Discovery and Design of Family VIII Carboxylesterases as Highly Efficient Acyltransferases

Article abstract of DOI:10.1002/anie.202014169

Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity-based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure–function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a “hydrolase” into an “acyltransferase”.

Full text of DOI:10.1002/anie.202014169

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Accepted Article
Title: Discovery and Design of Family VIII Carboxylesterases as Highly
Efficient Acyltransferases
Authors: Henrik Müller, Simon P. Godehard, Gottfried J. Palm, Leona
Berndt, Christoffel P. S. Badenhorst, Ann-Kristin Becker,
Michael Lammers, and Uwe Bornscheuer
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202014169
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Discovery and Design of Family VIII Carboxylesterases as Highly Efficient
Acyltransferases
Henrik Müller[a], Simon P. Godehard[a], Gottfried J. Palm[b], Leona Berndt[b], Christoffel P. S.
Badenhorst[a], Ann-Kristin Becker[c] Michael Lammers[b], and Uwe T. Bornscheuer*[a]
,
This article is dedicated to Prof. Manfred Schneider on the occasion of his 80th birthday.
[a]
M.Sc. H. Müller, M.Sc. S. P. Godehard, Dr. C. P. S. Badenhorst, Prof. Dr. U. T. Bornscheuer,
Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry
University of Greifswald
17487 Greifswald, Germany
E-mail: uwe.bornscheuer@uni-greifswald.de
Dr. G. Palm, L. Berndt, Prof. Dr. M. Lammers,
Department of Synthetic and Structural Biochemistry, Institute of Biochemistry
University of Greifswald
[b]
17487 Greifswald, Germany
[c]
M.Sc. A-K. Becker
Institute of Bioinformatics
University Medicine Greifswald
17487 Greifswald, Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: Promiscuous acyltransferase activity is the ability of certain
hydrolases to preferentially catalyze acyl transfer over hydrolysis,
even in bulk water. However, poor enantioselectivity, low transfer ef-
ficiency, significant product hydrolysis, and limited substrate scope
represent considerable drawbacks for their application. By activity-
based screening of several hydrolases, we identified the family VIII
carboxylesterase, EstCE1, as an unprecedentedly efficient acyltrans-
ferase. EstCE1 catalyzes the irreversible amidation and car-
bamoylation of amines in water, which enabled the synthesis of the
drug moclobemide from methyl 4-chlorobenzoate and 4-(2-ami-
noethyl)morpholine (~20% conversion). We solved the crystal struc-
ture of EstCE1 and detailed structure-function analysis revealed a
three-amino acid motif important for promiscuous acyltransferase ac-
tivity. Introducing this motif into an esterase without acetyltransferase
activity transformed a ‘hydrolase’ into an ‘acyltransferase’.
used in cascade reactions with other enzymes that are not active
or stable in the presence of organic solvents.[4] Since being re-
ported in 2007, the promiscuous acyltransferase MsAcT from My-
cobacterium smegmatis had been unrivalled in terms of transfer
efficiency.[5–7] However, we recently demonstrated that many es-
terases from the bacterial hormone-sensitive lipase family have
promiscuous acyltransferase activity, some comparable to that of
MsAcT, suggesting that this phenomenon might be more wide-
spread than previously thought.[8] However, enzymatic product
hydrolysis and low transfer efficiency has always been a major
drawback for application on industrial scale. In this study, we re-
port the discovery that exceptional promiscuous acyltransferase
activity is prominent in family VIII carboxylesterases.
We previously showed that the acyltransferase-catalyzed for-
mation of oligocarbonates from dimethyl carbonate and 1,6-hex-
anediol opacifies an initially transparent, aqueous solution.[9]
However, not all promiscuous acyltransferases are expected to
produce oligocarbonates from these substrates. In this study, we
used a general precipitation-based assay, employing 2-phe-
nylethanol and vinyl acetate as substrates, to screen the hydro-
lases available in our laboratory. Acyltransferase activity is indi-
cated by the occurrence of turbidity, which results from the low
water solubility of the reaction product, 2-phenylethyl acetate.[10]
This approach led to the discovery that EstCE1[11], a family VIII
carboxylesterase, has promiscuous acyltransferase activity with a
preference for aromatic acyl-acceptor substrates (Fig. S1).
We found that EstCE1 can catalyze not only the formation of
esters, but also the formation of amides, carbonates, and carba-
mates in water (Fig. 1).The ester formation from the reaction of
benzyl alcohol with a fourfold excess of vinyl acetate is very fast,
reaching >90% conversion within seconds. However, the benzyl
Enzymes are generally considered to be highly specific. However,
exposure to non-natural substrates can sometimes result in a re-
action as well. ‘Substrate promiscuity’ occurs if the chemical
transformation complies with the enzyme's typical catalytic func-
tion. More interestingly, enzymes may also catalyze distinctly dif-
ferent chemical transformations.[1] This phenomenon is referred
to as ‘catalytic promiscuity’ and, along with gene duplication and
divergence, marks the starting point for the evolution of new en-
zymes.[2] Combined with protein engineering, promiscuous en-
zymes represent attractive alternatives to conventional chemical
catalysts.[3] Promiscuous acyltransferases for catalyzing bond-
forming reactions in aqueous solutions are of increasing interest
as they represent an attractive alternative to conventional biocat-
alytic or chemical routes by making the use of expensive and less
sustainable organic solvents dispensable. Moreover, they can be
1
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acetate formed is hydrolyzed again within two hours. Benzyl me-
thyl carbonate formation is slower, but the product is also more
slowly hydrolyzed, with roughly 50% remaining after three hours.
Furthermore, EstCE1 can catalyze the formation of N-benzyl ac-
etamide and methyl N-benzylcarbamate. Almost full conversion is
rapidly reached, and the reactions appear to be irreversible. Even
after 24 hours, no product hydrolysis was detectable. Carbamates
are essential intermediates in the synthesis of pharmaceuticals
and are commonly used as protective groups.[12] The phosgene-
independent carbamoylation of amines presented here provides
a much more environmentally friendly and more selective alterna-
tive to conventional routes. It is noteworthy that EstCE1's syn-
thetic potential goes beyond the structurally rather simple model
compounds shown in Fig. 1.
promiscuous acyltransferases, suggesting an interesting avenue
for future research.
To investigate whether promiscuous acyltransferase activity
is as common in family VIII carboxylesterases as we suspected,
we expressed, purified, and assayed several EstCE1 homologues.
This demonstrated that promiscuous acyltransferase activity is in-
deed widespread in this enzyme family (Fig. 2). With two excep-
tions, the family VIII carboxylesterases showed significantly
higher relative activities in the presence of benzyl alcohol than
MsAcT and the previously characterized bHSLs.[6,8] EstCE1 cata-
lyzes acetyl transfer to benzyl alcohol 66 times faster than hydrol-
ysis of the acyl donor, thereby outperforming even one of the most
efficient MsAcT variants (K97A; 50-fold faster) recently reported
by our group.[6] Strikingly, EstM2[15] catalyzes the acetylation of
benzyl alcohol 149 times faster than donor hydrolysis, making it
roughly 20-times more efficient than wild-type MsAcT. Only EstA
(PDB 3ZYT)[16] did not show increased relative activity in the pres-
ence of benzyl alcohol, suggesting that it is not capable of cata-
lyzing the acetylation of benzyl alcohol. Except for 3ZYT, none of
the analyzed enzymes suffered from significant substrate inhibi-
tion or stability issues, even at 150 mM benzyl alcohol.
Figure 1. EstCE1-catalyzed synthesis of benzyl acetate (grey), benzyl methyl
carbonate (cyan blue), methyl N-benzylcarbamate (violet), and N-benzyla-
cetamide (black) in aqueous buffer. The acceptor concentration was 50 mM for
all reactions. For the ester formation, a fourfold excess of vinyl acetate was
used. For amide synthesis, a tenfold excess of ethyl acetate was used. Car-
bonate and carbamate formation were performed using a tenfold excess of di-
methyl carbonate. Dotted lines indicate the formation of the products in the ab-
sence of EstCE1.
As proof of concept, we used EstCE1 to synthesize the antide-
pressant moclobemide from methyl 4-chlorobenzoate (200 mM)
as acyl donor and 4-(2-aminoethyl)morpholine (50 mM) as acyl
acceptor (Fig. S4, ~20% conversion).
Moreover, we used EstCE1 for the acetylation of several ‘dif-
ficult-to-resolve’ secondary alcohols (Tab. S2).[13] EstCE1 exhibits
enantioselectivity and, most strikingly, appears to be perfectly se-
lective in the acetylation of (+)-menthol. This finding is in good
agreement with EstCE1’s high selectivity in the hydrolysis of
(+)-menthyl acetate.[11]
Interestingly, the hydrolysis of pNP-esters by many family VIII
carboxylesterases appears to be stimulated by methanol.[14] We
suspect that these enzymes actually catalyze the acylation of
methanol under the described conditions. The acyl-enzyme inter-
mediate of a promiscuous acyltransferase would be degraded
more rapidly in the presence of an organic nucleophile like meth-
anol, resulting in more rapid release of p-nitrophenol and higher
apparent hydrolysis activities. This is the principle behind our re-
cently published pNP-AcT assay.[8] For EstCE1, we could demon-
strate an eightfold increase in apparent pNPA hydrolysis at only
6.3% (v/v) methanol (Fig. S1). To confirm that the accelerated re-
lease of pNP is a result of transesterification to methanol, we con-
firmed the EstCE1-catalyzed formation of methyl butyrate from
pNP-butyrate and methanol by gas chromatography-mass spec-
trometry (GC-MS, Fig. S5). This suggests that many of the meth-
anol-stimulated hydrolases reported in literature may actually be
Figure 2. Relative acyltransferase activities of EstCE1 homologs, plotted on a
logarithmic scale, as a function of benzyl alcohol concentration. Relative activi-
ties were determined in triplicates using the pNP-AcT assay and the highest
value for each enzyme is noted. In these reactions, pNPA is the acyl donor and
benzyl alcohol the acyl acceptor.
It is well known that the conformation of the acyl group in the acyl-
enzyme intermediate has a high impact on the efficiency of the
deacylation step.[17,18] Therefore, the acyl donor scope of the fam-
ily VIII carboxylesterases was investigated, using the pNP-AcT
assay and pNP-esters of different chain-lengths as acyl donors
(Tab. 1 and Tab. S3). Almost all the enzymes examined turned
out to be valuable for the acylation of benzyl alcohol with chain-
lengths ranging from C2 to C8. In general, the highest activities
and AT:H ratios were measured for chain-lengths from C2 to C6.
EstCE1 and EstM2 have remarkably high acyltransferase activity
in the kU/(mg enzyme) range (Tab. 1). EstSRT1 (PDB 5GMX)[19]
and EstU1 (PDB 4IVI)[17,18] turned out to be superior in catalyzing
transfer of a C8 chain, with AT:H ratios (4.6 and 5.6) in the range
of the ratio of MsAcT for acetylation (Tab. S3). Extensive protein
engineering was necessary for MsAcT to accept C8 chains, with
even the best variant (F154A/I194V) having an AT:H ratio below
3.[6] While 3ZYT shows no transacetylase activity, moderate acyl-
transferase activity emerges in reactions with donors of longer
2
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10.1002/anie.202014169
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chain-lengths (Tab. S3). This may be explained by the hydropho-
bic aliphatic chains excluding water from the binding-pocket, pre-
venting hydrolysis and favoring binding of organic acceptors.[20]
R2 subsites.[18] In contrast to many structural homologs, the
Ω-loop in EstCE1 is significantly larger and therefore blocks the
central part of the R1 subsite. Analysis of the ligand-binding site
revealed the presence of a hydrophobic cavity (Fig 3B). Induced-
fit docking of benzyl alcohol into the modeled acetyl-enzyme in-
termediate suggests a unique role of F243, which is part of the
extended Ω-loop in EstCE1 (Fig. 3B). Via π-π-stacking, F243
helps to position the benzyl alcohol in a productive pose near the
carbonyl of the acyl-enzyme intermediate. Structural alignment
revealed that there is no equivalent residue in EstY-29 (PDB
5ZWQ)[21], 4IVI, 5GMX, or 3ZYT. Homology modelling suggests
that F244 of EstM2 corresponds to F243 of EstCE1 (Fig. S9). This
may explain why EstCE1 and EstM2 show significantly higher ac-
tivity in the acetylation of benzyl alcohol compared to their homo-
logs. Moreover, we used covalent docking to shed light on the
donor chain-length preference of EstCE1. In good agreement with
the experimental data shown in Tab. 1, the modeling suggests
that linear aliphatic chains with more than four carbons are likely
to clash with the benzyl alcohol binding site (Fig. 3B).
Based on sequence alignments, the separation of family VIII
carboxylesterases into three subclasses was previously sug-
gested.[15] EstCE1, EstM2, 5ZWQ, 5GMX, and 4IVI belong either
to subclass VIII.1 or VIII.2, both sharing a conserved WGG motif
(WSG for 5ZWQ) in the region where the so-called KTG-box ex-
ists in class C β-lactamases (Fig. S6).[22] Due to its proximity to
the catalytic triad, this motif appears to largely influence the phys-
ical properties of the ligand-binding site. Most interestingly, tryp-
tophan in the first position (W339 in EstCE1) largely contributes
Table 1. Acyl donor scope of EstCE1 and EstM2 determined by pNP-AcT as-
says with benzyl alcohol as acyl acceptor. ATmax represents the maximum acyl-
transferase activity measured in a range of 0 to 150 mM benzyl alcohol.
Enzyme
Donor
ATmax in U/mg
Hydrolysis in U/mg
AT:H
EstCE1
C2
C4
C6
C8
2605 ± 86
1081 ± 28
26.1 ± 0.9
0.29 ± 0.05
40.1 ± 2.2
38.7 ± 3.3
0.22 ± 0.03
0.13 ± 0.01
65
28
119
2.2
EstM2
C2
C4
C6
C8
5583 ± 221
361 ± 33
181.2 ± 17.3
0.013 ± 0.01
37.7 ± 1.2
54.2 ± 5.5
23.7 ± 2.3
0.02 ± 0.01
148
6.7
7.6
0.7
We solved the crystal structure of EstCE1 (PDB 7ATL), the
first discovered and so far most versatile acyltransferase from this
enzyme family, in order to gain a more in-depth insight into struc-
tural features contributing to high acyltransferase activity. Typical
for family VIII carboxylesterases, EstCE1 adopts a β-lactamase
fold (Fig. 3A) with the catalytic triad (S65, K68, and Y171) and the
active site placed at the interface of an α/β-subdomain and a
structurally more flexible helical subdomain. As observed in sev-
eral family VIII carboxylesterases, EstCE1's active site pocket is
covered by a so-called Ω-loop. The substrate-binding cavity
(Fig. 3A, grey transparent volume) can be divided into the R1 and
Figure 3. A) Ribbon diagram of EstCE1 with the Ω-loop and the R1, R2 and R2’ segments highlighted. Hydrophobic volumes within the ligand-binding site are
shown in yellow with transparent surfaces. B) Active site of EstCE1. The acyl-enzyme intermediates formed in the reactions summarized in Tab. 1 were modeled
by covalent docking. The acyl acceptor, benzyl alcohol, was modeled into the structure using induced-fit docking. Via π-π-stacking with F243 in the Ω-loop, benzyl
alcohol is placed in a hydrophobic cavity near the nucleophilic center. The acyl-enzyme intermediates of C6 and C8 are shown to clash with the putative benzyl
alcohol binding site. C) Mutation of D323 to glycine heavily decreases the polarity of the substrate-binding pocket (Fig. S11) and introduces transacetylation activity
into 3ZYT. D) and E) The acetyl-enzyme intermediates of 3ZYT and 4IVI were modeled via covalent docking, and benzyl alcohol was placed in the acceptor-binding
site by rigid receptor docking. Hydrophobic regions are illustrated by yellow, transparent volumes. In the family VIII.1/2 esterase 4IVI, W381 of the WGG-motif
significantly contributes to the proper positioning of the acyl acceptor in a 3.8 Å distance to the acyl-enzyme intermediate. In contrast, D323 in the HDG motif in
3ZYT leads to active repulsion of the acyl acceptor now placed more than 7 Å away from the acyl-enzyme intermediate. F) Acyltransferase activity of several EstCE1
variants determined using the pNPA-AcT assay, with benzyl alcohol as acyl acceptor. Specific acyltransferase activity refers to the maximum activity measured in
a range of benzyl alcohol concentrations from 0 to 150 mM. The maximum AT:H ratios are shown for each variant.
3
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Keywords: Acyl transfer • Acyltransferase • Biocatalysis • Fam-
ily VIII carboxylesterase • Transesterification
to the formation of the hydrophobic cavity in family VIII.1 and VIII.2
carboxylesterases and thereby facilitates the binding and posi-
tioning of organic nucleophiles, as shown for EstCE1 and 4IVI
(Fig. 3B,E). In 3ZYT, a member of the subclass VIII.3 (H-x-x motif),
HDG is found in place of the WGG of EstCE1, causing a dramatic
decrease in active site hydrophobicity (Fig. 3D). This may explain
why 3ZYT is the only one of the investigated family VIII carboxy-
lesterases that does not exhibit any transacetylase activity.
Therefore, we had a closer look at the role of this motif in promis-
cuous acyltransferase activity. By mutation of the HDG of 3ZYT
to HGG or WGG (Fig. 3C), we rationally transformed a ‘hydrolase’
into an ‘acyltransferase’ comparable to MsAcT, the current bench-
mark for promiscuous acyltransferase activity.[5,6]
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form 3ZYT, an esterase without promiscuous acetyltransferase
activity, into an acetyltransferase. To the best of our knowledge,
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Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft
(DFG, German Research Foundation) 231396381/GRK1947 and
the Bundesministerium für Bildung und Forschung (Grant No.
031B0354B). We thank the B.R.A.I.N. AG (Zwingenberg, Ger-
many) for providing the expression plasmid for EstCE1. We also
thank Prof. Wolfgang Streit (Univ. Hamburg) and Dr. Klaus
Liebeton (B.R.A.I.N. AG) for useful discussions. We thank Schrö-
dinger Maestro for providing a trial license of their software. We
acknowledge access to beamline BL14.2 of the BESSY II storage
ring (Berlin, Germany) via the Joint Berlin MX-Laboratory spon-
sored by the Helmholtz Zentrum Berlin für Materialien und Ener-
gie, the Freie Universität Berlin, the Humboldt-Universität zu Ber-
lin, the Max-Delbrück Centrum, and the Leibniz-Institut für Mole-
kulare Pharmakologie.
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4
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Entry for the Table of Contents
From hydrolase to acyltransferase by a single mutation. Promiscuous acyltransferase activity is widespread in family VIII carboxy-
lesterases. A detailed structure-function analysis improved understanding of this remarkable phenomenon and enabled us to rationally
transform a hydrolase into an acyltransferase by introducing a single mutation.
Institute and/or researcher Twitter usernames: @Wissen_lockt
5
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