Rational Engineered C-Acyltransferase Transforms Sterically Demanding Acyl Donors

Article abstract of DOI:10.1021/acscatal.9b04617

The biocatalytic Friedel-Crafts acylation has been identified recently for the acetylation of resorcinol using activated acetic acid esters for the synthesis of acetophenone derivatives catalyzed by an acyltransferase. Because the wild-type enzyme is limited to acetic and propionic derivatives as the substrate, variants were designed to extend the substrate scope of this enzyme. By rational protein engineering, the key residue in the active site was identified which can be replaced to allow binding of bulkier acyl moieties. The single-point variant F148V enabled the transformation of previously inaccessible medium chain length alkyl and alkoxyalkyl carboxylic esters as donor substrates with up to 99% conversion and up to >99% isolated yield.

Full text of DOI:10.1021/acscatal.9b04617

Research Article
pubs.acs.org/acscatalysis
Cite This: ACS Catal. 2020, 10, 1094−1101
Rational Engineered C‑Acyltransferase Transforms Sterically
Demanding Acyl Donors
†
†,‡
Anna Ządło-Dobrowolska, Lucas Hammerer, Tea Pavkov-Keller,§ Karl Gruber,§
̇
and Wolfgang Kroutil*,†,‡
†Institute of Chemistry, University of Graz, NAWI Graz, BioTechMed Graz, Heinrichstrasse 28, 8010 Graz, Austria
‡ACIB GmbH, Petersgasse 14, 8010 Graz, Austria
§Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50, 8010 Graz, Austria
S
* Supporting Information
ABSTRACT: The biocatalytic Friedel−Crafts acylation has been identified recently for the acetylation of resorcinol using
activated acetic acid esters for the synthesis of acetophenone derivatives catalyzed by an acyltransferase. Because the wild-type
enzyme is limited to acetic and propionic derivatives as the substrate, variants were designed to extend the substrate scope of
this enzyme. By rational protein engineering, the key residue in the active site was identified which can be replaced to allow
binding of bulkier acyl moieties. The single-point variant F148V enabled the transformation of previously inaccessible medium
chain length alkyl and alkoxyalkyl carboxylic esters as donor substrates with up to 99% conversion and up to >99% isolated
yield.
KEYWORDS: Friedel−Crafts reaction, acylation, biocatalysis, biotransformation, enzyme engineering, side-directed mutagenesis
Friedel−Crafts acylation based on a natural disproportiona-
tion12 has been published recently for the C-acylation of
phloroglucinol and resorcinol derivatives bearing alkyl, alkoxy,
and halo-functionalities by a recombinant acyltransferase from
Pseudomonas protegens (PpATaseCH).13 The biocatalytic
reaction proceeded with exquisite regioselectivity at the C6
position under ambient reaction conditions using various non-
natural donor substrates,13,14 to form dihydroxyphenyl ketones
which are key motifs found in many therapeutic targets, fine
chemicals, and natural products.3,15 Furthermore, ATase
exhibited reaction promiscuity and accepted aniline derivatives
as substrates for amide bond formation.16 Elucidation of the
crystal structure of the biocatalyst and soaking experiments
indicated the involvement of a cysteine (C88) in the subunit
PhlC in the mechanism reacting with acetyl donors to give a
thioester as an intermediate.17 The carbonyl oxygen of the
C88-bound acetyl group is positioned in the oxyanion hole,
while the methyl group of acetyl is directed toward a small
hydrophobic cavity formed by F148, L209, and L300.
INTRODUCTION
■
Carbon−carbon bond formation by the Friedel−Crafts
reaction1 is state-of-the-art in organic synthetic chemistry.2
Various catalysts and different types of activated molecules
have been employed.3 As an extension to established protocols
and because of environmental awareness and increasingly legal
regulations, alternative concepts are investigated including
biocatalytic approaches.4 Research on Friedel−Crafts alkyla-
tion using enzymes for methyl-, prenyl-, and glycosylation has
recently been published.5 For instance, S-adenosyl-L-methio-
nine-dependent C-methyltransferases were identified to modify
flavonoid and alkaloid scaffolds to yield products with a
specific methylation pattern.6 Prenyltransferases (PTases) were
reported to catalyze cofactor-independent transfer reactions of
prenyl moieties from isoprenoid donors to the aromatic ring of
prenyl acceptors, leading to a wide range of terpenoids.7,8
Wild-type PTases typically exhibited a strict specificity for
prenyl donors. Extended engineering studies on a recently
discovered PTase (AtaPT, from Aspergillus terreus) allowed
tuning of both the substrate scope and the prenylation
selectivity.9
The scope of acyl donors was previously restricted to acetyl
and propionyl groups. Here, structure-based molecular
modeling allowed us to expand significantly the scope of acyl
Sequence and structure-guided mutagenesis has been
successfully applied to O-glycosyltransferases, which resulted
in replacing the O-glycosylation to C-glycosylation activity.10,11
All the above mentioned biocatalytic reactions represent
alkylations; in contrast, a biocatalytic equivalent to the classical
Received: October 25, 2019
Revised: December 12, 2019
© XXXX American Chemical Society
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a
Scheme 1. Biocatalytic Friedel−Crafts Acylation Investigated
a
Acyl donors 3a−3e were selected as initial probes to investigate acyltransferase variants to accept sterically more demanding substrates. Donors
3f−3s were investigated subsequently.
donors to transform medium-chain length acyl and alkox-
yacetyl moieties.
to identify residues which might sterically interfere with the
binding of longer acyl chains. Based on this analysis, F148 was
subsequently replaced by valine and alanine (Figure 1). Also,
RESULTS AND DISCUSSION
■
The acyltransferase from P. protegens transfers an acetyl group
or a propionyl group to resorcinol (1); however, no activity
was detected toward acyl substrates bearing longer side-chains,
such as butanoyl.13 To investigate the transfer of sterically
more demanding acyl groups, 2,6-diacylated phloroglucinol
derivatives mimicking the native donor substrate diacetyl-
phloroglucinol were synthesized. Thus, butanoyl (3a),
isobutanoyl (3b), hexanoyl (3c), benzoyl (3d), or phenylacetyl
(3e) were attached to the phloroglucinol scaffold (Scheme 1).
To identify residues in the enzyme which might be
responsible for the limited substrate scope, a rational approach
was chosen. From the crystal structure of PpATaseCH (PDB
codes 5M3K, 5MG5) (Figure S2, Supporting Information), it
was deduced that residues F148, L300, L383, Y386, and Y298
located in the environment of the acetyl group may limit the
size of the cavity for substrate binding. The acetyl group
covalently attached to C88 in the crystal structure of the
enzyme was manually modeled to a butanoyl moiety in order
Figure 1. Crystal structure with the butanoyl residue modeled into
the active site by extending the acetyl moiety manually and
introducing mutation F148V by PyMOL mutation wizard. Figures
were prepared using PyMOL.
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Table 1. Conversions of ATase Variants with Acyl Donor Substrates 3a−3e
a
conv. [%]
b
entry
PpATaseCH variant
3a
3b
<0.1
6.1 0.1
8.8 3.6
<0.1
2.8 0.8
<0.1
4.3 1.2
2.9 0.8
2.2 1.0
<0.1
3c
<0.1
3d
<0.1
3e
specific activity [mU/mg]
1
2
3
4
5
6
7
8
wild type
F148A
F148V
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
55.4 4.1
8.6 1.1
10.2 0.8
7.4 1.7
9.1 0.8
8.5 3.5
4.0 0.8
4.9 1.1
9.3 0.2
12.5 1.4
19.1 1.8
10.7 1.5
8.7 0.9
c
c
52.5 2.0
66.8 1.5
2.3 0.6
35.4 2.7
2.3 0.6
37.5 2.0
21.3 1.3
29.2 0.7
30.8 0.5
22.5 2.2
8.5 1.3
2.2 0.2
54.8 0.9
24.0 0.9
19.1 2.0
45.7 5.1
14.6 0.4
16.9 1.2
10.4 1.4
17.7 3.7
21.2 1.9
19.2 0.9
6.6 0.5
1.0 1.7
1.0 1.7
0.6 0.5
0.8 0.2
0.9 0.8
2.3 0.3
0.7 0.7
0.6 0.6
2.8 0.2
1.2 1.0
1.0 0.9
<0.1
F148A/L383A
F148A/L383V
F148V/L383A
F148V/L383V
F148V/Y386A
F148V/Y386V
F148V/L300A
F148V/L300V
F148V/W211A
F148V/W211A/Y386A
9
10
11
12
13
<0.1
2.0 0.9
1.1 0.1
1.8 1.2
a
Conditions: cell-free Escherichia coli extract containing ATase variant (1.5 mg total protein) in K-phosphate-buffer (100 mM, pH 7.5, total volume
1 mL), resorcinol (1, 0.01 mmol), donor 3a−3e (0.015 mmol), imidazole (100 mM added from a 1 M stock solution prepared in the reaction
buffer), 35 °C, 750 rpm, 24 h. Conversions were determined based on three experiments by high performance liquid chromatography (HPLC)
b
using calibration curves with authentic samples. Specific activity refers to the activity of the enzyme for the disproportionation of
c
monoacetylphloroglucinol into diacetylphloroglucinol and phloroglucinol. Cell-free E. coli extract containing ATase variant (3 mg total protein).
a
Table 2. Acyltransferase F148V-Catalyzed Acylation of 1 with Butanoyl Donors (3f−3h) under Varied Conditions
entry
donor
donor conc. [mM]
acceptor conc. [mM]
additives
enzyme loading [mg]
conv. [%]
1
2
3
4
5
6
7
8
3f
100
100
100
100
100
100
100
15
10
10
10
10
10
10
10
10
10
10
10
Im 100 mM
Im 100 mM
Im 100 mM
Im 100 mM
Im 100 mM
DMSO (10% v/v)
1.5
1.5
1.5
3
18.2 5.4
13.7 2.1
30.4 2.8
34.5 2.8
53.7 0.2
32.1 3.0
27.3 2.3
13.3 1.2
28.5 1.0
31.4 3.1
34.5 0.6
3g
3h
3h
3h
3h
3h
3h
3h
3h
3h
6
1.5
1.5
1.5
1.5
1.5
1.5
Im 100 mM
Im 100 mM
Im 100 mM; Triton X-100
Im 100 mM; Tween 40
9
10
11
50
100
100
a
Reaction conditions: cell-free E. coli extract containing ATase F148A (1.5−6 mg total protein) in KPi-buffer (100 mM, pH 7.5, total volume 1
mL), resorcinol (1, 0.01 mmol), donor 3d−3h (0.1 mmol) with or without imidazole (Im, 100 mM added from a 1 M stock solution prepared in
the reaction buffer), or with DMSO (10% v/v), or Triton X-100 (0.25 mM), or Tween 40 (0.03 mM), 35 °C, 750 rpm, 24 h.
the region of the lid, besides the substrate binding region, is
involved in modulating activity, selectivity, and stability in
selected enzymes (e.g., lipases and kinases).18 Studies have
demonstrated that an access to the enzyme active site is
controlled by the position of the lid, that undergoes a
transition between an open and a closed conformation as the
enzyme binds the substrate in the catalytic cycle. It was shown
that protein engineering of lid structures might provide either
enzymes with new properties or reduction in the enzyme
activity.19 To investigate if residue W211, possessing the lid
function, impedes the entrance of bulky substrates to the active
site, it was replaced by smaller residues.
The selected amino acids were replaced by side-directed
mutagenesis for sterically less demanding amino acids such as
alanine or valine. Consequently, a focused library consisting of
11 single variants (F148A, F148V, W211A, W211F, Y298A,
Y298V, L300A, L300V, L383A, L383V, and Y386V) was
prepared and examined to catalyze the acylation of 1 using 3a−
3e as acyl donor substrates.
the exchange of F148 to either alanine or valine (F148A/V)
enabled to transfer sterically more demanding acyl moieties
(Table 1). For instance, using 3a as the substrate, the butanoyl
moiety was transferred to resorcinol with 53 and 67%
conversion using F148A and F148V, respectively (Table 1,
entry 2−3). Side-chain elongation of the acyl group to six
carbon atoms (3c) resulted still in 55 and 24% conversion, and
for a branched side-chain (3b), 6 and 9% conversion,
respectively, was obtained. Even the benzoyl moiety (3d)
was accepted, although just 1% conversion was reached under
the conditions employed. In contrast, substrate 3e bearing the
phenylacetyl group was not transformed. Residue F148 was
clearly identified as the key position for acyl donor selectivity,
and the mutation of this residue to either alanine or valine
enables the acceptance of enlarged acyl donors.
Consequently, variants F148V and F148A were chosen as
the base for further variants, in which the variants F148A/V
were combined with the other residues mentioned before
(W211, L300, L383, and Y386), leading to the preparation of
nine double variants (Table 1) and one triple variant (F148V/
W211A/Y386A).
All of the tested variants were functional proteins and
displayed reduced specific activity toward the native substrate
MAPG (Supporting Information, Table S1). Importantly, only
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a
Scheme 2. Biocatalytic Friedel−Crafts Acylation of Resorcinol Using Phenyl Esters as Donors
a
Values before brackets represent conversions performed on an analytical scale. Values in brackets represent isolated yield from reaction performed
on 0.25 mmol scale for 1. Reaction conditions for semi-preparative scale: ATase F148V, 30 °C, 120 rpm, 24 h, horizontal tube position.
All double variants and the triple variant were able to
catalyze the acyl transfer from donor substrates 3a and 3c;
however, only a few of them accepted 3b and 3d and again
none of them converted 3e (Table 1, entries 4−13). Actually,
all double/triple variants investigated displayed lower con-
versions than the single variant F148V. Although we expected
initially that an enlarged active site would even more easily
accommodate bulky acyl donors, the results may indicate that
probably besides steric restraints, also positive interactions are
responsible to ensure the transformation of a substrate.
Concluding, for the probes 3a−d investigated, F148V led to
the highest conversion.
Thermal stability experiments (Supporting Information
Figures S4−S7) were performed for the most interesting
variants (F148A, F148V, and F148V/L383A) and the native
enzyme. For all catalysts, immediate thermal denaturation was
observed at 70 °C. The native enzyme displayed high stability
in a temperature range of 30−50 °C, maintaining >85%
activity after 240 min. Enzyme variants showed lower stability
and may be used at 35 °C with half-life around 50 min for
F148A, 140 min for F148V, and 240 min for F148V/L383A.
Based on previous studies on acetyl derivatives,14 it was
expected that other activated acyl donors may be suited as well.
Consequently, the reactivity of the F148V variant was assayed
using vinyl butyrate (3f), methyl thiobutyrate (3g), and phenyl
butyrate (3h). Because the transformation with these donors
led to reduced conversion when using the same reaction
conditions as for 3a, the influence of the donor concentration
and the influence of additives such as imidazole, detergents, or
cosolvents was investigated to obtain improved conversion
(Table 2). Imidazole was previously identified to improve the
reaction with 2-propenyl acetate.14b
It turned out that an excess of acyl donor had a beneficial
effect on the conversion (entry 3 vs 8−9). Moreover, the
increase in enzyme loading to 6 mg improved product
conversion from 30 to 54% (entry 3 vs 4). Subsequently, the
influence of various additives was investigated. Compared to
reaction performed without additives (27%, entry 7), the
addition of imidazole (30%, entry 3) or dimethyl sulfoxide
(DMSO) (32%, entry 6) led to a small improvement. Likewise,
surfactants (31, 35%, entries 10−11) slightly promoted the
reaction. Finally, phenyl esters were chosen as the most
attractive type of acyl donors because not only did they lead to
highest conversion (30%, Table 2, entry 3 vs entries 1 and 2)
but also many are commercially or accessible via simple
synthetic methods.
To provide further insight into the scope of donors, the
variant F148V was tested with a broad range of phenyl esters
with varied side chains as donor substrates (3i−3s, Scheme 2).
Compared to the wild-type, the F148V variant clearly showed
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Table 3. Kinetic Parameters for Wild-Type PpATaseCH and Variant F148V in the Biocatalytic Friedel−Crafts-Type Acylation
substrate
enzyme variant
KM [mM]
Vmax [mM min−1
]
kcat [s−1
72
]
k
cat/KM [mM−1 s−1
]
a
MAPG
PpATaseCH
F148V
PpATaseCH
F148V
F148V
F148V
0.29 0.04
0.24 0.09
4.83 0.57
0.82 0.15
8
251 15
a
MAPG
7.2 1.9
31
4
a
c
MBPG
<0.014
a
MBPG
0.54 0.07
3.2 0.1
5.0 1.0
0.62 0.11
0.04 0.02
0.58 0.23
5.3 0.6
0.012 0.002
0.188 0.016
9.9 0.8
0.0037 0.0006
0.0387 0.0085
b
3h
b
3k
a
Spectrophotometric assay: potassium phosphate buffer (100 mM, pH 7.5) and substrate 0.04−1.6 mM (added from a 40 mM stock solution
prepared in DMSO) were added to a cuvette and preheated to 35 °C. The reaction (1 mL total volume) was started by the addition of purified
enzyme (0.18 mg). The reaction was followed for over 1 min. An increase in absorption is recorded because of the formation of DAPG (λ = 370
nm) or DBPG (λ = 370 nm). All reactions were performed in triplicates. HPLC assay: potassium phosphate buffer (100 mM, pH 7.5), donor 3h
b
or 3k 0.5−25 mM (added from a 50 mM stock solution prepared in DMSO), resorcinol (1, 0.275 mg, 10 mM), imidazole (100 mM, added from a
1 M stock solution in phosphate buffer) were added to an Eppendorf tube. The reaction (0.25 mL total volume) was started by the addition of
purified enzyme (0.75 mg). The reaction mixture was shaken (5 min for 3k and 60 min for 3h) at 35 °C and 750 rpm in an orbital shaker.
Reactions were quenched by addition of acetonitrile (0.25 mL). The precipitated protein was removed by centrifugation (20 min, 14 000 rpm), and
c
the supernatant was subjected to HPLC for determination of conversions. Reaction rates were lower than the detection limit, V < 0.014 mmol
min−1.
a significant extended substrate scope. Donor scaffolds bearing
functional groups, such as double bonds or hydroxyl groups,
were investigated. For instance, phenyl pent-4-enoate (3i) led
to conversion up to 35%, while neither phenyl (E)-but-2-
enoate nor phenyl 3-methylbut-2-enoate was transformed
(both possessing a conjugated double bond, Supporting
Information, Scheme S2). Short-chain substrate 3j bearing a
hydroxyl group in position 2 was poorly accepted by F148V
(5%), but significant activity was observed for the wild-type
enzyme (55%). When the hydroxyl moiety was protected as
methyl ether, wild-type ATase displayed no measurable
conversion for the corresponding phenyl 2-methoxyacetate
(3k), which is consistent with previous observations, that the
native enzyme accepts only short acyl moieties. Interestingly,
the same acyl donor 3k was well accepted by the F148V
variant, reaching completion at 10 mmol substrate concen-
tration within 24 h. Thus, compared to the aliphatic donor
(3h) with the same number of atoms, the 2-alkoxy compound
(3k) led to the significantly higher conversion (35 vs >99%).
Since 1990s, alkyl methoxyacetates have been used as an
efficient source of acyl moieties for lipase-catalyzed resolutions
of chiral amines.20 The presence of a methoxy substituent
accelerated the reaction, so the initial acylation rate was 100
times faster than the corresponding reaction with ethyl
butyrate.21 Interaction between the β-oxygen atom in
methoxyacetate and the amine nitrogen atom was found to
be a key factor in the rate enhancement. High preference for
the alkoxyacetyl moiety had also been identified for an
acyltransferase in amide bond formation.22
and potential adjuvants for the treatment of Staphylococcus
aureus-derived diseases.23
Because oxygen in the side chain showed a tremendous
influence on the conversion, we wondered what might be the
effect if the oxygen atom in, for example, donor 3k gets
exchanged with sulfur (donor 3r). The side chains of the O-
and S-donors differ only slightly in their sizes, but sulfur is a
weaker hydrogen bond acceptor.24 The corresponding product
2r was obtained with 31% conversion, which represents a
lower value compared to the analogue compound 2k (>99%
conv.). Obviously, the oxygen at position 2 has a significant
effect; however, the exact binding could not be elucidated
using docking experiments.
Interestingly, other acyl donors possessing an aromatic ring,
such as benzoyl phenylacetyl or phenylpropionyl were not
transformed (see Supporting Information, Scheme S2).
Medium-chain length donor 3s led to similar conversion as
for native-substrate mimicking analogue 3c.
To demonstrate the applicability of the biotransformation,
semipreparative transformations were performed starting with
0.25 mmol of substrate 1. Reactions were run for 24 h, and
products were isolated with high yields with up to 99%
(Scheme 2, values in brackets). Generally, isolated product
yields were comparable to the conversions obtained in
analytical-scale experiments. In few cases, isolated product
yield values were even higher because of the slightly altered
reaction conditions including lower reaction temperature (30
°C instead of 35 °C), shaking speed (120 rpm instead of 750
rpm), and horizontal tube positioning while shaking (instead
of vertical). In selected cases, hydrolysis of O-acetylated
coproduct was required. As previously shown,13 reaction with
carboxylic acid esters may give the Friedel−Crafts product or
lead to spontaneous O-acylation, whereby the O-acylated
products may serve again as the acyl donor; this is in contrast
to phloroglucinol derivatives for which no O-acetylation was
detected.
Finally, to evaluate the effect of the mutation F148V, the
kinetic parameters of the variant and the wild-type enzyme
PpATaseCH were compared for the disproportionation of
monoacetylphlorogucinol (MAPG) and monobutanoylphlor-
ogucinol (MBPG) into the corresponding products, diacetyl-
phloroglucinol (DAPG) and 3a, respectively. Initial velocities
were measured at substrate concentrations of 0.04−1.6 mM.
Variant F148V showed 10 times lower catalytic efficiency
Consequently, various alkoxyacetyl donor substrates were
subjected to the Friedel−Crafts bioacylation. Elongation of the
alkoxy side chain from methoxy to ethoxy (substrate 3l)
allowed again to achieve >99% conversion, and also for groups
(3m, 70%), good conversion was reached. However, when
shifting the oxygen atom in the acyl moiety from position 2 to
position 3 resulted in reduced conversion, such as 8% for the
butanoyl analogue (3n) and the unprotected hydroxyl groups
(3o, <0.1%). On introduction of a second oxygen atom in the
side chain of the well accepted donor 3m, donor 3p led to a
comparable conversion of 73%.
Even the donor bearing a phenyl group linked to the oxygen
atom in 2-position was accepted with reasonable conversion
(3q, 41% conv.). This may be of importance because 2q
constitutes the core structure of various antivirulence agents
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toward the native substrate MAPG than the wild type as
indicated by kcat/KM (Table 3). The lowered kcat/KM value
results mainly of a 10-fold decrease in kcat. The comparable KM
values indicate that binding is similar. In the case of the more
bulky compound MBPG, the wild type showed no detectable
activity, while F148V led to kinetic parameters similar as
observed for the variant with MAPG.
Because a significant effect of an oxygen atom in position 2
of the acyl donor was noted, kinetic parameters for 3h and 3k
were determined in the acylation of resorcinol 1. The amount
of resorcinol was kept constant (10 mM), while the initial
velocities were measured at different concentrations of donors
(0.5−25 mM). The transformation of each donor into the
corresponding product was monitored by measuring the
increase in product concentration.
Karl Gruber: 0000-0002-3485-9740
Wolfgang Kroutil: 0000-0002-2151-6394
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was financed by the Austrian Science Fund (FWF)
Lise Meitner Fellowship grant M 2172-B21.
■
ABBREVIATIONS
■
ATase, acyltransferase; PpATaseCH, acyltransferase produced
by codon-harmonized construct from P. protegens; Im,
imidazole; DMSO, dimethyl sulfoxide; MAPG, monoacetyl-
phloroglucinol; DAPG, 2,4-diacetylphloroglucinol; MBPG,
monobutyrylphloroglucinol; DBPG, 2,4-dibutyrylphloroguci-
nol; DPPG, diisobutyrylphloroglucinol; HPLC, high perform-
ance liquid chromatography; E. coli, Escherichia coli
The results show a significant difference in the kcat values of
the butanoyl and methoxyacetyl side chains; for the
methoxyacetyl side chain, the kcat was 15.7-fold higher than
for butanoyl.
CONCLUSIONS
■
For extending the spectrum of acyl groups accepted by the
wild-type ATase, rational enzyme engineering was used to
identify potential amino acid exchanges, which may generate
more space in the active site pocket. Enzyme variants
generated by side-directed mutagenesis were screened first
toward a set of diacylated phloroglucinols as donor substrates,
being closest to the native substrate. The results showed that
phenylalanine 148 is a key position for mutagenesis, whereby
F148V turned out to be suited best. Additional mutations to
increase the cavity of the active site even further did not lead to
the desired effect. Investigation of different types of the
activated butanoyl group indicated that phenyl esters are highly
suitable substrates. A broad set of various functionalized
phenyl esters was investigated as acyl donors. Interestingly, acyl
donors possessing an oxygen atom in position 2 turned out to
be well accepted substrates, leading to significant higher
conversion than the corresponding methylene substrates; for
instance, methoxyacetyl led to >99% conversion, while
butanoyl led to 35% conversion under comparable conditions.
This methodology allowed us to use ATase for the synthesis of
1-(2,4-dihydroxyphenyl)-2-phenoxyethan-1-one (2q), which
constitutes the core structure of various antivirulence agents.
REFERENCES
■
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(2) (a) Vekariya, R. H.; Aube, J. Hexafluoro-2-propanol-promoted
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.9b04617.
■
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AUTHOR INFORMATION
Corresponding Author
*E-mail: wolfgang.kroutil@uni-graz.at.
ORCID
■
Tea Pavkov-Keller: 0000-0001-7871-6680
1099
DOI: 10.1021/acscatal.9b04617
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ACS Catalysis
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