Selectivity in the Cyclization of Citronellal Introduced by Squalene Hopene Cyclase Variants

Article abstract of DOI:10.1002/cctc.201700734

The squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC) is a highly efficient enzyme catalyst for stereoselective Br?nsted acid catalysis. We engineered AacSHC to catalyze the selective Prins cyclization of citronellal. Four active site variants were identified for the diastereoselective cyclization of (S)-citronellal to stereoisomers (?)-iso-isopulegol, (+)-isopulegol and (?)-neo-isopulegol, respectively. The replacement of active site residues resulted in two triple variants that catalyzed the transformation of (R)-citronellal to give the isomers (+)-neo-isopulegol and (?)-isopulegol with up to >99 % de, respectively. The newly designed library of functionally diverse active site geometries exhibits high selective control during citronellal cyclization, leading exclusively to a single diastereomer of the desired isopulegol. Whereas the cyclization of citronellal with chemical catalysts was observed to produce the isopulegol isomer with the lowest energy, the reaction with AacSHC variants proceeded with higher product selectivity. The results of this study show that variants of AacSHC are excellent catalysts for the highly selective formation of isopulegol stereoisomers.

Full text of DOI:10.1002/cctc.201700734

Accepted Article
Title: Enabling New Selectivities in the Cyclization of Citronellal by
Squalene Hopene Cyclase Variants
Authors: Silke A Bastian, Stephan C Hammer, Nico Kreß, Bettina M
Nestl, and Bernhard Hauer
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10.1002/cctc.201700734
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Enabling New Selectivities in the Cyclization of Citronellal by
Squalene Hopene Cyclase Variants
Silke A. Bastian,[a] Stephan C. Hammer,[a] Nico Kreß,[a] Bettina M. Nestl,[a] and Bernhard Hauer*[a]
Abstract: The squalene hopene cyclase from Alicyclobacillus
acidocaldarius (AacSHC) is a highly efficient enzyme catalyst for
stereoselective Brønsted acid catalysis. We engineered AacSHC to
catalyze the selective Prins cyclization of citronellal. Four active site
variants catalyzed the diastereoselective cyclization of (S)-citronellal
to stereoisomers (-)-iso-isopulegol, (+)-isopulegol and (-)-neo-
isopulegol, respectively. The replacement of active site residues
resulted in two triple variants that catalyzed the transformation of (R)-
citronellal to give the isomers (+)-neo-isopulegol and (-)-isopulegol
with up to >99% de, respectively. The newly designed library of
functionally diverse active site geometries exhibits high selective
control during citronellal cyclization, leading exclusively to a single
diastereomer of the desired isopulegol. While the cyclization of
citronellal with chemical catalysts was observed to produce the
isopulegol isomer with the lowest energy, the reaction with AacSHC
variants proceeded with higher product selectivity. The results of this
study show that variants of AacSHC are excellent catalysts for the
highly selective formation of isopulegol stereoisomers.
isopulegol and neoiso-isopulegol. The selectivity of organic
reactions for which multiple products can be formed is directed by
the energies of different transition state structures that lead to the
energy minimum for each product. In the Prins reaction of 1 to 2,
several reactive conformations could in principle occur that differ
in their relative energies. These are attributable to the steric
hindrance of the substituent during substrate folding.
Conformations with the lowest repulsive energies possess the
lowest relative energies and are therefore energetically most
favored. In an achiral surrounding, the stereoselectivities or
relative abundance of isomers can be estimated using the
Boltzmann distribution. The ratio of isopulegol formation at
ambient temperature is 0.77 (2), 0.15 (neo-2), 0.07 (iso-2) and
0.01 (neoiso-2).[2,3] The formed carbocation intermediates have
the same structure as the four possible isopulegol stereoisomers
and the deprotonation of each carbocation species leads to a
corresponding isopulegol isomer.
Introduction
The Prins reaction is a synthetically very useful reaction since it
enables the formation of a C-C bond with the construction of two
vicinal stereocenters. One of the most studied intramolecular
version of this reaction is the cyclization of citronellal affording
isopulegol. The acid-catalyzed cyclization of (R)-citronellal (1) to
(-)-isopulegol (2) is an important step in the synthesis of (-)-
menthol, extensively used in pharmaceuticals, agrochemicals,
cosmetics and natural compounds with biological activities.[1] The
reaction is catalyzed by the protonation of the carbonyl group of
citronellal and proceeds via an intramolecular rearrangement
followed by a deprotonation leading to the formation of isopulegol
(Scheme 1). The presence of three stereogenic centers causes
the formation of four stereoisomers of isopulegol, each of which
occurs as a pair of enantiomers: isopulegol, neo-isopulegol, iso-
Scheme 1. Schematic representation of the cyclization of (R)-citronellal (R)-1
to isopulegol isomers (-)-isopulegol (-)-2, (+)-neo-isopulegol (+)-neo-2, (+)-
neoiso-isopulegol (+)-neoiso-2 and (+)-iso-isopulegol (+)-iso-2.
Chemical approaches including the cyclization of
1 over
homogeneous and heterogeneous catalysts were investigated
producing 2 as the predominant isomer followed by neo-2, iso-2
and neoiso-2. Isopulegol (2) having the substituents of the
cyclohexane ring in an equatorial position is about 15 kJ mol-1
more stable than neoiso-2.[2] High isopulegol (2) yields were
obtained with most catalysts, but the ratio of isopulegol
diastereomers differed strongly. By using heterogeneous
catalysts including hydrous zirconia, zeolites, mesoporous silica
catalysts and other solid acids fractions of isopulegol (2) of about
70 - 75% were obtained representing the thermodynamic
equilibrium.[2,4–7] Higher 2 ratios > 90 % have been reported with
homogenous catalysts such as ZnBr2[8] and Sc(OTf)3[9]. With novel
Lewis acids like Sn-Beta[10] and Zr-Beta zeolites[11] the
[a]
Dr. S. A. Bastian, Dr. S. C. Hammer, Dr. B. M. Nestl, Prof. Dr. B.
Hauer
Institute of Technical Biochemistry
Universitaet Stuttgart
Allmandring 31, 70569 Stuttgart, Germany
E-mail: bernhard.hauer@itb.uni-stuttgart.de
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/cctc.201700734
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diastereoselectivity to 2 was further increased to 93 %. In addition
to the desired cyclization of 1 to 2, side product formation such as
dimerizations of citronellal (1) and isopulegol (2) and the
defunctionalization of 2 was observed. In recent years, the field of
supramolecular catalysis has also progressed towards the Prins
cyclization of citronellal (1) and related substrates.[12–14]
Furthermore, the enantioselective Prins cyclization of achiral
citronellal analogues was enabled using enantiopure
supramolecular scaffolds in up to 69 % ee. These synthetic hosts
contain well-defined cavities for stabilization of high-energy
intermediates and transition states. The proposed mechanism of
catalysis is similar to that employed by terpene cyclase enzymes,
which control reaction outcomes by selectively stabilizing cationic
intermediates and transition structures through multiple
interactions with aromatic residues lining the active site.[15–18]
variants to control reactive citronellal conformers to yield different
isopulegol isomeric products.
Results and Discussion
The binding pocket of AacSHC is able to control the reactivity of
high-energy intermediates through a series of non-covalent
interactions and substrate preorganization in order to selectively
form molecules that would be difficult to access in bulk solution.
Taking advantage of the evolvability of AacSHC, we started to
examine how many modifications within the active site of AacSHC
are needed to bind and activate (1) in different reactive
conformations leading to different isopulegol isomers. 19 active
site residues (L36, W169, I261, S307, W312, F365, Q366, A419,
Y420, V448, W489, G490, Y495, G600, F601, F605, L607, Y609
and Y612) were chosen as the target amino acid residues for
mutagenesis (see Figure 1). In order to retain the hydrophobicity
of the active site, we introduced small to large hydrophobic amino
acids (A, G, F, L, V and W) with varying side chains. Our previous
work revealed that I261 and Y420 are crucial for the selective
formation of (-)-iso-2 from (S)-1, with residue I261 being located
14 Å from the proton source D376.
Squalene hopene cyclases (SHCs) have attracted interest due to
their ability to synthesize bioactive polycyclic compounds that are
challenging to access through conventional chemical
synthesis.[19] Various non-natural substrates have been
selectively converted to important polycyclic molecules.[15,20–22]
A
conserved aspartic acid acts as Brønsted acid for the protonation
of linear (poly)isoprene compounds. The active site shape plays
a tremendously important role during catalysis, as it displays the
ideal chiral hydrophobic environment that is necessary to
productively bind the substrate into an active site-defined
arrangement while simultaneously stabilizing the evolving cationic
key intermediates.[23,24] In earlier work, Siedenburg et al. reported
the investigation of twelve SHCs to catalyze the cyclization of
citronellal 1 via the protonation of the carbonyl functionality.[25,26]
They discovered the SHC1 from the bacterium Zymomonas
mobilis (ZmoSHC1) to form isopulegol (2) with 1 as substrate.
Furthermore, two variants (F486C and W555Y) were identified as
important for increased (2) formation. However, the selectivities
for these biotransformations were low resulting in the formation of
a mixture of isopulegol isomers.
We and others disclosed the conversion of non-natural
monoterpenoid substrates in water by engineering the active site
of the SHC from Alicyclobacillus acidocaldarius (AacSHC) to
create novel hydrophobic active site templates for chiral Brønsted
acid catalysis.[3,27,28] We targeted residues located within 15 Å of
the natural squalene binding site for site-directed mutagenesis
(PDB code: 1UMP[29]). We obtained an engineered variant of
AacSHC (I261A) that produced exclusively (-)-iso-2 from (S)-1
with moderate conversion. The excellent diastereoselectivity of
> 99 % de highlighted the exclusive conformational control by
binding the substrate in one specific reactive conformation,
forcing the methyl group into the unfavored axial orientation. As
the results obtained from the engineering of AacSHC indicated
that generating active site mutations is efficient in converting the
(S)-enantiomer of citronellal (1) into (-)-iso-2, we further
engineered the active site of AacSHC. We thus aimed to obtain
Figure 1. Mutagenesis of the active site of AacSHC for the selective formation
of isopulegol isomers from citronellal (1). Representation of the residues lining
the active site of AacSHC. The catalytic aspartic acid (D376) is shown in green
sticks, the residues targeted by mutagenesis are shown in black sticks with
diastereoselective variants in bold. The average distance between the carboxyl
group of the catalytic D376 and the hydrophobic residues of active variants
(A419, Y609, F365, Y420, G600, W169, I261) is 6.7 to 13.7 Å. The distance
between D376 and W312 is 4.3 Å.
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chromatography. The activity of the wildtype and designed
variants for the cyclization of 1 was investigated by monitoring the
formation of 2 using gas chromatography. Activities of the
different variants are shown in Table 1 and Supporting Information
Figures S1-S4. Several variants emerged that displayed
moderate monoterpenoid cyclase activity with high selectivities
> 99 % de and initial activities up to 8% conversion (Table 1 and
Tables S1-S2). Four AacSHC variants exhibited preference
towards in each case (only) one isomer of 2 (Figure 2). Three of
these positive variants had single point mutations to alanine
(I261A, W169A, Y609A), while the tryptophan at position 312 was
mutated to glycine. The modifications of amino acids located in
close proximity to the reactively bound substrate within AacSHC’s
active site cavity, might directly influence the mode of substrate
binding and folding. We assume that the creation of additional
space through the introduction of small hydrophobic side chains
might promote a certain preorganization of the substrate molecule.
The most active variants I261A and W169A catalyzed the reaction
to give (-)-iso-2 in > 99 % de. Similar to the best variant I261A
from our first mutant library, residue W169 is located at a
neighboring position at the back end of the enzyme’s active site
pocket (see Figure 1). Substitutions W312G and Y609A resulted
in the formation of (-)-neo-2 and (+)-2, respectively, whereby
diastereoselectivities of > 99 % de and up to 2-fold increased
conversions compared to the wildtype were obtained. Both
residues are in close proximity to the catalytic D376 and the
bound substrate and thus will have an active influence on binding
and folding of the substrate in the active site. The screening of the
library, however, failed to yield any mutant for the formation of (-)-
neoiso-2.
Table 1. Product formations [%] in the conversion of citronellal enantiomers
(S)- and (R)-1 with AacSHC wildtype and variants after 40 h.
(S)-1
wildtype
I261A[3]
W169A[3]
Y609A
(+)-2
(-)-iso-2
1.15 ± 0.01
7.70 ± 0.22
7.90 ± 0.23
(-)-neo-2
(-)-neoiso-2
2.65 ± 0.19
W312G
0.47 ± 0.07
(R)-1
wildtype
Y609G
(-)-2
(+)-iso-2
(+)-neo-2
0.11 ± 0.09
3.36 ± 0.10
4.64 ± 0.18
+)-neoiso-2
0.33 ± 0.14
2.59 ± 0.38
2.58 ± 0.11
The screening of the AacSHC mutant library with (R)-1 has shown
that while the reaction of the (S)-enantiomer is facilitated by single
point mutagenesis, its impact is smaller for the (R)-enantiomer.
Similar conversions were obtained in the cyclization of the (R)-
enantiomer using single point variants (see Supporting
Information Tables S4-S5). However, no variant was detected
that demonstrated excellent product selectivity. In contrast, the
best variants A419G, Y420C and Y609G provided a mixture of (-)-
2, (+)-neo-2 and (+)-iso-2 (Table 1 and Table S4). This suggests
that single amino acid exchanges in the active site of AacSHC are
not able to stabilize the reactive binding of the substrate in one
specific productive conformation. To investigate how such a
product specific AacSHC variant could be crafted, the active site
mutant library was further extended.
F365A/
Y420W/
G600F
A419G/
Y420C/
G600Aa
9.09 ± 0.03
1.26 ± 0.03
1.55 ± 0.14
A419G/
Y420C/
G600Ab
13.25 ± 0.42
[a] Reaction performed at 30°C using 2 mM (R)-citronellal, 0.2 % (v/v) Triton
X-100 and 3-5 mg/ml purified protein. [b] Reaction performed at optimized
reaction conditions at 20°C with 2 mM (R)-citronellal, 0.1 % (v/v) Triton X-100
and 2 mg/ml purified protein.
We have observed that substitution of isoleucine at position 261
to smaller alanine or glycine residues resulted in enhanced
enzyme activity most likely by inducing a reorientation of the
neighboring amino acid side chains.[3] Individual mutations were
introduced into the AacSHC gene by site-directed mutagenesis.
AacSHC wildtype, variants plus deleterious variant (D376C) were
produced in Escherichia coli and purified by ion exchange
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We generated combination mutants of active site residues to
examine synergistic effects of these mutations on the activity and
selectivity in the cyclization of the (R)-enantiomer of 1. By
combining mutations at positions A419, Y420, F365 and G600,
eleven double as well as triple variants were created (Table S3).
Combinations of mutations of active site residues had beneficial
effects on the selectivity of AacSHC towards (-)-2 generating
novel diastereoselective cyclase variants. The generation of triple
variants improved the selectivity of AacSHC in the cyclization of
high degree of conservation.[32] In enzyme superfamilies, small
changes in the composition of amino acids of the active site can
create differing activities.[33,34] As active sites provide highly
optimized environments for catalysis, changes at these centers
can have large effects on e.g. enzyme activity or substrate
specificity.[35] It is striking that exchanges of several of these highly
conserved amino acid residues led to remarkably improved
variants for the cyclization of citronellal (1) with excellent
diastereoselectivity.
(R)-1.
Further
analysis
revealed
that
variant
A419G/Y420C/G600A was the most active variant (10-fold
increased conversion) and highly selective generating a mixture
of (-)-2 and (+)-neo-2 in a 9:1 ratio (Table 1 and Figure 2). Due to
the fact that we have observed in preliminary work that
temperature and the concentration of detergent used had
significant advantages in the cyclization of non-natural
compounds, we optimized the conditions for the cyclization of (R)-
1 using the most active variant A419G/Y420C/G600A. We noted
an 1.5-fold increase of product formation in cyclization reactions
containing less detergent (0.1 % (v/v) Triton X-100) and
decreasing reaction temperature from 30°C to 20°C. Utilizing half
the concentration of purified protein under optimized reaction
conditions yielded in
Conclusions
Our results demonstrate the feasibility of tailoring the central
active site pocket of AacSHC for the complex and highly selective
Prins reaction of citronellal (1) to isopulegol (2). With only few
mutations within the enzyme´s active site, amino acid positions
could be identified that play a crucial role in the specific binding of
the non-natural substrate and for the control of high energy
intermediates during the reaction. By screening the active site
library, several single and triple variants were obtained that shifted
the product distribution towards a targeted isomeric selectivity. In
contrast to chemical catalysts, the control of high-energy
carbocations in the cyclization of 1 were selective, leading to
specific isopulegol isomers. In the future, random mutagenesis
and screening will be used to identify amino acid substitutions to
increase the activity of AacSHC variants while maintaining their
high product selectivity.
Notably, variant F365A/Y420W/G600F selectively protonated the
(R)-enantiomer leading to the formation of (+)-neo-2 with an
excellent diastereoselectivity (>99% de) and 10-fold enhanced
conversion.
The high levels of selectivity achieved in the SHC-catalyzed Prins
reaction of 1 are the results of precise control over the substrate
conformation and its interaction with catalytic functional groups
within the active site. The observed product selectivity of active
site variants may be attributed to the enzyme’s innate
hydrophobic active site cavity that stabilizes the formation and
preservation of highly reactive carbocation species and shield
them from quenching by water molecules. Furthermore, the
directing effect of the substrate’s methyl group allows the
functional positioning and reactive folding of the substrate
molecule within the active site for cyclization. In certain active site
contours, the required positioning of this methyl group prior to
catalysis might be sterically impeded by surrounding amino acid
residues and thus might prevent the substrate enantiomer from
being productively bound. This observation is in accordance with
findings described in previous work showing that the positioning
of the methyl group of a terpene substrate plays an important role
for the convertibility by SHCs.[30,31] Single point mutations in the
active site of AacSHC are sufficient to entail a distinctive product
specificity through the selective binding and activation of the
substrate in differing reactive conformations. Bioinformatic
analyses, in particular of sequences of terpene cyclases, have
shown that active site residues of SHCs reveal an exceptionally
Experimental Section
Overexpression and solubilisation of AacSHC wildtype and variants
AacSHC wildtype and variants were overexpressed from a pET-22b
plasmid construct containing the AacSHC gene under control of an IPTG-
inducible T7 promotor (lac-operon). To overexpress the target enzymes,
TB medium containing 100 mg/ml ampicillin was inoculated with cultures
of recombinant E. coli BL21(DE3) and shaken overnight (16 h) at 37 °C
(180 rpm), whereby expression was started by auto-induction. Cells were
harvested by centrifugation at 7000 g at 4 °C and the cell pellet was
resuspended in 3x volume of lysis buffer containing DNAse I and 1 µM
phenylmethylsulfonyl fluoride. Cell lysis was done by ultrasonication on ice.
After centrifugation at 7000 g for 20 min, the supernatant was discharged,
the cell debris pellet was resuspended in the same volume (w/v) of
solubilization buffer (60 mM citrate pH 6.0, 1 % detergent Triton X-100)
and incubated over night while shaking at 4 °C. The suspension was heat-
shocked at 50 °C for 30 min and centrifuged for 20 min at 7000 g to partially
purify the thermostable cyclase enzymes from residual E. coli proteins.
Subsequently, the supernatant was further purified using anion exchange
chromatography.
Purification of AacSHC wildtype and variants via ion-exchange
chromatography
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Prior to usage, the column material diethylaminoethyl–sephacel (DEAE-
Sephacel) was freshly regenerated with 5 column volumes (CV) of
regeneration buffer (12 mM citrate, pH 6.0, 0.2 % Triton X-100, 500 mM
NaCl) and equilibrated with 5 CV wash buffer (12 mM citrate, pH 6.0, 0.2 %
Triton X-100). The enzyme-containing supernatant from heat-shock
purification was diluted with the four fold volume of Milli-Q water (to reduce
the salt concentration within the preparation), filtered through 0.2 µm filters
and loaded on the DEAE-Sephacel resin. The loaded sample was washed
with 5 CV of wash buffer (12 mM citrate, pH 6.0, 0.2 % Triton X-100) and
eluted with 5(-10) CV elution buffer (12 mM citrate, pH 6.0, 0.2 % Triton X-
100, 200 mM NaCl). Protein concentration was determinded via Bradford
Assay using the BradfordUltra reagent (expedeon).
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citrate, pH 6.0, 0.2 % Triton X-100, 200 mM NaCl) and 2 mM substrate for
40 h at 30 °C while shaking with 600 rpm. Experiments were performed in
triplicates. Reaction setups with pure buffer and with AacSHC variant
D376C served as negative control (to monitor background activity). Prior
to chromatographic analysis 1 mM of 1-decanol was added to the
biotransformation as internal standard and the reaction mixtures were
extracted twice with 500 μl ethyl acetate. The organic phases were dried
over Na2SO4 and subsequently analyzed via GC-FID/MS.
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GC-FID/MS analyses were performed on an Agilent GC/FID system 7890A
with inert MSD 5975C, equipped with a DB-5ht column (30 m × 0.25 mm
× 0.1 μm) and a quadrupole mass analyzer. Separation method: 1 μl
injection volume, injection temperature: 250 °C, detector temperature:
320 °C. Gradient: column temperature set at 75 °C for 3 min, then
increased to 120°C at 6 °C/min, then to 150 °C at 10 °C/min and to 300 °C
at 30 °C/min. Total run time was 18.5 min. Citronellal (1) and isopulegol
(2) isomers were separated as depicted in Figures S1 and S2. Since
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This project has received funding from the European Union
Seventh Framework Programme (FP7/2007-2013) under grant
agreement n°289646 and the Ministry for Science, Research and
the Arts of Baden-Württemberg, Germany (Baden-Württemberg
Biosynthesis netWork BW2). We gratefully acknowledge the
Fonds der Chemischen Industrie for a Kekulé scholarship to SCH
(grant number K187/01).
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Keywords: biocatalysis • squalene hopene cyclase • active site
mutation • isopulegol • mutagenesis
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S. C. Hammer, P.-O. Syrén, M. Seitz, B. M. Nestl, B. Hauer, Curr.
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10.1002/cctc.201700734
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SHC as selectivity inspector:
Squalene hopene cyclase variants
are highly efficient Brønsted acid
catalysts for the asymmetric
Silke A. Bastian, Stephan C. Hammer,
Bettina M. Nestl, Bernhard Hauer*
Page No. – Page No.
cyclization of citronellal enantiomers.
The excellent isomeric selectivity is
facilitated by controlling high energy
intermediates during the reaction.
Enabling New Selectivities in the
Cyclization of Citronellal by
Squalene Hopene Cyclase Variants
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