Carboxylic Acid Reductase Can Catalyze Ester Synthesis in Aqueous Environments

Article abstract of DOI:10.1002/anie.202013962

Most of the well-known enzymes catalyzing esterification require the minimization of water or activated substrates for activity. This work reports a new reaction catalyzed by carboxylic acid reductase (CAR), an enzyme known to transform a broad spectrum of carboxylic acids into aldehydes, with the use of ATP, Mg²⁺, and NADPH as co-substrates. When NADPH was replaced by a nucleophilic alcohol, CAR from Mycobacterium marinum can catalyze esterification under aqueous conditions at room temperature. Addition of imidazole, especially at pH 10.0, significantly enhanced ester production. In comparison to other esterification enzymes such as acyltransferase and lipase, CAR gave higher esterification yields in direct esterification under aqueous conditions. The scalability of CAR catalyzed esterification was demonstrated for the synthesis of cinoxate, an active ingredient in sunscreen. The CAR esterification offers a new method for green esterification under high water content conditions.

Full text of DOI:10.1002/anie.202013962

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Carboxylic Acid Reductase Can Catalyze Ester Synthesis in
Aqueous Environments
Authors: Pornkanok Pongpamorn, Cholpisit Kiattisewee, Narongyot
Kittipanukul, Juthamas Jaroensuk, Duangthip Trisrivirat,
Somchart Maenpuen, and Pimchai Chaiyen
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013962
Link to VoR: https://doi.org/10.1002/anie.202013962

10.1002/anie.202013962
Angewandte Chemie International Edition
COMMUNICATION
Carboxylic Acid Reductase Can Catalyze Ester Synthesis in
Aqueous Environments
Pornkanok Pongpamorn†[a,b], Cholpisit Kiattisewee†[a], Narongyot Kittipanukul†[a], Juthamas Jaroensuk[a],
Duangthip Trisrivirat[a], Somchart Maenpuen[c], and Pimchai Chaiyen*[a]
Abstract: Enzymatic esterification reactions under aqueous
conditions are not common, as most of the well-known enzymes
catalyzing esterification require the minimization of water or
activated substrates for activity. This work reports a new reaction
catalyzed by carboxylic acid reductase (CAR), an enzyme known
to transform a broad spectrum of carboxylic acids into aldehydes,
with the use of ATP, Mg2+, and NADPH as co-substrates. When
NADPH was replaced by a nucleophilic alcohol, CAR from
Mycobacterium marinum can catalyze esterification under
aqueous conditions at room temperature. Addition of imidazole,
especially at pH 10.0, significantly enhanced ester production. In
comparison to other esterification enzymes such as
acyltransferase and lipase, CAR gave higher esterification yields
in direct esterification under aqueous conditions. The scalability
of CAR catalyzed esterification was demonstrated for the
synthesis of cinoxate, an active ingredient in sunscreen, with an
isolated yield of 32%. The CAR esterification reported here offers
a new method for green esterification under high water content
conditions.
metabolically engineered cells are generally accepted as
sustainable means for production of fine chemicals in the future,
the identification of new enzymatic reactions to catalyze
esterification in aqueous environments will pave the way for
performing esterification with metabolically engineered cells.[5]
Up until now, reports of efficient enzymatic esterification reactions
in aqueous environments are limited.[6] For examples, although
non-ribosomal polypeptide synthetase (NRPS) can catalyze
esterification and amidation, the use of NRPS as a biocatalyst is
challenging, as NRPS reactions are substrate-specific and
require a defined reaction order, thus, limiting the range of
convertible carboxylic acids.[7] CoA-dependent acyltransferases
and acetyltransferases are also known to play a role in lipid
biosynthesis. Despite their capability to synthesize ester, the
reported enzymes are mostly membrane-bound and their
substrate scopes are specific towards fatty acyl CoAs or acetyl
CoA.[8]
CAR catalyzes the reduction of carboxylic acids to
aldehydes.[9] It is a useful enzyme for the synthesis of many high-
value compounds.[9b, 10] The structure and mechanism of CAR are
similar to those of NRPS.[9b, 11] In the first catalytic step, ATP is
used for the adenylation of the carboxylic acid to form the acyl-
AMP intermediate. Through thiolation, the acyl-AMP intermediate
is attached to CAR’s phosphopantetheine (PCP) group. This PCP
moiety then transfers the thioester intermediate to the reductase
Esters are economically important chemicals because of their
wide applications as fragrances, flavor agents, cosmetics, soaps,
medicines, and herbicides/pesticides.[1] Esters can be prepared
chemically by reacting a carboxylic acid with an alcohol in the
presence of an acid or base catalyst, such as in the Fischer[2] and
Steglich[3] esterifications. Although existing catalytic technologies
employed in esterification are efficient, these reactions are not
environmentally friendly because they require the use of organic
domain, where NADPH is used in the reduction, liberating the
11d, 12]
aldehyde product from the protein.[9b,
Flitsch’s group
solvents,
non-biodegradable
catalysts,
and
elevated
demonstrated that CAR can catalyze amidation instead of
reduction with substitution of an amine.[13] The reaction
mechanism of carboxylic acid amidation by CAR is thought to be
similar to the mechanism of NPRS.[14]
temperatures. Recently, the ester synthesis capability of
acyltransferase from Mycobacterium smegmatis (MsAcT) in water
was reported. However, it was shown to require a pre-activated
acid for transesterification.[4] As biocatalysis by enzymes or
Inspired by the work mentioned above, we aimed to explore
whether CAR can catalyze esterification in water, in addition to
carboxylic acid reduction and amidation, for future application in
metabolically engineered cells. Previously, CAR has also been
shown to be involved in the chemo-enzymatic synthesis of esters
by reducing carboxylic acids into aldehyde substrates for the
subsequent Wittig reaction to generate α,β-unsaturated ester
products.[15] However, the ability of CAR alone to catalyze
esterification has not been reported.
[a]
Dr. P. Pongpamorn, C. Kiattisewee, N. Kittipanukul, Dr. J.
Jaroensuk, D. Trisrivirat, Prof. Dr. P. Chaiyen
School of Biomolecular Science and Engineering
Vidyasirimedhi Institute of Science and Technology (VISTEC)
Wangchan Valley, Rayong, 21210, Thailand
E-mail: pimchai.chaiyen@vistec.ac.th
[b]
Dr. P. Pongpamorn
National Science and Technology Development Agency (NSTDA)
111 Thailand Science Park, Phahonyothin Road,
Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand
Asst. Prof. Dr. S. Maenpuen
Department of Biochemistry, Faculty of Science,
Burapha University, Chonburi 20131, Thailand
These authors contributed equally.
We first carried out the standard CAR reaction with benzoic
acid as the substrate in methanol. CAR used in our experiments
was of the highest purity (>99%, SI, Figure S1) to avoid
promiscuous activities from other contaminating enzymes. Apart
from the formation of the benzaldehyde product, a side product,
identified as methyl benzoate was also detected. This discovery
led us to further explore the condensation between carboxylic
acids and alcohols catalyzed by CAR. When NADPH was omitted
from the reaction, more methyl benzoate product was observed,
but was still only found in trace amounts (data not shown).
Therefore, the conditions were adjusted to maximize the ester
[c]
†
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10.1002/anie.202013962
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production by CAR. The dependence on pH was evaluated by
initially varying the pH of the buffer system. The preliminary
esterification was conducted at 37ºC and consisted of 0.78 µM
Mycobacterium marinum CAR (MmCAR), 1 mM benzoic acid, 4%
methanol, 5 mM of ATP, and 10 mM of MgCl2 at their final
concentrations in either 100 mM Tris-HCl or 100 mM Na2CO3
buffer at pH 7.5-10.5. Methyl benzoate was detected using GC-
MS and the yields were calculated from a calibration curve based
on methyl benzoate standards. The highest yield of methyl
benzoate, 0.59%, was obtained in the 100 mM Na2CO3 buffer
system at a pH of 10.0 (Supporting Information (SI), Table S1).
Because the expression level in bacteria of the apo form of
MmCAR was higher than that of the holo form, which requires co-
expression of the Sfp protein to attach a PCP group, and because
both forms of CAR gave similar yields of methyl benzoate product
(results not shown), all subsequent experiments were performed
using only apo MmCAR. Although, the apo MmCAR prepared
may be a mixture of apo or holo forms due to plausible
modification by E. coli, this issue does not affect CAR-catalyzed
esterification because PCP is not involved in the esterification
mechanism (see later).
Figure 1. The proposed mechanism of apo CAR esterification enhanced by
imidazole additive. R1 = alkyl (C5H11, C6H13, C7H15, C9H19), Ph; R2 = alkyl
(CH3, C4H9, C6H13, C8H17, C9H19), Bn (SI, Table S3).
efficiently at a mild temperature such as at 25⁰C (SI, Figure S5).
The effect of alcohol was also evaluated for the production of
other esters (SI, Figures S6 and S7).
Methanol, at a
We then further explored the effects of additives which might
be mechanistically advantageous for enhancing the esterification.
According to the mechanism of CAR, the first step of the reaction
is the formation of acyl-AMP and acyl-PCP to activate the
carbonyl groups such that they are more electrophilic than in the
carboxylic acid form, making them more susceptible to
nucleophilic attack by a hydride from NADPH. Most chemical
esterification reactions also require activation of the carboxylic
acid to promote nucleophilic addition. Various additives that could
help activate the carboxylic acid were supplemented into the 100
mM Na2CO3 pH 10.0 buffer. All reactions were monitored for the
conversion of cinnamic acid to methyl cinnamate (SI, Table S2).
concentration higher than 5% decreased the yield of methyl
cinnamate, possibly due to the inhibitory effects on CAR (SI,
Figure S7). Details of product identifications are shown in SI
Figures S8-S13.
We investigated the mechanistic role of imidazole in
enhancing CAR-catalyzed esterification (SI, Figure S14-S19). We
hypothesized that imidazole either reacts with the acyl-AMP
intermediate to form acyl-imidazole which may somehow speed
up esterification or serve as an acid/base catalyst for the
esterification. We found that that CAR cannot convert cinnamoyl-
imidazole and alcohol into an ester in the absence of imidazole,
eliminating the possibility that acyl-imidazole is an intermediate in
the CAR-catalyzed esterification. The data suggest that imidazole
likely serves as a general acid/base catalyst to mediate proton
transfer during esterification (Figure 1).
The first group of additives used were thiol containing
compounds, to see whether any PCP mimics would enhance
ester production (SI, Table S2, Entry 1-3). The other groups of
additives were chosen based on the basic principles of chemical
catalysis.[16] Guanidine and imidazole can mimic functions of
To elucidate the involvement of the reductase domain and
PCP group in CAR-catalyzed esterification, we compared
esterification of methyl cinnamate using apoCAR, the truncated
CAR in which the reductase domain was removed, and the S685A
variants of apoCAR and truncated CAR in which PCP cannot be
attached.[12] Results (SI, Figure S20) showed that all enzymes
exhibited similar activities, indicating that the reductase domain
and the PCP group is not involved in esterification (Figure 1).
general acid and base catalysts commonly found in enzymes[16c]
.
The bifunctional 2-hydroxypyridine can act as a concerted acid
and base catalyst[16c], which can also help generate a carboxylate
anion. Thiamine[16b] and imidazole[16c] can serve as nucleophilic
catalysts. Enhancement effects from imidazole should be the
most promising, because histidine serves as a general acid/base
catalyst (catalytic residue) in many enzymes.[17]
Indeed, the results showed that among the seven additives,
imidazole at the concentration of 100 mM was the most effective
compound for improving the production of methyl cinnamate (SI,
Table S2, Entry 7). Since imidazole significantly enhanced the
production of methyl cinnamate, it was evaluated as a standalone
buffer. The optimum concentration of imidazole as a standalone
buffer was 200 mM (SI, Figures S3 and S4). When CAR was not
included in the 200 mM imidazole buffer system, no methyl
cinnamate peak was detected in the HPLC-DAD chromatogram.
Next, the effect of temperature on the production of methyl
cinnamate was examined in the original 100 mM Na2CO3 and in
the new 200 mM imidazole buffer systems. CAR worked most
With the optimized conditions, steady-state kinetics parameters of
CAR-catalyzed esterification of acetic acid, cinnamic acid, and
benzoic acid were investigated. Among these substrates, CAR
was most efficient at converting cinnamic acid to methyl
cinnamate, with the highest turnover number of 21.2 h-1 (Figure 2
and SI, Figures S21-S27).
In order to explore substrate scopes and preference in
reaction conditions of CAR in comparison to other enzymes
known to catalyze reactions in aqueous environments, we
explored esterification by CAR versus lipase (Novozyme 435) [17b,
18] and MsAcT under similar conditions (SI, Figures S28-36, Table
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10.1002/anie.202013962
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COMMUNICATION
S4-S5). As these enzymes operate via different reaction
mechanisms, their requirement of substrate scopes and reaction
conditions would be different.
In comparison to the production of amides previously
reported[13], without adjustment of reaction buffer to imidazole,
esterification behaves poorly (less than 1% conversion of methyl
benzoate compared to 11% conversion of benzamide). However,
with imidazole enhancement, methyl cinnamate was produced up
to 41% in 24 hours (Table 1) compared to 22% conversion of
cinnamamide at 24 hours. An improved esterification exhibits
comparable efficiency to that of amidation. However, the highest
percentage conversion of amide synthesis (71% conversion to
ilepcimide at 24 hours) is still higher than any tested esterification
condition (46% yield of phenylacetic acid in Table S5).
While lipase performed best in the neat condition with minimal
water content (yielded up to 62.5% methyl cinnamate, SI, Figure
S33), it could not catalyze efficient esterification under aqueous
environment (0-2% of product formation). CAR could produce
decent yields (41 % methyl cinnamate) of these ester products,
as shown in Table 1, which included the anti-inflammatory agent
(methyl ferulate)[19], an ester with tobacco mosaic virus inhibitory
activity (methyl 4-hydroxyphenylacetate)[20], a halogen containing
ester (methyl 2,4-dichlorophenoxyacetate),
and
the FDA
approved sunscreen active ingredient (cinoxate)[21] (Table 1; SI,
Figure S28). CAR can also use a variety of alcohols (SI, Figure
29). Cinoxate could also be produced with an isolated yield of
32% from the scaled up synthesis by CAR (Table 1 and SI,
Figures S30-S32). CAR esterification was also tested at a milder
pH of 7.5 and it outperformed lipase in all reactions for the
production of various flavor and fragrance esters in aqueous
solutions (SI, Table S4-S5).
We then further compared the esterification reaction of CAR
with that of MsAcT (SI, Figures S34-S36). MsAcT efficiently
catalyzes transesterification of vinyl acetate with benzyl alcohol (a
yield of greater than 50% within 1 hour, data from Figure S35).
However, its ability to perform direct esterification from acetic acid
is limited; the yield reached the highest level (~25%) after the first
hour and decreased to be less than 20% after 24 hours. This
observation suggests that MsAcT catalyzes the reverse
hydrolysis reaction as well as esterification. For direct
esterification of acetic acid and benzyl alcohol, CAR could not
catalyze direct esterification well either (yield of 5.6%, Table 1).
However, CAR could catalyze much better esterification of bulkier
acids with the product yield up 43% (Table 1 and Figure 2) while
direct esterification of these bulkier acids was significantly
impaired (<1% yield) in the reaction by MsAct (Table S5).
Figure 2. Steady-state kinetic parameters of CAR for the substrates: acetic
acid, cinnamic acid, and benzoic acid. CAR concentrations used were 20
µM for acetic acid and 5 µM for other acids.
In conclusion, carboxylic acid reductase enabled the
transformation of carboxylic acids into esters under mild aqueous
conditions at 25 ⁰C with a broad substrate range for carboxylic
acids and alcohols. This new activity of CAR might even allow it
to be used in a whole cell biocatalyst or in metabolic engineering
applications for the synthesis of esters, as it also worked well at
pH 7.5.
These results demonstrated that, in aqueous conditions, CAR
had
a clear advantage over lipase and MsAcT in direct
esterification, especially with bulky aromatic acid as substrates.
Esterification of some cinnamic acid derivatives, by Novozyme
435 requires high temperatures[22] and the minimization of the
amount of water to give high percentages of conversion[22d], while
MsAcT requires an activated ester as a substrate. Esterification
by CAR is advantageous in that it is thermodynamically
irreversible, as the presence of water does not result in the
reverse reaction of converting the product back to the acid form.
A plausible explanation could be that CAR does not contain a
catalytic triad like lipase or other transferase enzymes, where the
amino acids of the catalytic triad can also promote the nucleophilic
attack of water. [15a] However, CAR has the disadvantage of using
costly ATP as a co-substrate which requires further process
optimization in the future, possibly using an ATP regenerating
Acknowledgements
†
P.P., C.K. and N.K. shared equal contributions. This work was
supported by a Global Partnership grant from the Thailand
Science Research and Innovation and PMU-C and a postdoctoral
and research assistant fellowship from the Vidyasirimedhi
Institute of Science and Technology (VISTEC). We thank Dr.
Pirom Chenprakhon from Mahidol University for suggestions and
for providing us with the Novozyme 435.
Keywords: Carboxylic acid reductase • esterification • imidazole
buffer • green chemistry • biocatalyst
system to convert AMP back to ATP by
polyphosphate..[23]
a low cost
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10.1002/anie.202013962
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COMMUNICATION
Table 1. CAR in the Synthesis of Esters.
[5]
aH.-J. Cha, S.-Y. Hwang, D.-S. Lee, R. K.
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U. Bornscheuer, F. Hollmann, D.-K. Oh,
J.-B. Park, n/a; bS. R. Derrington, N. J.
Turner, S. P. France, Journal of
O
CAR
O
R2
+
R2
HO
R1 OH
R1
O
Mg2+, ATP
Imidazole buffer
Yield [%][a]
biotechnology 2019, 304, 78-88.
Acid
Alcohol
Ester
[6]
[7]
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Natl Acad Sci U S A 2009, 106, 4183-
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19[d]
8
43[d]
46
14
4
[8]
13[d]
10[d]
(32)[e]
41[f]
ND
3.5[g]
5.6[h]
ND
ND
Reaction contained 1 mM acid, 5 mM ATP, 10 mM MgCl2, CAR, alcohol,
and 200 mM imidazole buffer. [a] Yield determined by GC-MS after shaking
at 60 rpm, 25ºC. [b] Reaction perform in imidazole buffer pH 10.0. [c]
Reaction perform in imidazole buffer pH 7.5 [d] 50 µM CAR, 123.5 mM
MeOH or 51.6 mM 2-ethoxyethanol, 24 h. [e] Isolated yield after 45 h from
a 20 mL reaction scale. [f] 10 µM CAR, 1.24 M MeOH, 24h. [g] 5 µM
CAR, 1.24 M MeOH, 8h. [h] 20 µM CAR, 96.54 mM benzyl alcohol, 4h. ND
= not determined.
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Entry for the Table of Contents
Layout 1:
COMMUNICATION
An efficient and green method for the
synthesis of esters using carboxylic
acid reductase as a biocatalyst under
mild aqueous conditions.
Pornkanok Pongpamorn, Cholpisit
Kiattisewee, Narongyot Kittipanuku,
Juthamas Jaroensuk, Duangthip
Trisrivirat, Somchart Maenpuen, and
Pimchai Chaiyen*
Page No. – Page No.
Carboxylic Acid Reductase can
Catalyze Ester Synthesis in Aqueous
Environments
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