A Rational Active-Site Redesign Converts a Decarboxylase into a C=C Hydratase: "tethered Acetate" Supports Enantioselective Hydration of 4-Hydroxystyrenes

Article abstract of DOI:10.1021/acscatal.7b04293

The promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes catalyzed by ferulic acid decarboxylase from Enterobacter sp. (FDC-Es) depends on bicarbonate bound in the active site, which serves as a proton relay activating a water molecule fo

Full text of DOI:10.1021/acscatal.7b04293

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Article
Rational Active-Site Redesign Converts a Decarboxylase
into a C=C Hydratase: 'Tethered Acetate' Supports
Enantioselective Hydration of 4-Hydroxystyrenes
Stefan Payer, Hannah Pollak, Silvia Maria Glueck, and Kurt Faber
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b04293 • Publication Date (Web): 07 Feb 2018
Downloaded from http://pubs.acs.org on February 7, 2018
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ACS Catalysis
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Rational Active-Site Redesign Converts a Decarboxylase into a C=C
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-Hydroxystyrenes
†
†
†,‡
Stefan E. Payer, Hannah Pollak, Silvia M. Glueck and Kurt Faber†*
‡
Austrian Centre of Industrial Biotechnology c/o
†
Department of Chemistry, University of Graz, Heinrichstrasse 28/2, 8010 Graz, Austria
ABSTRACT: The promiscuous regioꢀ and stereoselective hydration of 4ꢀhydroxystyrenes catalyzed by ferulic acid deꢀ
carboxylase from Enterobacter sp. (FDC_Es) depends on bicarbonate bound in the active site, which serves as proton
relay activating a water molecule for nucleophilic attack onto a quinone methide electrophile. This 'coꢀfactor' is crucial
to achieve improved conversions and high stereoselectivities for (S)ꢀconfigured benzylic alcohol products. Similar efꢀ
fects were observed with simple aliphatic carboxylic acids as additives. Rational redesign of the active site by replacing
the bicarbonate or acetate 'coꢀfactor' by a newly introduced sideꢀchain carboxylate from an adjacent amino acid yielded
mutants which efficiently acted as C=C hydratase. A single point mutation of valine 46 to glutamate or aspartate imꢀ
proved the hydration activity by 40% and boosted the stereoselectivity 39ꢀfold in the absence of bicarbonate or acetate.
Keywords: Biocatalysis, Hydration, Enzyme Engineering, Decarboxylase, Hydratase
The ability of an enzyme to catalyze a reaction other
than its annotated 'natural' activity is known as catalytic
nitriles and thioethers without the need for bicarbonate
(Scheme 1b).1
9
1ꢀ3
promiscuity and is the result of evolutionary processes
upon the encounter of 'nonꢀnatural' substrates and the
The biocatalytic hydration of 4ꢀhydroxystyrenes opens
an easy access to the (S)ꢀ1ꢀ(4ꢀhydroxyphenyl)ethanol
structural motif (2) from renewable resources in a stereꢀ
oselective fashion without the need for protecting
groups. The product hydrate is a substructure of bioacꢀ
4ꢀ6
organism's strive for survival. This phenomenon is an
important criterium for the selection of enzymes for the
development of biocatalysts for the selective transforꢀ
mation of synthetic compounds, using rationally guided
20
21ꢀ22
tive molecules exerting herbicidal,
insecticidal,
7
or random based directed evolution protocols. Coumaric
23
antiꢀproliferative and (hepatitis Cꢀ) protease inhibition
acids and their derivatives constitute monomeric units of
24ꢀ25
activities.
Though little is known about the active
8
lignin in plant cell walls and are a major waste product
enantiomeric forms of these congeners, an improvement
of the biocatalytic styrene hydration would be of practiꢀ
cal interest to facilitate the exploitation of the benzylic
alcohols as chiral building blocks.
9ꢀ10
of palm oil manufacture.
is achieved by ferulicꢀ and phenolic acid decarboxylases
FDCs and PADs, respectively). These enzymes cleave
their substrates into CO and the respective 4ꢀ
In nature, their degradation
(
2
hydroxystyrenes (1, Scheme 1a), which constitute undeꢀ
11
sired offꢀflavor components in beer and wine but have
also found application as renewable building blocks for
polymers with interesting dielectric properties.1
2ꢀ14
By
supplying an excess of CO in the form of bicarbonate,
2
the process can be run in the reverse βꢀcarboxylation for
the production of substituted coumaric acid derivatives
15ꢀ17
(
Scheme 1a).
During studies on the regioselective
carboxylation, the promiscuous hydration of 4ꢀ
hydroxystyrene derivatives by FDCs and PADs in the
presence of bicarbonate was discovered (Scheme 1b),
best results were obtained with a ferulic acid decarboxꢀ
18
ylase from Enterobacter sp. (FDC_Es). The nucleophile
scope apart from water was extended to methoxyamine,
cyanide and propanethiol, which add via an analogous
mechanism to furnish (S)ꢀconfigured benzylic amines,
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Scheme 1. a) Reversible (de)carboxylation of coumaric
acids and b) promiscuous nucleophile addition to 4ꢀ
hydroxystyrenes catalyzed by phenolicꢀ and ferulic acid
decarboxylases.
1
2
3
4
5
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7
8
9
1
1
1
1
1
1
1
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Thr74
Thr74
Thr74
H
O
H
H
H
O
O
O
H
O
H
O
H
H
H
NH2
NH
NH
2
2
NH
2
H
H
N
H
O
O
O
O
N
N
O
Glu72
Arg49
O
Glu72
O
Glu72
Arg49
Arg49
NH2
NH2
O
O
O
O
O
O
H
O
H
H
O
H
H
H
H
H
O
H
O
H
Si-face attack
of water
H
tautomerization
H
H
H
O
H
O
Tyr27
O
Tyr27
O
Tyr27
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
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0
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8
9
0
OH
OH
OH
H
O
H
H
O
O
O
H
O
H
O
H
O
Tyr19
Tyr39
Tyr39
Tyr21
O
Tyr19
Tyr21
Tyr39
O
Tyr19
Tyr21
Scheme 2. Mechanism of the FDC_Es catalyzed hydration of 4ꢀhydroxystyrene with bicarbonate acting as 'coꢀfactor' for
protonꢀtransfer.
18
Experimental data and quantum mechanical calculaꢀ
nificant positive effects on the stereoselectivity were
detected with all aliphatic carboxylates, depending on
the side chain size (Et > Pr > Bu ≈ Me ≈ H) with a maxꢀ
imum for propanoate (74 % e.e.). Trifluoroacetate neiꢀ
ther enhanced conversion nor the e.e., due to its low pK ,
which disables it to act as catalytic acid in proton transꢀ
fer (c.f. Scheme 2). Due to the formation of an unidentiꢀ
fied side product, only 60% of the starting material was
recovered in presence of formate.
2
6
tions suggest, that the promiscuous hydration of 4ꢀ
hydroxystyrene requires a bicarbonate anion as 'cofacꢀ
tor', which is bound onto Arg49 in the active site of the
decarboxylase to achieve good conversion and high e.e.
of the benzylic alcohol product (S)ꢀ2 (Scheme 2). Potasꢀ
sium bicarbonate (0.5 M, pH 8.5) promotes the hydraꢀ
tion of 4ꢀvinylphenol (1) with ~65% conversion and
80% e.e. using FDC_Es as biocatalyst. In the absence
of bicarbonate, however, a water molecule occupies its
position, resulting in (almost complete) loss of stereoseꢀ
i
t
a
18
~
2
6
lectivity.
Consequently, supplementation of bicarꢀ
bonate (>0.5 M) is required to maintain stereoselectivity,
which renders this process economically and operationꢀ
ally less attractive.
In initial studies, the compensability of bicarbonate as
cofactor' was investigated by replacing it with different
anionic) additives (Figure 1). Potassium phosphate
'
(
buffer (50 mM) was used as reaction buffer and the pH
was adjusted to 8.0 after dissolving the additive salts to
avoid bicarbonate decomposition (pKaH = 7.7) and to
allow for direct comparison of additive effects. Control
reactions in neat phosphate buffer gave moderate conꢀ
version (~60%) but completely lack stereoselectivity.
–
2–
2–
Addition of Cl , HPO
4
or SO
4
improved the converꢀ
sion slighty, while imidazole had adverse effects, again
without stereoinduction. Borate gave low e.e. (15%),
while conversion and e.e. were significantly improved by
bicarbonate. Remarkably, best values for conversion and
e.e. were obtained in the presence of acetate. Next, the
concentration of bicarbonate and acetate was varied
within a range of 10 and 500 mM (Figure S3). While the
conversion profiles for both ions showed a similar behavꢀ
ior at increasing ion concentration (leveling off at ~90–
9
5% conv. and 250 and 300 mM), the product e.e. profile
with bicarbonate differs from that with acetate in a shalꢀ
low maximum of 33% e.e. (at ~100 mM), while the e.e.
rises linear with increased acetate concentration, reachꢀ
ing an e.e. of 45% at 500 mM. Since the carboxylate moiꢀ
ety appears to be an ideal promoter for stereoselective
hydration, a set of carboxylic acid sodium salts with
varying chain lengths, substituents and branching patꢀ
terns was compared at neutral pH (7.0) (Figure 1b). Sigꢀ
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Figure 1. Asymmetric hydration in presence of (anionic)
additives. Conditions: substrate 1 (10 mM, from 10 % w/w
propylene glycol solution) in potassium phosphate buffer
FDC_Es (20 mg/mL, 32 U). Effect of (a) various anions (0.5
M, pH 8.0) and (b) different carboxylates (0.5 M, pH 7.0)
on the enzymatic hydration (additives are shown in the
respective protonation state at the corresponding pH).
1
2
3
4
5
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7
8
9
(50 mM), lyophilized E. coli cells containing wild type
1
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1
1
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Figure 2. FDC_Es variants with tethered bicarbonate/acetate surrogates in positions Val46 and Arg49 (indicated by dashed
circles). The quinoneꢀmethide form of substrate 4ꢀvinylphenol (green) was docked into the active site of FDC_Es (PDB ID
4
UU3) using the UCSF Chimera AutoDock Vina plugin. Catalytic key residues are shown in blue, Val46 in red and others in
gray. Dotted red spheres indicate water molecules in the superimposed apoꢀstructure. Surrogate residues at positions 46 and
9 are shown in their protonation state at pH 7.0. Conditions: substrate 1 (10 mM) in potassium phosphate buffer (50 mM, pH
7.0), lyophilized E. coli whole cells containing FDC_Es variants (20 mg/mL).
4
Based on these promising results, we envisioned the
design of mutants bearing a 'tethered carboxylate' acting
as catalytic residue in the active site to mediate the proꢀ
ton transfer, thereby waiving the need for an external
bicarbonate/carboxylate 'coꢀfactor'. The amino acid resiꢀ
dues introduced should meet two crucial requirements:
vate it as catalytic base within the hydrogen bond netꢀ
work of Tyr27, Glu72 and Arg49 (Figure 3). Changing the
latter into a glutamate residue (thereby inverting the
positive charge at this position into a negative one) could
gain some additional hydration activity (86 % conv.) but
yielded racemic product. Tyr39 was suggested to dictate
the (S)ꢀstereoselectivity through steric repulsion between
27
(
i) the spatial arrangement in the active site needs to
mimic bound bicarbonate, suggesting residues near the
bicarbonate bindingꢀsite as targets for mutagenesis, and
its aromatic ring and C of the quinone methide interꢀ
β
mediate, which consequently presents its Siꢀface to the
water nucleophile located above the plane (Scheme 2).2
A double variant having both the beneficial Val46Glu
and a neutral Arg49Met mutation did not affect the conꢀ
version but showed a decrease in stereoselectivity comꢀ
pared to the single mutant. Removal of the hydrogen
bond donor Arg49 weakens the bonding network and
renders Tyr39 less rigid, which is responsible for the
lower e.e. of the product. The Val46Glu mutant was also
tested with other nucleophiles (methoxyamine, cyaꢀ
nide),1 which gave improved stereoselectivities comꢀ
pared to the wildꢀtype enzyme (Table S3).
6
(
ii) residues must be chemically competent surrogates of
bicarbonate/carboxylate to shuttle protons between the
substrate C and Glu72.
β
After detailed inspection of activeꢀsite residues of
FDC_Es, Val46 located on a mobile loop in vicinity to the
bicarbonate bindingꢀsite was selected for mutations.
Based on their ability to act as threeꢀatom proton shuttle,
Val46 was exchanged to Glu, Gln, Asp, His and Arg (Figꢀ
ure 2). The validity of this strategy was proven by the fact
that the introduction of a carboxylate group in position
9
4
6 by aspartate (Val46Asp) or glutamate (Val46Glu)
enhanced the stereoselectivity 39ꢀfold and boosted the
conversion by ~40%. The slightly higher stereoselectivity
(
ca. 6%) for the Asp versus the Glu mutant may be exꢀ
plained by the one CH ꢀunit shorter tether, which is
2
pulling the water nucleophile further out of the plane of
symmetry towards the Siꢀface of the substrate (Figure 3).
Apparently, only a carboxylate moiety is an efficient
bicarbonate/acetate mimic, since mutations to histidine,
glutamine and arginine were not beneficial (max. 57%
conv. for Val46Arg). However, despite its lower activity,
the Val46His mutant showed slight stereoselectivity (up
to 17% e.e.).
A homology model of the Val46Asp/Glu mutants reꢀ
veals that the additional carboxylate groups are well
positioned in proximity to a water molecule (W1) to actiꢀ
Figure 3. Overlay of the active site structures of the
FDC_Es Val46Glu and Val46Asp variant (Swissmodel webꢀ
server). Mutated residues Asp/Glu46 are highlighted in
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orange and the docked quinone methide form of the subꢀ
We thank Georg Steinkellner for his advice in structural
biology and Fahmi Himo for fruitful discussions on the
reaction mechanism and thermodynamics.
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strate 4ꢀvinylphenol is shown in green (Autodock Vina
plugin, UCSF Chimera). Water molecules shown as red
spheres are derived from the superimposed wildꢀtype
FDC_Es crystal structure (PDB ID: 4UU3). Distances are
given in Å.
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scale with 0.8 mol% of FDC_Es V46E to afford 68 mg of
S)ꢀhydrate product (60% yield) with high enantiomer
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(
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Gopalan, N.; RodríguezꢀDuran, L. V.; Saucedoꢀ
In conclusion, prompted by the observation of bicarꢀ
bonateꢀ and acetateꢀassisted asymmetric hydration of
hydroxystyrenes catalysed by ferulic acid decarboxylase
we rationally designed a hydratase from this decarboxꢀ
ylase through mutation of an Aspꢀ or Gluꢀcarboxylate
moiety into the active site, which efficiently functions as
protonꢀshuttle. The mutants showed 40% higher activity
and 39ꢀfold improved stereoselectivity, which allowed
preparativeꢀscale transformations with turnover numꢀ
bers of up to 220.
Castaneda, G.; Madhavan Nampoothiri, K., Review on
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website.
Experimental details, preparation of variants, docking and
details on structural biology as well as supporting screening
results (PDF).
1
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AUTHOR INFORMATION
Corresponding Author
Kurt.Faber@uniꢀgraz.at, phone: +43ꢀ316ꢀ380ꢀ5332, fax:
43ꢀ316ꢀ380ꢀ9840
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Jin, X.; Zhang, S.; Runt, J., Dielectric Studies of
Blends of Poly(ethylene oxide) and Poly(styreneꢀcoꢀpꢀ
hydroxystyrene). Semicrystalline Blends. Macromolecules
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+
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1
5. Wuensch, C.; PavkovꢀKeller, T.; Steinkellner, G.;
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
Gross, J.; Fuchs, M.; Hromic, A.; Lyskowski, A.; Fauland, K.;
Gruber, K.; Glueck, S. M.; Faber, K., Regioselective Enzymatic
betaꢀCarboxylation of paraꢀHydroxystyrene Derivatives
Catalyzed by Phenolic Acid Decarboxylases. Adv. Synth. Catal.
2015, 357, 1909ꢀ1918.
Funding Sources
16. Wuensch, C.; Glueck, S. M.; Gross, J.; Koszelewski,
D.; Schober, M.; Faber, K., Regioselective Enzymatic
Carboxylation of Phenols and Hydroxystyrene Derivatives. Org.
Lett. 2012, 14, 1974ꢀ1977.
Funding by the Austrian Science Fund (FWF project
P26863) and the Austrian BMWFW, BMVIT, SFG, Standorꢀ
tagentur Tirol, Government of Lower Austria and ZIT
through the Austrian FFGꢀCOMETꢀFunding Program is
gratefully acknowledged.
1
7.
Wuensch, C.; Schmidt, N.; Gross, J.; Grischek, B.;
Glueck, S. M.; Faber, K., Pushing the Equilibrium of Regioꢀ
complementary Carboxylation of Phenols and Hydroxystyrene
Derivatives. J. Biotechnol. 2013, 168, 264ꢀ270.
ACKNOWLEDGMENT
18.
Wuensch, C.; Gross, J.; Steinkellner, G.; Gruber, K.;
Glueck, S. M.; Faber, K., Asymmetric Enzymatic Hydration of
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ACS Catalysis
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