Evolving the Promiscuity of Elizabethkingia meningoseptica Oleate Hydratase for the Regio- and Stereoselective Hydration of Oleic Acid Derivatives

Article abstract of DOI:10.1002/anie.201901462

The addition of water to non-activated carbon–carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio- and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D-structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18-fold improved conversion of OA derivatives, while retaining the excellent regio- and stereoselectivity in the olefin hydration reaction.

Full text of DOI:10.1002/anie.201901462

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Accepted Article
Title: Evolving the Promiscuity of Elizabethkingia meningoseptica
Oleate Hydratase for the Regio- and Stereoselective Hydration of
Oleic Acid Derivatives
Authors: Matthias Engleder, Gernot A Strohmeier, Hansjörg Weber,
Georg Steinkellner, Erich Leitner, Monika Müller, Daniel Mink,
Martin Schürmann, Karl Gruber, and Harald Pichler
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901462
Angew. Chem. 10.1002/ange.201901462
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10.1002/anie.201901462
Angewandte Chemie International Edition
COMMUNICATION
Evolving the Promiscuity of Elizabethkingia meningoseptica
Oleate Hydratase for the Regio- and Stereoselective Hydration of
Oleic Acid Derivatives
Matthias Engleder,[b]+ Gernot A. Strohmeier,[b,c]+ Hansjoerg Weber,[c] Georg Steinkellner,[b,d] Erich
Leitner,[e] Monika Mueller,[f] Daniel Mink,[f] Martin Schuermann,[f] Karl Gruber,[b,g] and Harald Pichler[a,b]
*
Abstract: The addition of water to non-activated carbon-carbon Fatty acid hydratases (FAHYs; EC 4.2.1.X) catalyze the formation
double bonds catalyzed by fatty acid hydratases (FAHYs) allows for of medium- and long-chain hydroxylated fatty acids by addition of
highly regio- and stereoselective oxyfunctionalization of renewable oil water to isolated carbon-carbon double bonds of free mono- or
feedstock. So far, the applicability of FAHYs has been limited to free polyunsaturated fatty acids.[1–5] As such, they provide access to
fatty acids, mainly owing to the requirement of a carboxylate function secondary and tertiary alcohols, which makes them valuable tools
for substrate recognition and binding. Here, we describe for the first for the production of a variety of chemicals, including flavor
time the hydration of oleic acid (OA) derivatives lacking this free additives,[6-8] surfactants, lubricants and precursors in polymer
carboxylate by the oleate hydratase from Elizabethkingia chemistry.[9–13] The use of FAHYs in synthesis promises obvious
meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D- advantages relating to their exquisite regio- and stereoselectivity,
structure and a sequence alignment uncovered conserved amino acid which permits reactions that are not possible with unselective
residues at the entrance of the substrate channel as target positions acid-catalyzed chemical hydrations.[14-21] Moreover, FAHYs are
for enzyme engineering. Exchange of selected amino acids gave rise not impeded by the common challenges of other enzymes applied
to OhyA variants which showed up to an 18-fold improved conversion in hydroxylation reactions, which are often limited by low
of OA derivatives, while retaining the excellent regio- and expression, poor stability and/or complex electron transport
stereoselectivity in the olefin hydration reaction. Our study expands chains for nucleotide cofactor regeneration.[1,2,20-27] This has led to
the range of applications of this enzyme class beyond the hydration a growing attention for this class of hydro-lyases for establishing
of free fatty acids and provides the first report on an engineered FAHY new routes in sustainable organic syntheses. Most known FAHYs
with enhanced catalytic activity for OA derivatives.
are highly regioselective in hydrating either the cis-9 or cis-12
double bond(s) of unsaturated fatty acids.[5,28-33] The only enzyme
described so far showing a broader substrate scope is a FAHY
isolated from Lactobacillus acidophilus NTV001 (FA-HY1). It
catalyzes the hydration of cis-9, cis-12, cis-13, cis-14 or cis-15
double bonds of free fatty acids with different chain lengths and
degrees of saturation, but not of the esters thereof,[34] which
highlights limitations of the substrate scope. In contrast, the
collection of 2046 putative FAHY sequences in a hydratase
[a]
Prof. Dr. H. Pichler
Institute of Molecular Biotechnology
Graz University of Technology, NAWI Graz, BioTechMed Graz
Petersgasse 14, 8010 Graz (Austria)
E-mail: harald.pichler@tugraz.at
engineering database (HyED) reflects
a hitherto largely
[b]
[c]
Dr. M. Engleder, Dr. G. A. Strohmeier, Dr. G. Steinkellner, Prof. Dr.
K. Gruber, Prof. Dr. H. Pichler
ACIB GmbH – Austrian Centre of Industrial Biotechnology
Petersgasse 14, 8010 Graz (Austria)
Dr. G. A. Strohmeier, Prof. Dr. H. Weber
Institute of Organic Chemistry
unexplored potential of this enzyme class to possibly catalyze
reactions that are unattainable with current synthetic
methodologies.[35]
The most thoroughly characterized FAHY to date is the
oleate hydratase from Elizabethkingia meningoseptica (OhyA, EC
4.2.1.53).[36–38] This enzyme catalyzes the regio- and
stereoselective hydration of oleic acid (OA), yielding (R)-10-
hydroxy stearic acid with an excellent ee of ≥ 98% and without the
need for co-factor recycling.[14,21,37,38] Currently, it is one of only
four FAHYs with a resolved X-ray crystal structure, and the only
one analyzed in complex with the essential, non-covalently bound
FAD cofactor.[33,38,39,40] By comparing the surrounding of the FAD
binding site of OhyA with other FAHY structures lacking a co-
crystallized cofactor, a structural role of the flavin for correct
arrangement of the active site was inferred.[5,32,38,39,40]
Despite the progress in research, the applicability of FAHYs
in an industrial setting is still limited, which can be mostly
attributed to their strict substrate requirements. All published work
demonstrated that a minimum distance of 7 carbons between the
essential carboxylate and the double bond in cis-configuration,
Graz University of Technology, NAWI Graz
Stremayrgasse 9, 8010 Graz (Austria)
Dr. G. Steinkellner
Innophore GmbH
Am Eisernen Tor 3, 8010 Graz (Austria)
Prof. Dr. E. Leitner
[d]
[e]
Institute of Analytical Chemistry and Food Chemistry
Graz University of Technology, NAWI Graz
Stremayrgasse 9, 8010 Graz (Austria)
Dr. M. Mueller, Dr. D. Mink, Dr. M. Schuermann
InnoSyn B.V.
[f]
Urmonderbaan 22, 6167 RD Geleen (The Netherlands)
Prof. Dr. K. Gruber
[g]
Institute of Molecular Biosciences
University of Graz, NAWI Graz, BioTechMed Graz
Humboldtstrasse 50, 8010 Graz (Austria)
These authors contributed equally to this work
[+]
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201901462
Angewandte Chemie International Edition
COMMUNICATION
along with a minimum fatty acid chain length of 11 carbons are
mandatory for conversion.[5,28,35,42] Hydration at a terminal carbon
in unsaturated fatty acids has also not been described so far, as
a partial positive charge at the primary carbon would not be
favored (Markovnikov’s rule).[38,43] Several structural requirements
for substrates to undergo OhyA-catalyzed hydrations were
recently bypassed by Demming and colleagues. They succeeded
in converting 1-decene to (S)-2-decanol in a reaction using whole
cells of Escherichia coli containing OhyA.[43] Strikingly, varying an
amino acid position (Ala248) located at the end of the alkyl binding
pocket to larger hydrophobic or aromatic residues allowed for
improved hydration of various short-chain 1-alkenes, and even
permitted the conversion of six out of 23 tested functionalized and
internal alkenes.[44] The activity of their catalytic system was,
however, strongly dependent on the addition of a carboxylic acid
as decoy molecule to arrange the binding of a non-covalently
joined “pseudo-fatty acid motif”, which is presumably formed in
situ by simultaneous alignment of the decoy carboxylic acid and
the terminal olefin to be hydrated.[43,44,45] These and own results
inspired us to further explore the promiscuity of oleate hydratases
based on applying OhyA as the model enzyme. Driven by our
primary focus on the role of the head group of a fatty acid in
substrate recognition, we performed conversion assays with nine
different OA derivatives (Scheme 1, entries 1a–1j). The rationale
behind our model substrate selection was to examine the
acceptance of head groups with different physiochemical
properties regarding size and hydrophobicity, as well as charge
and polarization, i.e. amine, amide, hydroxamic acid, alcohol and
short-chain ester groups. Additionally, we preferably selected OA
analogs that were already commonly used in various applications
and which could be easily obtained in more than 90% purity
through purification of commercially available materials or via
synthesis from appropriate precursors.
cells harboring recombinant OhyA. As indicated by characteristic
mass fragmentation patterns in GC-MS, non-physiological
substrates 1c–1g, as well as 1i were converted to the respective
10-hydroxy compounds 2c–2g and 2i (Figure S1–S7 and S9).
Control reactions using an OhyA-free E. coli strain showed no
hydration of any substrate and no side reactions (Figure S1–S10).
Products from biotransformations were extracted, purified and
verified by NMR analysis (Figure S11–S22, S25 and S26). We
were pleased to see that the (R)-alcohols of the hydrated fatty
acid derivatives were obtained with ee’s ≥ 95%. This confirmed
that the excellent stereoselectivities remained unchanged in
OhyA-catalyzed hydration reactions when using the selected non-
natural substrates. Whereas the OhyA-mediated conversion of
short-chain alkenes described by Demming et al.[43] required the
use of a carboxylic acid as decoy molecule, hydration of OA
derivatives catalyzed by the same enzyme was independent of
any co-substrate and worked in the absence of a free carboxylic
acid head group. Our study, therefore, presents the first hydration
of fatty acid derivatives devoid of a free carboxylate by the action
of an oleate hydratase, and, thus, breaks the paradigm of the
essential carboxylic acid function required in substrate recognition
by FAHYs.[5,22,28,34,42,43] In the tested reaction setups, only OA
derivatives 1b, 1h and 1j were not converted. We attribute the
non-conversion of 1b to the distinctly different properties of its
amine head group, which exists as a cationic ammonium salt
under our reaction conditions and, presumably, cannot bind to the
enzyme’s active site in a proper way to enable hydration. In case
of 1h and 1j, the hydrophobicity of the substrates combined with
the steric constraints of the entrance channel most likely
prohibited their conversion.
Even though we observed hydration of six out of nine tested
non-natural substrates, the enzyme was notably more active on
OA (1a). We considered this as a challenge for the development
of a strategy to improve enzyme activity on fatty acid derivatives
lacking a free carboxylate head group. To start this task, we
referred to our previous study,[38] in which we proposed that
several amino acid positions in OhyA (Gln265, Thr436, Asn438
and His442) close to the carboxylate of a docked OA molecule
mediate substrate binding. This was supported by an analysis of
the sequences compiled in the HyED, which showed that the
identified positions were conserved among most members of the
database (Figure S29).[35] Thus, we selected OhyA residues
Gln265, Thr436, Asn438 and His442 for rational amino acid
exchanges. Ultimately, our goal was to stabilize the interaction
between the enzyme and OA derivatives by introducing amino
At the beginning of our investigations, we incubated cell-
free lysate containing recombinantly expressed OhyA with the
series of non-natural substrates, but did not detect compound
peaks corresponding to hydrated products in GC-MS analyses.
Knowing that the previously reported enzyme activity for the
hydration of cis-9-undecenoic acid was also found low with cell-
free lysate, but could be greatly enhanced by using E. coli whole
cells as enzyme source, we decided to repeat the
biotransformations in a whole cell system and judge again the
enzyme’s activity on alternative substrates.[35,43]
Remarkably, after 96 h of biotransformation product from
most OA derivatives was indeed detected in assays with E. coli
E. coli whole cells expressing OhyA
0.2 mM FAD, 100 mM glucose
R
R
4
4
4
4
50 mM HEPES buffer pH 6.0
22 h, 30°C
1a-1j
(R
)-2a-2j
O
O
O
a
f
c
h
e
b
g
d
i
OH
NH2
O
OH
NH2
OH
N
H
O
O
O
O
O
j
O
O
O
O
Scheme 1. Regio- and stereoselective hydration of oleic acid (OA) and OA derivatives by E. meningoseptica oleate hydratase (OhyA). A whole cell E. coli biocatalyst
harboring OhyA was used in the biotransformation assays.
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10.1002/anie.201901462
Angewandte Chemie International Edition
COMMUNICATION
acid side chains that promote binding of the different head groups
(Table 1). In view of the advantages provided by a whole cell
system compared with cell-free lysates or purified enzymes, such
as the higher operational stability, easier handling and the
avoidance of tedious purification steps,[43,44] we decided to employ
recombinant E. coli whole cell biocatalysts throughout our work.
Hydration of 1c was improved by substituting the amide side
chains of Gln265 and Asn438 for hydroxyl or carboxyl functions.
Most prominently, we found that combining the substitutions in
OhyA variant Gln265Ser/Asn438Asp resulted in
a 2.7-fold
increase in formation of 2c (Figure S50). We explain this higher
activity of the double variant towards 1c with a more favorable
interaction between its head group compared to wild type-like
amino acids (Figure 1b). In case of 1d, we obtained a 3.6-fold
increase upon exchanging multiple substrate binding residues for
alanines in OhyA Gln265Ala/Thr436Ala/Asn438Ala (Figure S51),
whereas all enzyme variants harboring single amino acid
exchanges and most variants with two substitutions showed
Table 1. Conserved amino acid (AA) positions in the substrate binding
region of OhyA selected for the site-directed mutagenesis study. Amino
acids are colored according to the physiochemical properties of their side
chains: Hydrophobic: Black. Positively charged: Green. Negatively
charged: Red. Positively polarized, uncharged: Orange. Negatively
polarized, uncharged: Blue. Aromatic: Grey.
AA position
AA in wild type
enzyme
Exchanged for
265
436
438
442
Gln
Thr
Asn
His
Ala/Glu/Lys/Ser
Ala/Asn/Asp/Lys
Ala/Arg/Asp/Lys/Ser
Ala/Asn/Asp/Gln/Glu/Tyr
To unravel the involved complexity of oleyl amine (1b) and
oleamide (1c) binding, we introduced side chains providing
hydroxyl and carboxyl functions, while the acceptance of N-
hydroxy oleamide (1d) and oleyl alcohol (1e) was challenged by
selecting amide, amine and guanidinium groups, as well as
alanines. Binding of OA esters (1f–1j) was fostered by positioning
alanine residues at the apex of the substrate binding channel to
open up and increase the hydrophobicity at its entrance. In total,
we created 33 enzyme variants (19 single, 10 double, three triple,
and one quadruple variant) as authoritative subset to represent
various tactics in modifying the substrate recognition site. After
generation of the variants by site-directed mutagenesis, we
expressed them in E. coli BL21 Star (DE3) (see Table S2 for a
comprehensive list of all variants). The analysis of cell-free E.
coli lysates containing recombinant enzymes by SDS-PAGE
confirmed soluble and, overall, largely uniform expression of the
wild type enzyme and most variants (Figure S30). Only OhyA
His442Asp was prone to being expressed in insoluble fashion,
while Gln265Ala/Thr436Ala was not expressed at all and, thus,
not employed in activity assays.
We tested the activity of each variant for the physiological
substrate (1a) and, consequently, assessed the impact of the
amino acid exchanges on the conversion of OA derivatives (1b–
1j). While the highest hydration activity for 1a was still obtained
with the wild type enzyme (Figure S31), we were intrigued by the
fact that several substitutions affected the conversion of non-
natural substrates 1c–1j (Figure S33–S40), confirming our initial
selection of important residues for substrate binding (Figure 1a–f
and Figure S41). We noticed that the impact of each amino acid
exchange on enzyme activity was strongly dependent on its
location, which indicated a position specific effect of each
variation. We would also like to emphasize that the highly R-
selective hydration (ee ≥ 95%) was not affected for any of the
tested substrates, proving the broad utility of this approach for the
challenging asymmetric hydration of non-activated carbon-carbon
double bonds (Figure S42–S49).
Figure 1. Docking of oleic acid (1a) and oleic acid derivatives 1c–1j to the OhyA
3D structure after in silico mutagenesis of conserved substrate binding residues
265, 436, 438 and 442. Panels a–f show the enzyme variant – substrate
combination that resulted in the best conversion to provide structure-based
evidence for the impact of the amino acid exchanges. The hydrophobicity of the
enzyme cavity is represented by a color gradient from red (hydrophobic) to blue
(hydrophilic). Co-crystallized FAD (yellow) and the substrates in the best
docking mode are shown in stick representation. Substrate binding residues and
catalytic Glu122 and Tyr241 are highlighted. a) Docking of (1a) to the 3D
structure of OhyA wild type enzyme. b) Docking of oleamide (1c) to OhyA
Q265S/N438D. c) Docking of N-hydroxy oleamide (1d) to OhyA
Q265A/T436A/N438A. d) Docking of oleyl alcohol (1e) to OhyA
Q265A/T436A/N438A. e) Docking of methyl (1f), ethyl (1g) and n-propyl (1i)
oleate to OhyA Q265A/T436A/N438A. f) Docking of i-propyl (1h) and n-butyl (1j)
oleate to OhyA Q265A/T436A/N438A.
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10.1002/anie.201901462
Angewandte Chemie International Edition
COMMUNICATION
either
a substantially lower or wild type-like activity. We
Table 2. List of individually best OhyA-variants for the regio- and
stereoselective hydration of OA (1a) and OA derivatives (1c–1j) obtained
by site-directed mutagenesis of substrate binding residues.
hypothesize that the extended space in the substrate binding
region provided by the triple alanine variant resulted in a better
acceptance of the more sterically hindered 1d (Figure 1c).
Hydration of 1e was also 2-fold higher with the triple alanine
variant OhyA Gln265Ala/Thr436Ala/Asn438Ala (Figure S52). This
observation was somewhat counterintuitive to our initial
understanding of substrate recognition by OhyA, since we were
expecting that a larger opening of the substrate binding channel
would tend to destabilize the binding of the smaller head group of
1e. We therefore mainly attribute the higher conversion of 1e to
the introduction of a more hydrophobic environment, resulting in
a better compatibility of substrate and the binding region (Figure
1d).
Improvement
compared to
wild type[a]
Abs.
conf. at
C-10[c]
Best variant for
hydration reaction
Yield
/ %[b]
Compound
a
b
c
Wild type
-
-
93
0
R
-
No conversion
Gln265Ser/
Asn438Asp
2.7
3.6
2.0
5.2
8.8
54
8
R
R
R
R
R
R
R
R
d
e
f
Gln265Ala/Thr436Ala/
Asn438Ala
The activity of OhyA on short-chain OA esters 1f–1j could be
increased remarkably with variants harboring alanine residues at
the substrate entrance channel. While several single and double
alanine substitutions moderately improved ester conversions, the
triple alanine variant OhyA Gln265Ala/Thr436Ala/Asn438Ala
attracted our attention by showing a 5- to 18-fold improvement in
hydration of 1f, 1g and 1i (Figure S53, S54 and S56). Most likely,
this can be attributed to the larger and more hydrophobic opening
of the substrate binding channel, which was lined out by less
substrate-like amino acids in the wild type enzyme (Figure 1e).
Based on control experiments, we rule out that the hydration of
OA esters in E. coli cells occurred through sequential ester
hydrolysis,[46] oleic acid hydration and esterification of (R)-10-
hydroxy stearic acid. Co-incubation of 1f and 1g with methanol,
ethanol or i-propanol in bioconversions did not result in the
formation of any ester side product derived from a trans- or re-
esterification reaction (Figure S58 and S59). It is especially
noteworthy that selected OhyA double variants and the triple
alanine variant allowed the conversion of sterically demanding
fatty acid ester derivatives 1h and 1j (Figure 1f), whereas the wild
type enzyme was inactive on these structures (see Figure S8, S10,
S55 and S57 for GC-MS and GC-FID data, and Figure S23, S24,
S27 and S28 for NMR spectra). We also performed activity
assays with a respective quadruple alanine variant (OhyA
Gln265Ala/Thr436Ala/Asn438Ala/His442Ala), but did not end up
with conversion of any of the OA esters. This suggested that the
exchange of histidine at position 442 for an alanine was severely
affecting enzyme activity.
Gln265Ala/Thr436Ala/
Asn438Ala
30
6
Gln265Ala/Thr436Ala/
Asn438Ala
g
h
i
Gln265Ala/Thr436Ala/
Asn438Ala
12
1
Gln265Ala/Thr436Ala/
Asn438Ala
Wild type
inactive
Gln265Ala/Thr436Ala/
Asn438Ala
17.6
4
j
Gln265Ala/Thr436Ala/
Asn438Ala
Wild type
inactive
2
[a] Ratio of conversions obtained after 96 h compared to the wild-type
enzyme in application; quantified by GC-FID analysis. [b] Isolated yield after
chromatography. [c] ee‘s ≥ 95 % (R) in all cases as determined by 1H-NMR
after esterification of the 10-hydroxy group with (S)-(+)-O-acetylmandelic
acid.[38]
Acknowledgements
We thank Lhamo Yachungtsang, Michael Riedl and Myria
Bekerle-Bogner for excellent technical assistance. This work has
been supported by the Federal Ministry of Science, Research and
Economy (BMWFW), the Federal Ministry of Traffic, Innovation
and Technology (bmvit), the Styrian Business Promotion Agency
SFG, the Standortagentur Tirol, the Government of Lower Austria
and Business Agency Vienna through the COMET-Funding
Program managed by the Austrian Research Promotion Agency
FFG.
In summary, we describe the first regio- and stereoselective
addition of water to fatty acid derivatives without the prerequisite
of a free carboxylate. Stabilizing the interaction between the head
groups of OA derivatives and the substrate binding site of OhyA
by rational design markedly enhanced the activity on eight
different OA derivatives, including amide, hydroxamic acid,
alcohol and esters, while the highly R-selective hydration of the
cis-9 double bond (ee ≥ 95%) was retained in all cases (Table 2).
Our work allows for a deeper understanding of the substrate
recognition in this enzyme class and, to the best of our knowledge,
reports the first successful engineering of a FAHY towards
improved conversion of OA derivatives. This knowledge provides
a foundation for the development of FAHYs towards applications
beyond current limitations.
Keywords: Enzyme catalysis • hydro-lyases • fatty acid
hydratase • protein engineering • substrate promiscuity
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This article is protected by copyright. All rights reserved.

10.1002/anie.201901462
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Redefining the substrate spectrum
of oleate hydratases: We report on
highly regio- and stereoselective
hydration of oleic acid derivatives by
an oleate hydratase, and the design
and application of hydratase variants
with enhanced activity on such
Matthias Engleder,+ Gernot A.
Strohmeier,+ Hansjoerg Weber, Georg
Steinkellner, Erich Leitner, Monika
Mueller, Daniel Mink, Martin
Schuermann, Karl Gruber, and Harald
Pichler*
Page No. – Page No.
structures. Our study demonstrates
that the carboxylate of a free fatty
acid - previously considered essential
- is not mandatory for conversion,
which expands hydration biocatalysis
beyond inferred restrictions.
Evolving the Promiscuity of
Elizabethkingia meningoseptica
Oleate Hydratase for the Regio- and
Stereoselective Hydration of Oleic
Acid Derivatives
This article is protected by copyright. All rights reserved.