DMSPP), was
kept in the active site and assumed to be rigid. For docking
calculations with L-tyrosine, Lys-174 was mutated to phenyla-
lanine in silico. Docking calculations (genetic algorithm) were
prepared with ADT (33) using a grid that covers the complete
binding site. The results were visualized with PyMOL (48) and
verified for chemical sense.
FIGURE 2. Analysis of the overproduced and purified FgaPT2 and its 17
mutants on SDS-PAGE. The purified proteins were separated, together with
the low molecular weight calibration kit (GE Healthcare), on 12% SDS-poly-
acrylamide gels and stained with Coomassie Brilliant Blue G-250.
For isolation of the enzyme products, reactions were carried
out in large scales (10 ml) containing 50 mM Tris-HCl (pH 7.5),
5 mM CaCl2, 1 mM aromatic substrate, 1.5 mM DMAPP, and
Ϫ1
0.2–0.4 mg ml recombinant protein. After incubation at
37 °C for 16 h, the reactions were terminated by the addition of
10 ml of methanol. After removal of the precipitated protein by
centrifugation at 6000 rpm for 30 min, the reaction mixtures
were concentrated on a rotating vacuum evaporator at 35 °C to
a final volume of 1 ml before injection into HPLC.
HPLC Analysis and Isolation of the Enzyme Products—The
enzyme reaction mixtures were analyzed on HPLC (Agilent
series 1200, Bo¨blingen, Germany) by using a Multospher 120
RP-18 column (250 ϫ 4 mm, 5 ␮m, CS-Chromatographie Ser-
vice, Langerwehe, Germany) at a flow rate of 1 ml minϪ1. Water
(solvent A) and methanol (solvent B) were used as solvents. For
analysis of the enzyme products, a linear gradient of 30–100%
(v/v) solvent B in 20 min was used. The column was then washed
with 100% (v/v) solvent B for 5 min and equilibrated with 30% (v/v)
solvent B for 5 min. Detection was carried out with a photodiode
array detector and illustrated at 277 nm in this study. For isolation
of the enzyme products, the same HPLC equipment with a Mul-
RESULTS
Acceptance of L-Tyrosine and 4-Amino-L-phenylalanine by
tospher 120 RP-18 column (250 ϫ 10 mm, 5 ␮m, CS-Chromatog- FgaPT2—HPLC analysis of the incubation mixture of L-trypto-
raphie Service) was used. A linear gradient of 50–100% (v/v) of phan with 0.36 ␮M FgaPT2 showed a conversion yield of 58 Ϯ
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FIGURE3.HPLCanalysisofincubationmixturesofL-tryptophanandL-tyrosine(1a)withFgaPT2(A–D)anditsmutantFgaPT2_K174F(E–H).Theenzyme
assays contained 0.36 or 3.6 ␮M of the recombinant enzymes and were incubated at 37 °C for 16 h. Detection was carried out with a photodiode array detector
and illustrated for absorption at 277 nm. mAU, milliabsorbance units.
1.0% (Fig. 3A). Using the same amount of protein, a minor addi- 3-nitro-L-tyrosine (7a), 3,5-dibromo-L-tyrosine (8a), and 3,5-
tional peak with an approximate conversion of 1.8 Ϯ 0.15% was diiodo-L-tyrosine (9a) were incubated with 3.6 ␮M recombinant
detected in the incubation mixture of L-tyrosine (1a) (Fig. 3B). FgaPT2 at 37 °C for 16 h. As shown in Fig. 4, FgaPT2 showed a
By increasing the FgaPT2 concentration to 3.6 ␮M, a clear and relatively high substrate specificity toward L-tyrosine and its
unique product peak with a yield of 18 Ϯ 1.0% was observed in analogue 3a. Clear product formation was only observed in the
the HPLC chromatogram of 1a (Fig. 3D). Under this condition, reaction mixture of 3a (data for 2a and 4a–9a not shown). With
L-tryptophan was completely converted to 4-dimethylallyl a total product yield of 70 Ϯ 4.9%, 3a was even better accepted
tryptophan (4-DMAT) (Fig. 3C). No product formation was by FgaPT2 than 1a. Inspection of the HPLC chromatogram of
detected in the incubation mixtures of 1a with 3.6 ␮M the reaction mixture of 3a revealed the presence of two product
5-DMATS or 6-DMATSSa (data not shown). Interestingly, the peaks 3b and 3c at 9.7 and 12 min, with product yields of 67 Ϯ
retention time of the enzyme product of FgaPT2 with 1a was 4.5 and 3 Ϯ 0.46%, respectively (Fig. 4B).
found to be 10 min, which is 2 min shorter than that of the
Enzyme Product Characterization—For structure elucida-
O-prenylated derivative obtained from the 7-DMATS assay tion, the enzyme products 1b, 3b, and 3c were isolated from
under the same HPLC condition (data not shown) (30). This 10-ml enzyme reaction mixtures of 1a and 3a, respectively, and
indicated the presence of different products in the reaction subjected to MS and NMR analyses. The obtained NMR data
mixtures of 1a with both tryptophan prenyltransferases. To test are given in Table 2. MS data indicated a monoprenylation in
the substrate specificity of FgaPT2 toward tyrosine derivatives, the isolated products 1b, 3b, and 3c by detection of molecular
D-tyrosine (2a), 4-amino-L-phenylalanine (3a), ␣-methyl-L-ty- masses, which are 68 Da larger than those of the respective
rosine (4a), 3-fluoro-DL-tyrosine (5a), 3-iodo-L-tyrosine (6a), substrates (see “Experimental Procedures”). The 1H NMR spec-
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FIGURE 4. HPLC analysis of the reaction mixtures of 1a (A and C) and 3a (B and D) with FgaPT2 or FgaPT2_K174F as well as prenyl transfer reactions
catalyzed by both enzymes (E and F). The enzyme assays contained 3.6 ␮M of the recombinant enzymes and were incubated at 37 °C for 16 h. Detection was
carried out with a photodiode array detector and illustrated for absorption at 277 nm. mAU, milliabsorbance units.
trum of 3c corresponded perfectly to that of the regularly in silico docking of L-tyrosine (blue) into the binding pocket of
N-prenylated derivative of 3a, which had been identified as an L-tryptophan (Fig. 5). The final docking result is in good agree-
enzyme product of SirD previously (28).
ment with the observed catalytic activity and shows that the
Inspection of the 1H NMR spectra of 1b and 3b revealed the hydroxyl group occurs near a cavity occupied with water mol-
presence of only one product each. The signal of H-2Ј at 5.41 ecules (Fig. 5, red spheres). The C3-atom of 1a or 3a (Fig. 5, blue
ppm (triple septettes, 7.3, 1.4 Hz for 1b) or 5.34 ppm (triple sphere) can easily attack the C1-atom of DMSPP. This model
septettes, 7.2, 1.4 Hz for 3b) proved them to be regularly pre- explained well our experimental results that 1a and 3a were also
nylated products. Signals of three coupling aromatic protons substrates for FgaPT2 and that the resulting products were
were observed in spectra of both 1b and 3b, indicating the pre- C3-prenylated derivatives.
nylation at an aromatic carbon atom of 1a and 3a. The coupling
According to this model, Thr-102 was proposed to interact
patterns of one doublet with a small coupling constant of 2.0 with the hydroxyl group of 1a or amino group of 3a through
(1a) or 1.6 Hz (3b), one doublet with a large coupling constant hydrogen bond. Arg-244, Tyr-191, Leu-81, and Ile-80 tend to
of 8.1 Hz, and one double doublet with coupling constants of 8.1 stabilize the side chain. Tyr-413, Lys-187, Arg-100, Tyr-409,
and 2.0 Hz (1b) or 8.1 and 1.6 Hz (3b) also proved a C2- or Arg-404, Arg-257, Gln-343, and Lys-259 are involved in the
C3-prenylation in 1b and 3b (Table 2). Given the electron diphosphate binding sites. In a previous study for the FgaPT2
donating effects of the 4-hydroxyl in 1a or 4-amino group in 3a, reaction with L-tryptophan (21), Lys-174 was proposed to act as
the Friedel-Crafts alkylation should take place at C-3 of the a base for abstracting the proton at C-4 from an intermediate,
benzene ring. To prove this hypothesis, HSQC and HMBC which was formed after attacking of C-4 to the prenyl cation,
spectra of 1b and 3b were then taken for structure determina- and rebuilding the aromatic ring. Later, Luk et al. (35) reported
tion. In the HSQC spectrum of 1b, the chemical shifts of the that the FgaPT2 reaction with tryptophan might undergo a
three aromatic proton-bearing carbons are found at 130.7, reverse prenylation at C-3 followed by a Cope rearrangement
115.7, and 128.0 ppm, which were assigned to C-2, C-5, and C-6 and rearomatization. In both proposed mechanisms, Lys-174
of the C3-prenylated product, respectively (Table 2). Clear always acted as a base for regaining the aromatic ring. The func-
HMBC correlations between H-1Ј of the prenyl moiety and C-4 tion as a base is lost for K174F at this position, which is accom-
of the benzene ring as well as H-2 and C-1Ј proved unequivo- panied by the prenylation of L-tyrosine at its C-3 atom. In our
cally that 1b was the C3-prenylated product. Furthermore, the structural model, the carboxyl entity of Glu-89, which has an
1H NMR spectrum of 1b taken in CD3OD corresponded per- interaction with N-1 of
fectly to that of the natural product isolated from Streptomyces toward the C-3 of 1a or 3a aL-ntdrytphteorpehfoarne s(u2i1t,ab3le5)t,oifsunocrtiieonnteads
sp. IFM 10937 (34). Similar correlations were observed in the a base to abstract the proton from the ␴-complex.
HMBC spectrum of 3b, and the same conclusion can be there-
To prove the proposed mechanism, we determined the activ-
fore drawn for this compound. These results confirmed the ity of I80F, E89A, K174E, and K174Q obtained in a previous
C3-prenylation of 1a and 3a by FgaPT2 (Fig. 4, E and F) as the study (21) toward L-tryptophan and L-tyrosine by incubation at
unique or main reaction (95.7%). In the case of 3a, the N-pre- 37 °C for 6 h. As shown in Fig. 6, relative activities of 43.2 Ϯ 2.0,
nylation was only 4.3% of the total product formation.
2.3 Ϯ 0.21, 2.0 Ϯ 0.24, and 119.0 Ϯ 9.3% of that of FgaPT2 were
Molecular Modeling and Site-directed Mutagenesis of detected for I80F, E89A, K174E, and K174Q with L-tryptophan,
FgaPT2—To get more insights into the catalytic mechanism of respectively. The results for I80F, E89A, and K174E are similar
FgaPT2 toward 1a and 3a, we carried out a molecular modeling to those obtained in the previous study (21). Differing from that
study with FgaPT2 and 1a based on the crystal ternary complex observed by Metzger et al. (21), K174Q showed a slightly higher
of FgaPT2 with DMSPP and L-tryptophan (21). We performed activity than FgaPT2. This result indicated that Gln could elim-
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FIGURE 5. The stereo view of active sites and proposed reaction model of FgaPT2 with 1a and 3a. The active site of FgaPT2 is shown in a stick represen-
tation (gray). Three ␤-strands and Tyr-189 were removed to give a detailed view into the active site. The substrates 1a and 3a (blue; amino moiety or hydroxyl
group is marked as a green sphere) are docked into the active site of FgaPT2_K174F using AUTODOCK4 and superimposed very well with the natural substrate
tryptophan (black). This is possible because the binding site is designed to be occupied by a larger tryptophan residue. The hydroxyl or amino group of 1a and
3a, respectively, occurs near a cavity occupied with water molecules (red spheres). The C3-atom of L-tyrosine (blue sphere) can easily attack the C1-atom of
DMSPP (pink dotted lines). Amino acids that were mutated in our experiments are colored in magenta.
FIGURE 6. Relative activities of FgaPT2 and its mutants with L-tryptophan or L-tyrosine as aromatic substrate. The assays with L-tryptophan and
L-tyrosine contained 0.36 and 3.6 ␮M recombinant proteins, respectively, and were incubated at 37 °C for 6 h. The absolute product yield of FgaPT2 with
L-tryptophan (42 Ϯ 2.0%) was defined as 100% relative activity. For L-tyrosine with FgaPT2, this value was 19 Ϯ 1.6%. Error bars represent S.D. from three
independent measurements.
inate a proton, or that another amino acid acts as a base instead. in Table 1. We speculated that replacement of Lys-174 by an
Using L-tyrosine as prenyl acceptor, relative activities of 0.62 Ϯ aromatic amino acid residue would have more interaction with
0.05, 5.7 Ϯ 1.0, 3.2 Ϯ 0.26, and 9.8 Ϯ 0.89% of that of FgaPT2 the benzene ring of the substrate L-tyrosine and stabilize the
were detected for I80F, E89A, K174E, and K174Q, respectively. intermediates of the prenylation. Therefore, we prepared addi-
This indicated different behaviors of I80F and K174Q toward tional Lys-174 mutants FgaPT2_K174F, FgaPT2_K174Y, and
L-tryptophan and L-tyrosine and suggested that Lys-174 was FgaPT2_K174W.
important but not the base to abstract a proton. Meanwhile, the
In another previous study, we showed that the cyclic dipep-
significant activity decrease of E89A implied that it could be the tide brevianamide F prenyltransferase FtmPT1 from A. fumiga-
base and supported our molecular model for the FgaPT2 reac- tus shared a similar structure and reaction chamber with
tion with L-tyrosine.
FgaPT2 (36). However, the amino acid residues involved in the
For further investigation, we chose three amino acids resi- substrate binding differ slightly from each other. For example,
dues, Thr-102, Lys-174, and Arg-244 in the structure of FgaPT2 Gly-115 in FtmPT1 was proposed to be involved in the binding
as “hot spots” for site-directed mutagenesis using primers listed of brevianamide F (36). The corresponding Thr-102 in FgaPT2
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is likely not directly involved in the binding of L-tryptophan, but
located in the active site of the enzyme (21). Mutation of Gly-
115 in FtmPT1 to threonine resulted in a derivative, which cat-
alyzed a reverse C3- instead of a regular C2-prenylation of bre-
vianamide F (36). As shown in Fig. 5, Thr-102 in FgaPT2 is
suggested to be involved in the binding of L-tyrosine. Therefore,
we changed this residue to five different amino acids to get
FgaPT2_T102X (X ϭ Val, Arg, Cys, Gly, or Ser). In addition,
molecular modeling indicated the binding of the amino group
of the L-tyrosine side chain to Arg-244. Thus, different mutants
at this position, FgaPT2_R244X (X ϭ Glu, Asn, Asp, or Gln),
were constructed as described under “Experimental Proce-
dures.” In total, we designed 13 additional mutants (Table 1) on
the basis of the proposed mechanism as well as physical and
chemical properties of the amino acids for investigation on
enzyme activities.
Determination of Kinetic Parameters for FgaPT2 and Its
Mutant by Using L-Tyrosine as Substrate—To compare the cat-
alytic efficiencies of FgaPT2 and FgaPT2_K174F toward 1a,
kinetic parameters were determined by Eadie-Hofstee, Hanes-
Woolf, and Lineweaver-Burk plots as described under
“Experimental Procedures.” Km value of 0.58 Ϯ 0.0082 mM
was found for K174F, significantly lower than that of FgaPT2
at 0.82 Ϯ 0.070 mM. The turnover number of K174F toward
1a at 0.010 Ϯ 0.000049 sϪ1 was 3.4-fold of that of FgaPT2 at
0.0029 Ϯ 0.000034 sϪ1. As consequence, the catalytic effi-
ciency (kcat/Km) of K174F with 1a as substrate was improved
to 4.9-fold, in comparison with the non-mutated FgaPT2.
DISCUSSION
Prenylated aromatic compounds represent a large group of
secondary metabolites and are widely distributed in bacteria,
fungi, and plants (1, 3, 5, 37). In comparison with their non-
prenylated precursors, these compounds show distinct and in
general more potent biological and pharmacological activities
(2, 3, 38, 39). Prenylated tyrosine and derivatives build a small
group within the prenylated aromatic natural products (40, 41).
3-Dimethylallyl-L-tyrosine was isolated from Streptomyces sp.
IFM 10937 in 2008 during a screening program for TNF-related
apoptosis-inducing ligand (TRAIL) resistance-overcoming
activity, together with N-acetyl-3-prenyl-L-tyrosine (34). How-
ever, neither the biosynthetic pathway of this type of natural
products nor an enzyme for L-tyrosine C3-prenylation has been
reported until now.
Identification of the FgaPT2 Mutant Carrying Tyrosine C3-
Prenyltransferase but Almost No Tryptophan Prenyltransferase
Activity—To evaluate the activity of the resulting mutants, the
constructs obtained from site-directed mutagenesis experi-
ments were introduced into E. coli BL21 (DE3)pLysS cells.
After induction of the gene expression as described previously
(19), the overproduced proteins were purified on nickel-nitri-
lotriacetic acid-agarose and used for enzyme assays with L-tryp-
tophan or L-tyrosine under the same condition for FgaPT2
mentioned above (37 °C for 6 h). The obtained results for all of
the 17 mutants with L-tryptophan and L-tyrosine are illustrated
in Fig. 6 and compared with those of FgaPT2.
Prenyltransferases represent key enzymes in the biosynthesis
of prenylated natural products (1, 3, 4, 8, 42). Because of
genome sequencing and mining of biosynthetic pathways, sig-
nificant progress has been achieved on the genetics, enzymol-
ogy, and structure of prenyltransferases using various aromatic
substrates such as indoles including tryptophan, flavonoids,
coumarins, xanthones, and naphthalenes (1, 3, 4, 8, 43). A num-
ber of studies have demonstrated the high flexibility of prenyl-
transferases, especially the members of the DMATS superfam-
ily, toward their aromatic substrates (8). However, a switching
of substrate preference by structure-based rational engineering
has not been reported prior to this study.
As shown in Figs. 3 and 6, clearly different activity profiles
were observed for these mutants toward L-tryptophan and L-ty-
rosine. Similar or slightly higher, but in most cases, lower activ-
ity than that of non-mutated FgaPT2 was detected in the incu-
bation mixtures of L-tryptophan with 0.36 ␮M recombinant
mutants. Using L-tyrosine as substrate, most mutants showed
much lower activities than FgaPT2. No significant changes on
the activities were detected for T102C with L-tryptophan and
L-tyrosine. Substantial increase of the enzyme activity toward
L-tyrosine was detected for K174F. Approximate 3-fold activity
of that of FgaPT2 was calculated for this mutant (Fig. 3). On the
other hand, no conversion was detected in the incubation mix-
ture of L-tryptophan with 0.36 ␮M K174F, even after an incuba-
tion for 16 h (Fig. 3E). Increasing the protein concentration to
3.6 ␮M and incubation time to 16 h, a minor product peak with
a yield of 0.24 Ϯ 0.073% was detected for K174F (Fig. 3G). With
this protein concentration, a product yield of 50 Ϯ 3.5% was
calculated for K174F with 1a as substrate (Fig. 3H). Isolation
and structure elucidation confirmed the same enzyme product
as that identified from the incubation mixture of FgaPT2 with
1a. From these results, it can be concluded that K174F func-
tions as a tyrosine C3-prenyltransferase and not as tryptophan
prenyltransferase anymore. K174F in a concentration of 3.6 ␮M
was then incubated with eight tyrosine derivatives 2a–9a at
37 °C for 16 h. The product yield of 3a with K174F was slightly
increased to 76 Ϯ 4.4% (Fig. 4). No product formation was
detected in the incubation mixtures of other substrates (data
not shown).
Based on the fact that the L-tyrosine O-prenyltransferases
SirD and TyrPT catalyzed C7-prenylation of L-tryptophan and
the tryptophan C7-prenyltransferase 7-DMATS catalyzed the
O-prenylation of L-tyrosine (14, 28, 30), we detected in this
study the prenylation of L-tyrosine by the tryptophan C4-pre-
nyltransferase FgaPT2. Identification of enzyme product
revealed a C3-prenylation of L-tyrosine by FgaPT2, demonstrat-
ing the first enzymatic Friedel-Crafts alkylation of L-tyrosine as
free amino acid. Subsequent molecular modeling-guided site-
directed mutagenesis led to create an enzyme derivative K174F,
which showed practically no activity toward L-tryptophan,
whereas the acceptance of L-tyrosine by this enzyme was
improved significantly. With 3.6 ␮M enzyme, a product yield of
50 Ϯ 3.5% was detected for K174F after incubation at 37 °C for
16 h. Therefore, FgaPT2 and especially its mutant K174F rep-
resent the first series of L-tyrosine C-prenylating enzymes.
Meanwhile, this study demonstrated the possibility to change
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Creating a Specific Tyrosine C3-Prenylating Enzyme
FIGURE 7. Proposed mechanism for the conversion of 1a by FgaPT2 to the C3-prenylated product 1b. Interactions between the substrates and FgaPT2
were shown with dotted lines. The curved arrows show the direction of electron flow.
the substrate preference of a prenyltransferase by site-directed FgaPT2, FgaPT2_E89A showed only a relative activity of 5.7 Ϯ
mutagenesis.
1.0% toward L-tyrosine, providing strong support for this
C-Prenylated tyrosyl residue was found in cyclic peptides, e.g. hypothesis. It is also considerable that a reverse prenylation at
cyanobactins, from cyanobacteria (40, 44). A prenyltransferase C-1 is followed by a Cope rearrangement as proposed for
LynF in Lyngbya aestuarii from the TruF enzyme family was FgaPT2 with L-tryptophan (35).
proven to catalyze reverse O-prenylations of tyrosyl residues in
Using the molecular model mentioned above, the activities of
such ribosomal cyclic peptides. C-Prenylated tyrosine-contain- other mutants obtained in this study can also be interpreted.
ing derivatives are then formed from the O-prenylated tyrosyl For the mutation at Thr-102, substitution of Thr with similar
residue by Claisen rearrangement afterward (44, 45). To amino acids, such as Cys and Ser, (partly) retained the catalytic
exclude the possibility that the C3-prenylated products 1b and ability toward L-tryptophan, whereas T102C demonstrated
3b are rearrangement rather than enzyme products during the similar catalytic ability toward 1a as FgaPT2, 1.9-fold of that of
incubation procedure, we carried out incubations with 1a and T102V, and 7.1-fold of that of T102S. This indicates that both
3a for 10, 15, 30, 60, 120, and 960 min. Similar profiles of the the hydrogen donor and the methyl group are important for the
HPLC chromatograms were observed for different incubation activity of FgaPT2 toward 1a. Although the hydrogen bond is
times (data not shown). In the incubation mixtures of 1a, only not crucial for the reaction (T102V), steric collisions (T102R)
the product peak 1b was detected. In the incubation mixtures of abolish the reaction, whereas smaller side chains avoid the cor-
3a, the ratio of the peak area of 3b to that of 3c remained almost rect positioning of the aromatic ring systems (T102G). Interest-
constant after different incubation times (approximately 22 Ϯ ingly, mutant T102C slightly increases the catalytic turnover as
0.65:1). It seems that 3b and 3c are independently formed dur- the nucleophilic sulfur atom is more suitable to stabilize the
ing the incubation. To provide more details on stability of 3b partial positive charge of the cationic intermediate.
and 3c, 3a was incubated with FgaPT2 at 37 °C for 2 h. The
By substitution of Lys-174 with Glu, Tyr, or Trp, the activities
reaction mixture was then heated to 80 °C and maintained for toward both L-tryptophan and 1a were reduced or abolished,
3 h. No change was observed for the ratio of 3b and 3c in the whereas K174F demonstrated a specific and 4.9-fold catalytic
HPLC chromatogram of the treated sample, excluding the efficiency toward 1a, but almost complete loss of activity
heat-catalyzed rearrangement between 3b and 3c.
toward L-tryptophan. The rigidity and increased size of the phe-
Taking the results obtained from molecular modeling and nyl side chain of K174F seem to support the stabilization of a
mutagenesis experiments into consideration (Figs. 5 and 6), a smaller substrate (tyrosine) in a cavity that was designed to
mechanism was proposed for FgaPT2 reaction with 1a. incorporate a larger educt (tryptophan). Besides, the introduc-
According to this hypothesis, the residue Thr-102 in FgaPT2 tion of the mutation K174F is likely to induce minor conforma-
interacts with the 4-hydroxyl group of 1a in the reaction cavity tional rearrangements to its neighboring residues of the indole
and contributes to the stabilization of the intermediate. A num- binding site by steric restrains. The higher activity of K174F for
ber of basic amino acids and tyrosine residues mentioned 1a could be interpreted by much better interaction of the mod-
before are involved in the binding of DMAPP and responsible ified cavity to the phenyl moiety with the benzene ring of L-ty-
for the formation of the dimethylallyl carbon cation (21, 46). A rosine compared with the interaction with the indole ring of
detailed course of the reaction remained to be elucidated and L-tryptophan.
might start by a nucleophilic attack of electron-rich C-3 of the
All four mutated derivatives at Arg-244 showed poor conver-
benzene ring onto C-1Јof the dimethylallyl carbon cation, sion of L-tryptophan. Although FgaPT2 does accept the
resulting in the formation of an intermediate with a non-aro- removal of the positive charge by the Arg-244 head group
matic system. A plausible deprotonation to regain aromaticity (R224Q and R244N), the introduction of a negative charge by
might involve Glu-89 of FgaPT2, resulting in the formation of R244E would abolish the prenylation of tryptophan due to
C3-prenylated product 1b (Fig. 7). In comparison with that of charge-charge repulsion. The enzyme activities of these four
JANUARY 16, 2015•VOLUME 290•NUMBER 3
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Creating a Specific Tyrosine C3-Prenylating Enzyme
8. Yu, X., and Li, S.-M. (2012) Prenyltransferases of the dimethylallyltrypto-
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Arg-244 mutants toward L-tyrosine were abolished completely
(Fig. 6), demonstrating its essential role in the interaction of
FgaPT2 with the side chain of L-tyrosine. The absolute impor-
tance of this amino acid residue was also demonstrated by the
double mutant K174F_R244E.
10. Bonitz, T., Alva, V., Saleh, O., Lupas, A. N., and Heide, L. (2011) Evolu-
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In this study, we reported the C3-prenylation of L-tyrosine
and 4-amino-L-phenylalanine by the tryptophan C4-prenyl-
transferase FgaPT2 from the fungus A. fumigatus and demon-
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del-Crafts alkylation of tyrosine and derivative as free amino
acids. Our results provided additional evidence for the relation-
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mutants. K174F exhibited much higher catalytic efficiency
toward L-tyrosine than FgaPT2, whereas its activity toward
L-tryptophan was almost abolished. C3-Prenylated L-tyrosine
and 4-amino-L-phenylalanine remained unique or predomi-
nant product of the mutant. The ratio of the product yields of
L-tyrosine to L-tryptophan was increased from 1:31 with
FgaPT2 to 208:1 with K174F. This means that K174F does not
function as tryptophan prenyltransferase anymore. Moreover,
it acts as L-tyrosine C3-prenyltransferase and could serve as new
biocatalyst for C3-prenylation of L-tyrosine. Therefore, the
result provides an exciting example for creating biocatalysts by
mutation of known enzymes. It is also considerable that
enzymes for specific prenylation of flavonoids or hydroxynaph-
thalenes could be created by mutation of some members of the
DMATS superfamily because such compounds have already
been accepted by some of these enzymes as prenylation sub-
strates (20, 47). Meanwhile, this study presents an excellent
example of successful interdisciplinary cooperation and of the
importance of structure biology in the discovery and develop-
ment of novel biocatalysts.
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JANUARY 16, 2015•VOLUME 290•NUMBER 3
JOURNAL OF BIOLOGICAL CHEMISTRY 1373

Enzymology:
Site-directed Mutagenesis Switching a
Dimethylallyl Tryptophan Synthase to a
Specific Tyrosine C3-Prenylating Enzyme
Aili Fan, Georg Zocher, Edyta Stec, Thilo
Stehle and Shu-Ming Li
J. Biol. Chem. 2015, 290:1364-1373.
doi: 10.1074/jbc.M114.623413 originally published online December 4, 2014
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