Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants

Article abstract of DOI:10.1002/cbic.201700449

Squalene–hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl–farnesyl ether product from farnesol. Furthermore, the addition of small-chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and, thus, to significant alteration of the product pattern relative to that obtained with the wild type. It is proposed that the mutation of leucine at position 607 may facilitate premature quenching of the intermediate by small alcohol nucleophiles. This mutagenesis-based study opens the field for further intermolecular bond-forming reactions and the generation of non-natural products.

Full text of DOI:10.1002/cbic.201700449

Accepted Article
Title: Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene
Cyclase Variants
Authors: Lisa C Kühnel, Bettina M Nestl, and Bernhard Hauer
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10.1002/cbic.201700449
ChemBioChem
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Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene
Cyclase Variants
Lisa C. Kühnel, Bettina M. Nestl and Bernhard Hauer*[a]
Abstract: Squalene hopene cyclases (SHC) catalyze the
polycyclization of squalene into a mixture of hopene and hopanol.
Recently, amino acid residues lining the catalytic cavity of the SHC
from Alicyclobacillus acidocaldarius were replaced by small and large
hydrophobic amino acids. The alteration of leucine 607 to
phenylalanine resulted in increased enzymatic activity towards the
formation of an intermolecular farnesyl-farnesyl ether product from
farnesol. Furthermore, the addition of small chain alcohols acting as
nucleophiles led to the formation of non-natural ether-linked
terpenoids and thus, to a significant alteration of the product pattern
compared to wildtype. It is proposed that the mutation of Leu at
position 607 may facilitate premature quenching of the intermediate
by small alcohol nucleophiles. This mutagenesis-based study opens
the field for further intermolecular bond forming reactions and
generation of non-natural products.
small sesquiterpenes like farnesol (C15) up to C35 non-natural
polyprenoids were successfully converted.[4–9] SHCs can be also
utilized for carbon-carbon, carbon-oxygen and carbon-nitrogen
bond formation reactions using functionalities such as alcohols,
aromatic systems, carboxylic acids, ketones or amides.[10–12]
Previous work demonstrated that SHCs can perform Friedel–
Crafts alkylations with aromatics as well as hydroamidations
revealing the potential of these enzymes for chiral Brønsted acid
catalysis.[13,14]
Hoshino and coworkers observed in wildtype AacSHC some
degree of unspecificity in the cyclization of the truncated squalene
substrate farnesol (1).[5] A conversion of 64% was observed for 1
giving four products (2 – 5) that were formed from one common
bicyclic intermediate (Scheme 1). Along with deprotonated alkene
products albicanol (2) and drimenol (3) and the corresponding
alcohol drimane-8, 11-diol (4) as major product, the nucleophilic
attack of the hydroxyl group of a second farnesol molecule to the
bicyclic skeleton forming the ether linkage product 5 was detected.
The squalene-hopene cyclase from Alicyclobacillus
acidocaldarius (AacSHC) is a triterpene cyclase that catalyzes in
nature the cyclization of the linear triterpene squalene to hopene
and hopanol. This complex polycyclization is one of the most
demanding biochemical reactions forming five new C-C bonds
and nine stereocenters.[1] The reaction is initiated by binding the
substrate in a specific product-like conformation and triggering of
cation formation by protonation of a double bond. The highly
acidic Asp376 of AacSHC is the electrophilic residue that initiates
the cyclization by protonating the terminal isopropylidene of
squalene. The enzyme provides a highly conserved, hydrophobic
active site containing electron-rich amino acids that facilitate the
production of carbocations as well as the shielding of
carbocations from solvent.[2] AacSHC cyclase exhibits
a
remarkably broad substrate tolerance forming new C-C and C-X
bonds. Functionalized acyclic terpene derivatives can be used as
substrates by SHCs for the synthesis of numerous non-natural
carbopolycyclic and heteropolycyclic products in a stereoselective
manner. One commercial interesting example is the enzymatic
synthesis of the high valuable flavor compound ambroxan starting
from the simple alcohol precursor homofarnesol in the grams-
scale.[3] Various nucleophiles attacking the carbocationic
intermediates in the reaction enabled the stereoselective
syntheses of a multitude of polycyclic products. Starting from
Scheme 1. Cyclization reaction of farnesol (1) and resulting products (2-5).
Major product 4 (45%) is followed by minor products 2 (3%), 3 (7%) and 5
(9%).[5]
This was the first reported example where the carbocation
generated on the farnesol molecule was quenched by reacting
with an available substrate molecule to produce an ether. On the
basis of the crystal structure of the enzyme, the hydrophobic
active site cavity lined with aromatic amino acid residues appears
to have enough space to accept two molecules of farnesol. This
result prompted us to evaluate the manipulation of the enzyme
reaction by substrate analogues that would lead to chemically and
structurally novel, non-natural ether products.
[a]
Dr. Lisa C. Kühnel, Dr. Bettina M. Nestl, Prof. Dr. Bernhard Hauer
Institute of Biochemistry & 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/cbic.201700449
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We report herein the enzymatic cyclization of farnesol in which
the terpenoid unit is connected with geraniol or small alcohol
nucleophiles using AacSHC variants. In contrast to the cyclization
of farnesol, the incubation of geraniol with purified recombinant
AacSHC wildtype did afford very small amounts of its cyclization
product. This may reflect the fact that the active site of the enzyme
does not favour a reactive binding of the geraniol substrate.[5,12,15]
Our previous studies suggested that the alteration of the active
site pocket of AacSHC facilitated the conversion of truncated
terpene-like substrates without disturbing the important catalytic
machinery.[12,16]
the reactive carbocation intermediate from reacting with water or
other nucleophiles.
AacSHC variants with alanine or phenylalanine at position
607 displayed highest activities and selectivities towards the
farnesyl-farnesyl ether product 5. An approximate four-fold
increased formation of 5 was obtained with variant L607F
compared to the wildtype (Table 1). Up to 65.1% ± 5.4% of 1 was
successfully converted into the enzymatic products. Inspired by
these results, we further investigated the AacSHC mutant library
for increased formation of 5. Alternatively, we became interested
in expanding the reaction scope towards new cyclization-addition
products quenching the carbocation intermediate with geraniol (6)
and small alcohols as nucleophiles (9-12). To determine the
substrate specificity of the L607F variant towards the nucleophilic
addition of further terpenoids, biotransformations using farnesol
(1) with geraniol (6) were performed. GC analysis of the
incubation mixture of 1 and 1 % (v/v) 6 with AacSHC wildtype and
variant L607F afforded five product peaks. Table 1 shows the GC
profile of the product distribution pattern. Interestingly, in addition
to 2-4, two novel ether products 7 and 8, in which the geraniol unit
is connected to the drimane (7) and cyclogeraniol (8) skeletons,
were formed (Figure S2). An approximately 4-fold increased
formation of these ethers products was obtained with variant
L607F, while no farnesyl-farnesyl ether 5 was detected (Table 1).
This might be explained by the fact that an excess amount of 6
was utilized in biotransformations competing with the farnesol
substrate. Recent investigations of Syrén et al. on the cyclization
of geraniol and geranyl alkyl ethers by AacSHC revealed that
increased alkyl substituents provided increased activities for the
cyclization and selectivities towards the geranyl alkyl ether.[12]
Figure 1. Library screening of AacSHC wildtype and variants for the cyclization
of farnesol (1) towards products 2-5. Biotransformations were performed with
purified protein in triplicates.
Mutant design was approached by site-directed mutagenesis of
active site amino acid residues. The introduction of small
hydrophobic and aromatic residues that preserve the hydrophobic
nature of the active site was explored to fine-tune and re-size the
active site of the AacSHC. The analysis of the single mutant
library identified variants that were highly active towards the
truncated squalene substrate farnesol (conversion of 1 ≥50 %,
see also Supporting Information Figures S1 and S4). Selected
variants are shown in Figure 1. The analysis of the AacSHC
mutant library allowed us to identify regions in the active site
responsible for favouring either deprotonation or addition
products (Figure 2). Replacement of active site residues resulted
in small but important alterations in the precise positioning and
folding of the substrate within the active site. Further, these results
suggested that small changes in the active site geometries are
sufficient to alter the selectivity of the reaction. The synthesis of 4
as major product suggests that cation hydroxylation during the
annulation phase generally occurs by attack of water inside the
active site cavity. Remarkably, most variants altered the product
selectivity in favour of the deprotonation products 2 and 3. Three
variants demonstrated significant increases in the formation of
bicyclic products 2 and 3, influencing the cation deprotonation. An
excellent product selectivity (>99 %) towards 3 was observed by
variant I261W (Figure 1). The observation that most variants
showed an increased formation of cyclization products 2 and 3
can be explained by the re-sizing of the active site that prevented
Scheme 2. Reactions leading to novel ether-products (7, 8, 13-16) and other
cyclization products (2-5) using AacSHC wildtype and L607 variants
It is anticipated that aliphatic alcohols (ethanol 9, n-butanol 10,
n-pentanol 11 and n-hexanol 12) were subjected to the enzymatic
reaction with AacSHC wildtype and L607F in order to synthesize
novel non-natural ethers (Scheme 2). Reduced conversions of 1
were observed with 1% (v/v) 9 and 12 as nucleophiles
(conversions of 41.2% ± 10.6% and 22.5 ± 4.03%, respectively).
A similar observation about inhibition of AacSHC in the presence
of 2% ethanol was be made by Gärtner in 1987.[17] Whereas
farnesol was less converted in the presence of 9 and 12,
formation of
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Figure 2. Surface presentation of the AacSHC catalytic domain (PDB code 1UMP, grey surface) and farnesol (1) as sticks presented separately. The active
site aspartic acid D376 is shown as lines (red surface). (A) Replacement of residues W489 and L607 (shown in pink) provided increased formation of farnesyl-
farnesyl ether product 5 in the course of the cyclization of 1. (B) Active site of AacSHC was rotated 180° compared to their position in (A) around the x-axis.
Also shown in blue are the residues W312, Q366 and I261 that were replaced by alanine or tryptophan in order to favour the formation of elimination products
2 and 3.
farnesyl butyl- as well as pentyl ethers 14 and 15 was monitored
using 10 and 11 (Table 1, Figures S3 and S5). Farnesol (1)
incubated with 10 and 11 were competent substrates for the
enzyme and efficiently converted to their corresponding ether
products. 61.7% ± 2.3% of farnesol was converted in when
incubated with n-butanol (10) and 40.5 ± 6.95% with n-pentanol,
respectively. Again, improved ether formations were obtained by
variant L607A compared to the wildtype.
A raise in the concentration of n-butanol from 1% (v/v) to 2.5%
(v/v) led to 3-fold enhanced formation of 14, however, enhancing
the amount of 10 up to 2.5% (v/v) resulted in an abolished activity
towards farnesol. In particular, variant L607F generated up to
28% more of 14, while a drop in the formation of 4 was obtained.
Remarkably, while the increase of 14 occurred at the expense of
product 4, we have seen little differences in the formation of
deprotonation products 2 and 3 when adding the alcohols as
nucleophiles. These data suggest that n-butanol (10) and n-
pentanol (11) are competing with water in the active site to
perform the nucleophilic attack at the final bicyclic cation. This
insight aligns with observations that mutations of the active site of
AacSHC affect the cyclization reaction and the subsequent
quenching of the carbocation intermediate (nucleophilic addition
favored over deprotonation). Analogously, active site variants
have a great plasticity to produce non-natural products by
connecting a farnesol unit with small alcohols in an intermolecular
fashion.
In conclusion, we were able to modify the product specificity
by engineered variants of AacSHC that have altered specificity.
We demonstrated that the residue at position 607 in the active site
of AacSHC is important for the intermolecular coupling reaction of
farnesol with alcohol nucleophiles. We believe it is a combination
of the hydrophobic environment in the mutant active site,
displacement of water and L607 by phenylalanine, and the
conformation of the substrate being favourable for cyclization,
which led to the ether product formation. For further production of
non-natural products, manipulation of the enzyme reaction by
terpene analogues with different isoprene chain length and/or with
aromatic ring systems is now in progress in our laboratories.
Table 1. Product distribution [%] for the conversion of farnesol (1) with 1,
geraniol (6), n-butanol (10) and n-pentanol (11) using AacSHC wildtype
and variant
nucleo-
phile
variant
2
3
4
5
7
8
14
15
WT
4
7
85
61
55
41
66
43
36
4
1
6
L607F
WTa
6
19
17
14
15
27
19
14
0
17
4
2
8
9
L607Fa
WTa
0
33
14
14
14
1
4
10
11
L607Fa
L607Fb
7
9
3
28
WTa
15
14
17
24
57
43
1
2
10
17
L607Fa
Reaction conditions: farnesol (2 mM in DMSO), 1-2.5 % (v/v) nucleophile,
citrate buffer pH 6 (60 mM), 0.01 % CHAPS, 1 mg/mL purified catalyst,
65°C, 24 h [a] 1% (v/v) of geraniol (6), n-butanol (10) and n-pentanol (11)
[b] 2.5% (v/v) of n-butanol (10)
Experimental Section
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Overexpression and solubilisation of AacSHC wildtype and variants
EFPIA). Sebastian Löw is thanked for helpful comments on the
manuscript.
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,
400 mL TB medium containing 100 mg/ml ampicillin was inoculated with
cultures of recombinant E. coli BL21(DE3) and shaken overnight (18 h) at
37 °C (180 rpm), whereby expression was started by auto-induction. Cells
were harvested by centrifugation for 15 min at 7000 x g at 4 °C and the
cell pellet was resuspended in 3x volume of lysis buffer containing DNAse
I and 0.1 mM phenylmethylsulfonyl fluoride. Cell lysis was done by
ultrasonication on ice. After centrifugation for 20 min at 7000 x g and 4°C,
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 CHAPS) 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 x g and 4 °C to partially purify the thermostable cyclase
enzymes from residual E. coli proteins. Protein concentration was
determinded via Bradford Assay using the BradfordUltra reagent
(expedeon).
Keywords: squalene hopene cyclase • active site mutagenesis •
cyclization • intermolecular reaction • ether formation
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Enzymatic conversions were carried out in a 500 μl scale in glass vials
using 1 mg/ml partially purified AacSHC variant in elution buffer (60 mM
citrate pH 6.0, 1 % detergent CHAPS) and 2 mM substrate for 96 h at 60 °C
while shaking with 600 rpm. Experiments were performed in triplicates.
Reaction setups in buffer 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 methyl-tert-butyl ether (MTBE).
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with inert MSD 5975C, equipped with a DB-5HT column (30 m × 0.25 mm
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Separation method: 1 μl injection volume, injection temperature: 275 °C,
detector temperature: 410 °C. Gradient: column temperature set at 70 °C
for 2 min, then increased to 230 °C at 20 °C/min, to 240 °C at 20 °C/min,
to 270 °C at 20 °C/min, to 380 °C at 20 °C/min and held for 2 min.
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Acknowledgements
The research has been performed within project CHEM21, funded
by the IMI (agreement n°115360 within FP7/2007-2013 and
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Connected molecules: Non-natural
ether-linked terpenoids were
Lisa C. Kühnel, Bettina M. Nestl,
Bernhard Hauer*
generated by adding small chain
alcohols to farnesol. A variant of the
squalene hopene cyclase from
Alicyclobacillus acidocaldarius was
identified facilitating the connection
of the carbocation intermediate with
small alcohol nucleophiles.
Page No. – Page No.
Title
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