Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry

Article abstract of DOI:10.1002/cctc.201601249

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP⁺, adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

Full text of DOI:10.1002/cctc.201601249

Accepted Article
Title: Characterization of carboxylic acid reductases as enzymes in the
toolbox for synthetic chemistry
Authors: William Finnigan, Adam Thomas, Holly Cromar, Ben Gough,
Radka Snajdrova, Joseph P Adams, Jennifer A Littlechild, and
Nicholas Harmer
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
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To be cited as: ChemCatChem 10.1002/cctc.201601249
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ChemCatChem
10.1002/cctc.201601249
FULL PAPER
Characterization of carboxylic acid reductases as enzymes in the
toolbox for synthetic chemistry
[
a]
[a]
[a]
[a]
[b]
William Finnigan , Adam Thomas , Holly Cromar , Ben Gough , Radka Snajdrova , Joseph P.
[
b]
[a]
[a]
Adams , Jennifer A. Littlechild , Nicholas J. Harmer*
Abstract: Carboxylic acid reductase enzymes (CARs) meet the
demand in synthetic chemistry for a green and regio-specific route to
aldehydes from their respective carboxylic acids. However, relatively
few of these enzymes have been characterized. A sequence
alignment with members of the ANL superfamily of enzymes shed
light on CAR functional dynamics. Using a phylogenetic analysis of
Expanding the toolbox of available enzymes is essential for the
successful development of new synthetic routes and sustainable
manufacturing processes.[3]
An important opportunity that is ripe for exploitation is synthetic
routes based on organic acids. These compounds have a long
known and hypothetical CARs, four unstudied enzymes were selected, history of production by fermentation.[4]
Indeed, multiple
and for the first time, a thorough biochemical characterization carried
out. Kinetic analysis of these enzymes with various substrates shows
they have a broad, but similar substrate specificity. Electron rich acids
are favored, suggesting that the first step in the proposed reaction
mechanism, attack by the carboxylate on the -phosphate of ATP, is
the step determining substrate specificity and reaction kinetics. The
effects of pH and temperature provide a clear operational window for
carboxylic acids were identified to be “Top Value Added
Chemicals From Biomass”,[5] many of which are now being
produced industrially. Reduced products of these organic acids,
especially optically pure aldehydes and alcohols, are essential
building blocks for use in the chemical, pharmaceutical and food
industries.[6] However, chemical methods for the reduction of
carboxylic acids are limited, and require chemicals such as lithium
aluminium hydride and sodium borohydride in stoichiometric
the use of these CARs, while investigation of product inhibition by
+
[7]
NADP , AMP and pyrophosphate (PP
i
) indicates that binding of
amounts.
substrates at the adenylation domain is ordered with ATP binding first.
This paper consolidates CARs as important and exciting enzymes in
the toolbox for sustainable chemistry, providing specifications for their
use as a biocatalyst.
Two enzyme classes are capable of reduction of organic acids to
aldehydes, and a review of the biocatalytic reductions possible by
organisms harboring them published.[7]
The aldehyde
oxidoreductases (AORs) reversibly oxidize organic aldehydes to
their respective acids. The oxidized product is more
thermodynamically favorable, and so the equilibrium tends
Introduction
towards this product. AORs are therefore more useful for
syntheses that require the oxidation of aldehydes.[8,9] In contrast,
The demand for 'green chemistry' is of increasing global
importance, driven by the need to balance sustainable and
efficient resource utilization with the demands and increasing
consumption of a rising population.[1] Biological solutions to
chemistry challenges are a critical component in meeting this
demand. The use of isolated enzymes and cell-based systems
that produce negligible dangerous waste, often with higher yields,
offers an alternative to traditional chemical processes. In some
cases, biological alternatives are more rapid and cost effective
than their chemical counterpart.[2] Despite these potential
advantages, enzymes are still under-used in chemistry.
the carboxylic acid reductases (CARs) catalyze the reduction of a
carboxylic acid to an aldehyde at the expense of ATP and NADPH,
+
[6]
i
producing AMP, PP and NADP as by-products. The reduction
of carboxylic acids into aldehydes using CARs has previously
been confirmed by a number of studies, using GC-MS analysis.
[10,11]
Products other than the aldehyde have not been detected.
The hydrolysis of ATP makes the reduction of acids to aldehydes
by CARs strongly thermodynamically favorable, making their use
an attractive “green chemical“ route to aldehyde production.[7]
This synthesis can be coupled to other enzymes such as an
alcohol dehydrogenase which can provide a complete route to
the alcohol product.[10]
[
[
a]
W. Finnigan, A. Thomas, H. Cromar, B. Gough, J.A. Littlechild, N. J.
Harmer
Indeed, CARs have been employed in a number of synthetic
pathways. These include the production of the flavor vanillin by
yeast,[12] and a synthetic pathway for the production of propane
Biosciences, College of Life and Environmental Sciences
University of Exeter
Stocker Road, Devon, Exeter EX4 4QD, UK
E-mail: N.J.Harmer@exeter.ac.uk
in Escherichia coli.[13]
These examples both highlight the
potential of CARs as part of a toolbox for synthesis of fine
chemicals from non-oil-based chemical precursors.[5]
b]
R. Snajdrova, J. Adams
Synthetic Chemistry
AC - API Chem – UK
GlaxoSmithKline R&D Ltd
Medicines Research Centre, Gunnels Wood Road, Stevenage,
Hertfordshire, SG1 2NY, UK
CARs are relatively large, multidomain enzymes of around 130
kDa. They feature an N-terminal adenylation domain, a C-
terminal thioester reductase domain that likely adopts
Rossmann fold, and a central phosphopantetheine binding
domain (Figure 1).[14] A phosphopantetheine arm must be
a
Supporting information for this article is given via a link at the end of
the document.
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ChemCatChem
10.1002/cctc.201601249
FULL PAPER
covalently attached to a conserved serine in this central domain
through the action of a phosphopantetheine transferase for the
production of an active enzyme.[15] Fungal α-aminoadipate
reductases, which are responsible for the reduction of α-
aminoadipate to α-aminoadipate semialdehyde in lysine
biosynthesis, share this domain architecture, also with the
requirement for the loading of a central phosphopantetheine
prosthetic group.[16] However these enzymes have been shown
to have a different substrate specificity from CARs.[11]
Phosphopantetheine arms are most commonly associated with
acyl carrier proteins where they maintain an acyl chain in a
energetically active thioester bond, with the length and flexibility
of the arm allowing access to spatially distinct active sites.[17] In
CARs, the phosphopantetheine arm is believed to act in much the
same way, shuttling an attached acyl chain between the N- and
C-terminal domains. [15]
The proposed mechanism of CAR enzymes has four main steps
(
labelled 1 to 4; Figure 1). In the first two steps, the relatively
unreactive carboxylic acid is activated to form a thioester with the
phosphopantetheine arm at the N-terminal adenylation domain, in
a mechanism possibly similar to that of the ANL superfamily of
adenylating enzymes such as long chain fatty acid CoA
ligases.[18],[19] (1) ATP and a carboxylic acid enter the active site
of the adenylation domain where the α-phosphate of ATP is
attacked by an oxygen from the carboxylic acid, forming an AMP-
acyl phosphoester with the release of pyrophosphate.[19] (2) The
thiol group of the phosphopantetheine arm can then
nucleophilically attack the carbonyl carbon of the AMP-acyl
phosphoester intermediate, releasing AMP and forming an acyl
thioester with the phosphopantetheine arm.
(3) The
phosphopantetheine arm transfers to the C-terminal reductase
domain (4) where the thioester is reduced by NADPH, releasing
+
the aldehyde and NADP , and regenerating the thiol of the
phosphopantetheine arm in the process.[7]
Figure 1 – Proposed mechanism of CAR enzymes. 1: ATP and a
carboxylic acid enter the adenylation domain where
a phosphoester
Relatively few CARs have been explored to date. CARs were first
described in Neurospora crassa as an aryl-aldehyde: NADP
intermediate is formed releasing pyrophosphate in the process. 2: the thiol
of the phosphopantetheine arm nucleophilically attacks the carbonyl carbon
+
of this intermediate forming
a
thioester intermediate with the
oxidoreductase.[
20]
Recently this CAR has also been further
phosphopantetheine arm, releasing AMP. 3: the phosphopantetheine arm
transfers to the reduction domain where, 4: the thioester bond is reduced
by NADPH releasing an aldehyde product, regenerating the
characterized.[21] Subsequently, CARs were characterized from
Nocardia asteroides JCM 3016
[
22]
and later Nocardia iowensis
+
[
23]
phosphopantetheine thiol group and producing NADP .
(
referred to as niCAR here) when they were reclassified as
carboxylic acid reductases.[23] Characterization of the Nocardia
asteroides JCM 3016 CAR was performed by comparing the
relative activity of this enzyme towards various aromatic
substrates (1 mM concentration). This CAR was reported to
prefer 3-substituted benzoates and aliphatic acids that were
substituted with a phenyl group. No reaction of this CAR with
simple aliphatic acids was reported. The optimum pH for activity
substituted benzoates or ring-deactivating groups showed no or
very low levels of activity. The reduction of racemic ibuprofen by
whole Nocardia iowensis cells gave a enantiometic excess (ee) of
6
1.2 %, which has been attributed to enantio-selectivity by niCAR
based on kinetic data for its reduction of (S)-(+)-Ibuprofen and
R)-(-)-Ibuprofen enantiomers [23]. The requirement for the
(
presence of a phosphopantetheine transferase for the loading of
of this enzyme was pH 7.5, and the optimum temperature for
activity was 40 °C.[22]
a phosphopantetheine group onto the CAR enzyme was shown
for niCAR [
23]
and is presumed to be the case for all the CAR
enzymes.
The relative activities of the Nocardia iowensis CAR (niCAR)
against various aromatic substrates have also been reported. The
highest activity was achieved with indole-5-carboxylic acid, which
was the most activated carboxylic acid tested. Substrates with 2-
A CAR from Mycobacterium marinum has also been described
and its application for the reduction of fatty acids to fatty alcohols
explored. This CAR is active against fatty acids between two and
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ChemCatChem
10.1002/cctc.201601249
FULL PAPER
eighteen carbons in length.[10] CARs have also been reported in
the fungi Syncephalastrum racemosum[24] and Trametes
versicolor.[25]
CAR adenylation domains were aligned with a firefly luciferase, a
fatty acyl-CoA ligase and a reductase domain from a non-
ribosomal peptide synthetase, all from the ANL superfamily
(
Supplementary Figure 1). CARs share ~20% sequence
Recently a characterization of CARs from Nocardia iowensis,
homology with other ANL superfamily members. Members of the
ANL superfamily catalyze the initial adenylation of a carboxylic
acid to form an acyl-AMP intermediate, which is generally
followed by the formation of a thioester. The family name is based
on three of its sub families: Acyl-CoA synthetases, the non-
ribosomal peptide synthase (NRPS) adenylation domain, and the
Luciferase enzyme. [18] Previous alignment and crystallography
studies have identified ten motifs that are conserved within the
superfamily. Of the ten, five are strongly conserved within the
CARs, including the active site ppxTSGSTGxPK, rGxTE and TGD
motifs (where p=aliphatic and r=aromatic residues). These motifs
are considered “signature” to the ANL superfamily, and are
involved in the hydrolysis of ATP. [26] The remaining five motifs are
also present albeit with lower conservation.
Nocardia
brasiliensis,
Mycobacterium
marinum
and
Mycobacterium smegmatis showed CARs to prefer substrates
where the carboxylic acid was the only polar or charged group,
giving a useful insight into the substrate specificity of these
enzymes. Also, a model was developed for the prediction of CAR
reactivity using this and previous CAR data.[11] It is worth noting
that the CAR characterized by Moura et al. is distinct from msCAR
used in this study.
Here, we have produced a detailed phylogeny of the CARs and
identified four previously undescribed CARs for further study that
are broadly spread across this phylogeny. With the addition of
niCAR for comparison to earlier work, a thorough biochemical
characterization was carried out on each. We investigated the
effects of temperature and pH to identify suitable conditions for
the use of CARs in biocatalytic reactions. We further performed
a full kinetic analysis on a range of aromatic and aliphatic
substrates with these CARs to look for potential differences in
their substrate specificity and to examine the effects of various
functional groups on their kinetic parameters. Finally, we describe
potential issues of product inhibition with the CAR enzymes. Our
investigation provides a more thorough description of the factors
to be considered when using the CAR enzymes in industrial
biocatalysis.
A total of 48 unique sequences showing homology to known CAR
proteins were gathered using pBLAST, or mined directly from
GenBank by raw text searches (Supplementary Figure 2). All
sequences identified were solely from Subclass actinobacteridae.
Within this Subclass, sequences were obtained from families
Streptomycetaceae and Corynebacterinae.
A masked multiple sequence alignment of the dataset was shown
to be best fit to the Whelan and Goldman model of amino acid
substitution, with a discrete gamma distribution of mutation rates
and an assumed presence of invariant sites (WAG+I+G). This
model was implemented into
a
Bayesian phylogenetic
Table 1 – Carboxylic acid reductases chosen for this study.
reconstruction (Figure 2). According to 16S data, the
Streptomycetaceae are thought to have evolved before the
Corynebacterinae. However, rooting the tree on the
streptomycetes has poor parsimony as numerous gene loss
events would have had to have occurred for this to be the case.
Instead, due to an outgroup being unobtainable for this dataset,
we opted to root the tree on its midpoint. The tree is extremely
well supported, with all nodes possessing a confidence score of
Abbreviation
Source
NCBI Reference:
mpCAR
msCAR
niCAR
Mycobacterium phlei
Mycobacterium smegmatis
Nocardia iowensis
WP_003889896.1
AFP42026.1
[
27]
Q6RKB1.1
>
0.75, and only four of forty-six biologically relevant nodes being
noCAR
tpCAR
Nocardia otitidiscaviarum
Tsukamurella paurometabola
WP_029928026.1
WP_013126039.1
scored at below the highest possible confidence score of 1.
In order to better understand how CAR functionality differs across
clades, we selected sequences for characterization from a range
of host organisms that broadly cover distinct areas of the
phylogenetic tree.
Five carboxylic acid reductases were chosen for a thorough
biochemical characterization from a range of host organisms containing
putative CAR’s. niCAR has previously been characterized and was
[
11, 23]
chosen for comparison.
CAR abbreviations have been chosen to
reflect their source. NCBI ascension numbers are shown which may be
used to access the protein sequences. A table of the sequence
identities of these five orthologues is provided as Supplementary Table
1.
Results
Alignment and phylogenetic analysis
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ChemCatChem
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Mycobacterium_obuense
Mycobacterium_chlorophenolicum
Mycobacterium_vaccae
Mycobacterium_smegmatis3
Mycobacterium_smegmatis2
Nocardia_vulneris
1
1
1
1
1
0
.82
1
Mycobacterium_phlei
1
Mycobacterium_xenopi
Mycobacterium_mageritense
Mycobacterium_fortuitum
Mycobacterium_heraklionense
Mycobacterium_triplex
Mycobacterium_avium
Mycobacterium_colombiense
Mycobacterium_parascrofulaceum
Mycobacterium_leprae
Mycobacterium_lepromatosis
Mycobacterium_marinum
Mycobacterium_tuberculosis
Nocardia_paucivorans
1
1
1
1
1
1
1
1
1
1
1
1
Nocardia_brevicatena
1
1
Nocardia_rhamnosiphila
Nocardia_testacea
1
Streptomyces_rimosus
1
Streptomyces_aureofaciens
Streptomyces_celluloflavus
Streptomyces_griseofuscus
Nocardia_aobensis
Nocardia_mikamii
Nocardia_seriolae
1
1
0
.77
1
1
1
1
Nocardia_concava
0
.76
Nocardia_otitidiscaviarum
Nocardia_asteroides
Nocardia_thailandica
1
1
Nocardia_transvalensis
Nocardia_araoensis
Nocardia_gamkensis
Nocardia_iowensis
Nocardia_brasiliensis
1
1
1
1
1
Mycobacterium_genavense
Mycobacterium_smegmatis1
Mycobacterium_intracellulare
Mycobacterium_abscessus
Tsukamurella_spongiae_hyp
Tsukamurella_carboxydivorans_hyp
Tsukamurella_pseudospumae_hyp
Tsukamurella_paurometabola
Segniliparus_rotundus
1
1
1
1
0
.91
1
1
0
.07
Figure 2 – Phylogenetic tree of CAR enzymes. A midpoint rooted phylogeny of a masked alignment of 48 carboxylic acid reductase sequences retrieved from
GenBank. Phylogeny was constructed with MrBayes and visualized in FigTree. Node labels represent Bayesian posterior probabilities describing node reliability
(
with 1 being unequivocal) computed by MrBayes. Coloured branches represent CARs that have been studied: Blue – in previous research, Red – in this paper,
Purple – in both this paper and previous research.
Kinetic characterization of CAR enzymes
Expression and Purification
The CAR enzymes were characterized in terms of their substrate
specificity towards a range of aromatic carboxylic acids, a range
of aliphatic unsaturated fatty acids, and the cofactors ATP and
NADPH. Lists of the substrates with their chemical structure can
be found in Figure 3 and Supplementary Figure 4. For each CAR,
an initial assay at high substrate concentration (5 mM) was carried
out to identify compounds for which CAR had activity. For those
compounds where activity was detected, a full kinetic analysis
was performed (Supplementary Figures 5-9). All the CARs that
CAR enzymes (Table 1) were expressed in E. coli and purified
from the cell lysates by nickel affinity chromatography followed by
gel filtration in order to obtain a high level of purity (Supplementary
Figure 3). The optimum conditions for the expression of
Mycobacterium phlei CAR (mpCAR) in E. coli in LB media were
determined to be induction at OD600 0.6 with 150 M IPTG,
followed by incubation for approximately 18 hours at 20 °C, with
orbital shaking at 225 rpm (data not shown). Similar conditions
were assumed to be suitable for the expression of the other CAR
enzymes and indeed all CARs were well expressed. CARs were
co-expressed with the Sfp phosphopantetheinyl transferase from
Bacillus subtilis on a separate plasmid, as the loading of a
phosphopantetheine group onto CAR enzymes has been shown
to be essential for activity. [15]
M
were tested showed similar K values for NADPH and ATP. For
NADPH the K was between 24 and 36 M, whilst for ATP K
M
M
values of between 64 and 84 M were observed. These values
are both well within the physiological ranges for these cofactors
and in good agreement with previous studies. [6,10,23]. Production
of benzaldehyde and 4-methylbenzaldehyde from the derivative
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ChemCatChem
10.1002/cctc.201601249
FULL PAPER
acids was confirmed by HPLC, with no other products observed.
NADPH consumption was also confirmed as a good measure of
aldehyde production (Supplementary figures 10 and 11).
Investigating the effects of electronic density on aryl
substituted carboxylic acid substrates
All of the enzymes that we tested showed strong activity against
the classical CAR substrate benzoic acid (compound 1, Figure 3A,
Table 2), which all previously studied CARs have shown activity
against. [
6,10,11]
A series of substituents of varying electronic
configuration were tested (compounds 2-5, Figure 3A, Table 2).
Compounds with more electron rich systems generally lowered
K giving increased catalytic efficiency compared to benzoic acid.
M
Minimal activity was detected with 2- methoxybenzoic acid.
In contrast, compounds that incorporated an electron withdrawing
nitro group in the benzene ring (compounds 6-8, Figure 3B, Table
2) resulted in a large decrease in the turnover number of the
CARs, in most cases inhibiting activity all together. Again, there
was no detectable activity with a nitro group in the 2 position;
whilst in the para position only tpCAR showed a low level of
activity. However, all the CARs tested were active against 3-
nitrobenzoic acid but with a lower kcat than benzoic acid.
Absorbance at OD340nm by nitro compounds was shown not to
interfere with the assay (Supplementary Figure 12).
Investigating the effect of the aromatic unit on catalytic
activity
3-phenylpropionic acid (compound 9, Figure 3C, Table 3) has a
carboxylate group out of conjugation from the aryl ring, extended
away from the aryl ring by two carbons giving the carboxylate
group greater flexibility. When tested with the CARs, this change
caused a reduction in K
3
3
M
, with a similar or slightly lower kcat. (E)-
-phenylprop-2-enoic acid (cinnamic acid; compound 10, Figure
C, Table 3), being a conjugated system, was expected to have
activity between 9 and the model compound 1, benzoic acid. The
CAR activity against (E)-3-phenylprop-2-enoic acid showed a
substantial reduction in kcat compared to 3-phenylpropionic acid
M
or benzoic acid, with a slight further reduction in K .
Figure 3 – CAR activity for various benzoic acid derivatives, heterocycles
-
1
and fatty acids. The kcat (min ) determined for each enzyme against each
substrate displayed as follows: mpCAR , msCAR , tpCAR , noCAR
and niCAR . Below each substrate is its chemical structure. Error bars show
the standard error. A: Benzoic acid and derivatives with electron donating
groups. B: Derivatives with an electron withdrawing groups. C: Derivatives
with various substituents between the carboxylate group and benzene ring. D:
Heterocycles containing either an oxygen, sulfur or nitrogen. E: Fatty acids
between four and eighteen carbons in length.
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FULL PAPER
Table 2 – CAR activity against benzoic acid and its derivatives with electron donating and withdrawing groups
1
.
2. 4-
Methylbenzoic
acid
3. 4-
Methoxybenzoic
acid
4. 3-
Methoxy
benzoic
acid
5. 2-
Methoxy
benzoic
acid
6. 4-
Nitrobenzoic
acid
7. 3-
Nitrobenzoic
acid
8. 2-
Nitrobenzoic
acid
Benzoic
acid
Hammet sigma constants:
0
-0.17
-0.27
0.12
-
0.71
0.78
-
[
28]
-
1
mpCAR
k
cat (min )
140 ± 20*
122 ± 3
132 ± 4
104 ± 3
NA
NA
3.7 ± 0.5
NA
K
M
(mM)
20 ± 4*
7 ± 1*
3.7 ± 0.2
33 ± 2
2.8 ± 0.2
48 ± 5
3.0 ± 0.2
35 ± 3
NA
NA
NA
NA
0.3 ± 0.1
11 ± 4
NA
NA
(min-
1
k
cat / K
mM )
M
-
1
-
1
msCAR
k
cat (min )
197 ± 4
154 ± 6
179 ± 6
18 ± 1*
12 ± 1*
NA
NA
NA
NA
40 ± 10
NA
NA
K
M
(mM)
3.4 ± 0.2
0.16 ± 0.02
0.19 ± 0.02
0.5 ± 0.2
1
1
1
k
k
k
cat / K
mM )
M
(min-
57 ± 4
900 ± 100
930 ± 80
1.4 ± 0.2*
NA
NA
100 ± 50
NA
-
1
-
1
tpCAR
noCAR
niCAR
k
cat (min )
142 ± 3
2.0 ± 0.1
72 ± 6
152 ± 2
0.69 ± 0.03
220 ± 10
130 ± 2
0.45 ± 0.02
290 ± 10
186 ± 2
0.56 ± 0.02
334 ± 10
19 ± 3
9 ± 3
13 ± 1
0.6 ± 0.1
22 ± 6
33 ± 2
0.7 ± 0.1
44 ± 8
NA
NA
NA
K
M
(mM)
cat / K
mM )
M
(min-
2.2 ± 0.7
-
1
-
1
k
cat (min )
(mM)
183 ± 6
2.1 ± 0.2
89.1 ± 8
135 ± 5
1.2 ± 0.2
110 ± 20
138 ± 4
1.1 ± 0.1
130 ± 10
136 ± 5
0.9 ± 0.1
150 ± 10
NA
NA
NA
NA
NA
NA
59 ± 2
2.5 ± 0.3
24 ± 3
NA
NA
NA
K
M
cat / K
mM )
M
(min-
-
1
-
1
k
cat (min )
98 ± 7
94 ± 2
49 ± 1
93 ± 1
NA
NA
18 ± 1
NA
K
M
(mM)
0.9 ± 0.1
103 ± 9
1.0 ± 0.1
97 ± 6
0.25 ± 0.01
200 ± 10
0.68 ± 0.03
137 ± 6
NA
NA
NA
NA
5.6 ± 0.7
3.2 ± 0.4
NA
NA
(min-
1
k
cat / K
mM )
M
-
1
M
NA: no activity was detected with that substrate. *: K was unusually large and substrates concentrations could not reach a high enough concentration to
accurately determine kinetic constants. Errors represent the standard error. No Hammett constants are shown for 2-substituents as steric effects cannot be
properly accounted for.
Table 3 – CAR activity against benzoic acid derivatives with the carboxylic acid group extended from the ring.
9. 3-
10. (E)-3-
11. Phenylpropynoic acid
12. 3-oxo-3-
13. trans-2-
Phenylpropionic
acid
Phenylprop-
2-enoic acid
67 ± 2
0.3 ± 0.02
240 ± 2
Phenylpropanoic acid
Phenylcyclopropane-
1-carboxylic acid
-
1
mpCAR
msCAR
tpCAR
noCAR
niCAR
k
cat (min )
(mM)
(min-1 mM-1)
21.5 ± 0.7
3.0 ± 0.3
7.2 ± 0.7
184 ± 9
NA
NA
18 ± 2
3.8 ± 0.8
5 ± 1
20 ± 1
1.8 ± 0.2
12 ± 1
K
M
k
k
k
k
cat / K
M
NA
-
1
k
cat (min )
(mM)
(min-1 mM-1)
118 ± 2
NA
75 ± 2
2.2 ± 0.1
0.006 ± 0.0001
380 ± 20
43 ± 1
K
M
0.16 ± 0.02
1200 ± 200
158 ± 2
0.075 ± 0.004
1600 ± 500
38 ± 1
NA
0.27 ± 0.02
280 ± 20
85 ± 2
cat / K
M
NA
-
1
k
cat (min )
(mM)
(min-1 mM-1)
6 ± 4
0.09 ± 0.02
70 ± 40
NA
K
M
0.32 ± 0.01
500 ± 20
140 ± 4
0.310 ± 0.002
120 ± 2
0.55 ± 0.04
150 ± 10
63 ± 2
0.061 ± 0.005
700 ± 60
48 ± 1
cat / K
M
-
1
k
cat (min )
(mM)
(min-1 mM-1)
105 ± 3
K
M
2.7 ± 0.2
52 ± 4
0.72 ± 0.07
147 ± 15
7.7 ± 0.7
NA
0.29 ± 0.03
210 ± 20
37.1 ± 0.5
1 ± 0.1
cat / K
M
NA
46 ± 3
-
1
k
cat (min )
85.8 ± 0.9
7 ± 0.4
10.8 ± 0.3
K
M
(mM)
0.97 ± 0.03
88 ± 3
0.05 ± 0.02
170 ± 70
1.3 ± 0.2
5 ± 1
0.39 ± 0.02
94 ± 4
0.21 ± 0.02
51 ± 5
k
cat / K
M
(min-1 mM-1)
NA: no activity was detected with that substrate. Errors represent the standard error.
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FULL PAPER
Table 4 – CAR activity against heterocycles and fatty acids
1
4.
15. 1H-
Pyrrole-2-
carboxylic
acid
16. Furan-
2-
carboxylic
acid
17.
Thiophene-
2-
carboxylic
acid
18. C4 –
Butanoic
acid
19. C8 –
Octanoic
acid
20. C12 –
Dodecanoic
acid
21. C18 –
Octadecanoic
acid
Pyridine-2-
carboxylic
acid
-
1
mpCAR
k
cat (min )
NA
NA
NA
50 ± 20*
NA
58 ± 1
55 ± 2
3.7 ± 0.3
K
M
(mM)
NA
NA
NA
NA
NA
NA
50 ± 20*
NA
NA
2.0 ± 0.1
29 ± 2
0.09 ± 0.01
600 ± 70
0.09 ± 0.02
39 ± 9
k
cat / K
M
(min-1 mM-1)
1.1 ± 0.6*
-
1
msCAR
tpCAR
noCAR
niCAR
k
cat (min )
(mM)
(min-1 mM-1)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
50 ± 10
13 ± 4
4 ± 2
19 ± 1
4.7 ± 0.5
4.0 ± 0.4
NA
123 ± 4
3.3 ± 0.3
37 ± 3
129 ± 7*
7.9 ± 0.8*
16 ± 2*
296 ± 8
0.1 ± 0.01
3000 ± 300
219 ± 3
131 ± 5
0.05 ± 0.01
2700 ± 400
157 ± 5
46 ± 4
0.6 ± 0.09
80 ± 10
K
M
k
k
k
cat / K
M
NA
-
1
k
cat (min )
(mM)
(min-1 mM-1)
23 ± 3
24 ± 7
0.9 ± 0.3
76 ± 4
20 ± 2
3.9 ± 0.4
NA
82 ± 3
82 ± 3*
15 ± 1
K
M
3.3 ± 0.3
25 ± 3
5.0 ± 0.4*
17 ± 2*
0.2 ± 0.01
1140 ± 50
141 ± 2
0.04 ± 0.01
3600 ± 400
99 ± 3
0.12 ± 0.03
120 ± 30
11 ± 1
cat / K
M
-
1
k
cat (min )
(mM)
(min-1 mM-1)
135 ± 4
2.6 ± 0.2
52 ± 4
170 ± 20*
50 ± 8*
K
M
NA
0.2 ± 0.01
750 ± 30
233 ± 5
0.04 ± 0.01
2500 ± 300
157 ± 9
0.02 ± 0.01
500 ± 300
68 ± 7
cat / K
M
NA
3.4 ± 0.7*
260 ± 30*
32 ± 4*
-
1
k
cat (min )
NA
60.8 ± 0.9
1.00 ± 0.05
K
M
(mM)
NA
NA
0.2 ± 0.01
0.02 ± 0.01
0.7 ± 0.1
kcat / K
M
(min-1 mM-1)
NA
NA
NA
58 ± 3
8 ± 1*
1350 ± 90
7000 ± 2000
100 ± 20
M
NA: no activity was detected with that substrate. *: K was unusually large and substrates concentrations could not reach a high enough concentration to
accurately determine kinetic constants. Errors represent the standard error.
The cognate compound with a triple bond (phenylpropynoic acid;
compound 11, Figure 3C, Table 3) showed very low or no
detectable activity in the CAR reaction.
lower than the activity observed with benzoic acid as substrate.
In cases where no activity was detected it is possible that the KM
was outside the range of detection of the assay.
Two other compounds were tested: firstly, the -keto acid 3-oxo-
3
-phenylpropanoic acid (compound 12, Figure 3C, Table 3)
showed an increase in K with mpCAR, msCAR and tpCAR, but
a decrease in K with noCAR and niCAR in comparison to 3-
Fatty acids
M
M
All the CARs showed very high catalytic efficiency for fatty acids
between eight and twelve carbons in length, with low K values
M
compared to benzoic acid (compound 18-21, Figure 3E, Table 4).
Octadecanoic acid (21), with a carbon chain length of 18 carbons,
phenylpropionic acid. However, in all cases, the kcat was reduced
when compared to 3-phenylpropionic acid or benzoic acid, as it
was for (E)-3-phenylprop-2-enoic acid.
Finally, trans-2-
phenylcyclopropane-1-carboxylic acid (compound 13, Figure 3C,
Table 3) features a cyclopropane ring between the benzene ring
and carboxylate group. For all the CARs this modification resulted
in much lower K values, and a much lower kcat, compared to 3-
M
phenylpropionic acid or benzoic acid.
showed a similarly low K
except mpCAR were active against butanoic acid (compound 18,
Figure 3E, Table 4) but with a very large K , in most cases too
M
but a greatly reduced kcat. All CARs
M
large to characterize accurately. In general, mpCAR was much
less efficient with fatty acids than the other CAR enzymes.
Heterocycles
Effects of pH
Heterocycles containing nitrogen, oxygen or sulfur were tested
The activity of an enzyme at different pH values is an important
consideration for an industrial enzyme. Therefore, the effect of pH
on CAR activity was examined by measuring activity against
(
compounds 14-17, Figure 3D, Table 4). Generally, weak activity
was observed, with decreasing values for increasing
heteroatom size. Where there was activity, kcat was generally
K
M
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FULL PAPER
1
20
120
100
80
60
40
20
0
120
100
80
60
40
20
0
noCAR
mpCAR
tpCAR
1
00
8
6
4
2
0
0
0
0
0
5
6
7
8
9
10
5
6
7
8
9
10
5
6
7
8
9
10
pH
pH
pH
1
1
20
120
100
80
60
40
20
0
niCAR
msCAR
00
8
6
4
2
0
0
0
0
0
5
6
7
8
9
10
5
6
7
8
9
10
pH
pH
Figure 4 – The activity of CAR enzymes in response to pH. Overlapping buffers were used to cover a range from pH 5.6 to pH 9.0 in intervals of 0.2 and are
displayed as follows: MES-NaOH PIPES-NaOH , MOPS-NaOH , HEPES-NaOH , Tris-HCl Activity against 4-methylbenzoic acid acid is shown relative
to the highest activity at 100%. Errors bars show the combined standard deviation of three readings and three blank readings (with no enzyme) at each pH
benzoic acid at different pH values. mpCAR, niCAR, noCAR and
tpCAR all showed optimum activity at pH 7.5, whilst msCAR
showed an optimum activity at pH 7.8 (Figure 4). Both niCAR and
tpCAR show a sharp peak of activity around pH 7.5, with activity
quickly decreasing as the pH moved away from this point. In
contrast, mpCAR and noCAR show a slightly broader optimum
around pH 7.0 to 7.6. msCAR behaves very differently from the
other CARs. At more acidic pH values between pH 5.5 and 6.8 it
shows very low activity where the other CARs are more active.
However, it is also more active at more alkaline pH values where
the other CARs are less active.
temperatures beyond 44 °C, while msCAR is marginally more
thermostable and is able to retain some activity until 47 °C.
Activity at temperature was tested in a 10 minute reaction. The
more thermostable CARs, mpCAR, msCAR and niCAR all
showed an optimum activity of 42 °C (Figure 5B). Activity
decreased past this temperature at various degrees relative to the
thermostability of each enzyme. noCAR showed a slightly lower
optimum at 38 °C while tpCAR had a much lower optimum still, at
only 31 °C.
The half-life and degradation constant at 30 °C were calculated
by measuring activity at various time points over 120 hours. The
data were fitted to a one phase decay equation by non-linear least
Effects of temperature
squares regression.
mpCAR, a CAR from a moderate
thermophile, showed by far the longest half-life at 30 °C at 123.2
hours (Table 5). In contrast, tpCAR has a much shorter half-life
of only 25.0 hours. The half-lives of msCAR, niCAR and noCAR
fell between these extremes at 53.7, 42.9 and 35.3 hours
respectively. Total turnover numbers (TTN) for the three best
Thermostability was investigated by incubating the CAR enzymes
at various temperatures for half an hour and measuring residual
activity against 4-methylbenzoic acid relative to a control kept on
ice. tpCAR was the least thermostable CAR tested, being
completely inactive after half an hour at 42 °C (Figure 5A). In
contrast, mpCAR, a CAR from the moderate thermophile M. phlei,
retains 92% of its activity following the same incubation at 42 °C.
mpCAR was able to retain residual activity up to 50 °C making it
the most thermostable CAR identified to date. Both niCAR and
noCAR showed intermediate thermostability, denaturing at
D
substrates were calculated as kcat / K (Table 5).
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1
25
noCAR
mpCAR
msCAR
tpCAR
niCAR
1
1
25
00
A
B
1
00
75
50
25
0
7
5
2
5
0
5
0
30
35
40
45
50
30
35
40
45
50
Temperature oC
Temperature oC
Figure 5 – The effects of temperature on CAR enzymes. A – Thermostability of CAR enzymes. The residual activity of CAR enzymes against 4-methylbenzoic
acid after a 30-minute incubation at different temperatures is displayed. Activity is shown relative to a control sample kept at 4 oC with errors bars showing the
standard deviation of three readings. B – Activity of CAR enzymes at different temperatures. Activity is relative to the fastest rate at 100%. Error bars show the
combined standard deviation of three readings and three blank reading (with no substrate) at each temperature.
Product inhibition
Discussion
+
mpCAR was tested for product inhibition with AMP, NADP and
The CAR enzymes offer an excellent opportunity for green
chemistry: they offer the opportunity to reduce carboxylic acids
selectively to aldehydes, without the use of harsh reducing agents.
CARs also have the clear advantage over other enzymes capable
of carrying out this reaction of the reduced product being
thermodynamically favored, due to the hydrolysis of ATP.
Although previous studies have identified a few CARs from
different species, and demonstrated that they have activity
against diverse acids, none of these studies has provided a
detailed, kinetic comparison of diverse CARs. We therefore aimed
to thoroughly characterize example CARs from across the
known CAR family, together with the best-characterized
CAR from N. iowensis. Our aim was to demonstrate the
similarities and differences between these CARs, learn
+
PP
i
. NADP showed competitive inhibition with NADPH with a K
I
of 143 ± 8 M (Supplementary Figure 13) and AMP was a
competitive inhibitor of ATP with a K of 8200 ± 900 M
Supplementary Figure 14). PP showed mixed inhibition with
ATP with a K of 220 ± 50 M, and an  of 2.5 ± 1.4. Surprisingly,
PP also showed competitive inhibition with 4-methylbenzoic acid
with a K of 340 ± 40 M (Supplementary Figures 15 and 16).
I
(
i
I
i
I
Table 5 – Half-life and degradation constant K
D
of CAR enzymes when
o
incubated at 30 C.
TTN
Benzoic
acid
TTN 4-
Methylbenzoi
c acid
TTN 4-
Halflife
K
D
Enzyme
Methoxybenzo
ic acid
(hours)
(hrs -1)
more about the CAR mechanism, and highlight the
potential of these enzymes for biocatalysis.
0
.0056 ±
.004
.013 ±
30000 ±
20000
15000 ±
1000
20000 ±
20000
mpCAR
msCAR
niCAR
123.2
53.7
25
20000 ± 20000
14000 ± 1000
175 ± 4
0
0
10000 ± 1000
336 ± 8
0.001
The effect of the addition of electron donating or
withdrawing groups
0
.28 ±
350 ± 30
0
.002
.02 ±
.002
.016 ±
.002
0
9000 ±
1000
9000 ±
1000
noCAR
tpCAR
35.3
42.9
6800 ± 700
6000 ± 1000
6900 ± 700
8000 ± 1000
0
The reduction of carboxylic acids to aldehydes typically
involves a transfer of a ‘hydride’ to the carbonyl unit. We
therefore initially expected that electron withdrawing
groups, which make this carbon more electrophilic, would
be preferred substrates. However, our observation was that,
contrary to our expectation, electron donating groups were
preferred substrates (Figure 3, Table 2). The addition of electron
0
0
The halflife, K
D
and TTN for the three best substrates of CAR enzymes calculated
-K*X
from activity after incubation at 30 °C over time, fitted to Y=Y0*e . Standard error
for K
D
D
is shown. TTN has been calculated as kcat / K , with the combined error
shown.
M
donating groups to benzoic acid resulted in a reduction in K , and
so an increase in catalytic efficiency. We reasoned that these
groups would drive electrons into the -system, making the first
step of the reaction (attack by the negatively charged carboxylate
group on the  phosphate of ATP) more favorable. As two of the
other steps (2 and 4) involve nucleophilic attacks on the acid
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group carbon atom of the carboxyl group (which should favor
electron withdrawing groups), this strongly suggests that the first
step in the reaction has the greatest impact on substrate
inhibits activity due to a steric hindrance because of its close
proximity to the carboxylate group.
specificity and selection. It is possible that the reduced K
M
with
Previously it has been reported that 2-substituted benzoic acids
are poor substrates for niCAR, in good agreement with our
data.[23] However very low activity was observed with 8 previously,
which we did not detect. Substrates with the addition of electron
donating groups to benzoic acid were previously shown to be
good substrates for niCAR, in agreement with our results. The
activity of niCAR with electron withdrawing chloro and bromo 3-
substituted benzoic acids supports our reasoning that in the 3-
position the absence of a resonance effect allows better activity
with these substrates than in the other positions. [23]
electron donating substituents is a consequence of the acyl-AMP
intermediate forming more readily, although very detailed studies
of the kinetics of this individual step would be required to confirm
this. Indeed, previous studies of NRPSs have shown the
adenylation reaction to be a rate limiting step.[33] In long-chain
fatty acid ligases, the acyl-AMP intermediate has been shown to
be unable to leave the active site [19], so the addition of a group
which likely improves the formation of this intermediate might be
expected to cause a lower K
Moreover, when benzyl-AMP was used as a substrate with a CAR
from Nocardia asteroides it showed a K of 70 nM, compared to
60 nM for benzoic acid, suggesting that this intermediate binds
M
and greater catalytic efficiency.
M
2
Modification of the aromatic ring and unit on CAR activity.
[
22]
more tightly to the enzyme.
Furthermore, the
phosphopantetheine binding and C-terminal reductase domains
shows high sequence identity to that of other ANL superfamily
members that process very different substrates. For example a
NRPS from Mycobacterium intracellulare, WP_014382786.1, has
an average of 58 % identity to the CARs in Figure 2 for this C-
terminal region. This strongly suggests that substrate specificity
must be determined in the adenylation domain, likely at the
formation of the first intermediate.
The CARs generally showed less activity towards heterocycles
compared to a benzene ring. They showed a preference for
heterocycles containing a larger heteroatom, or with a less
aromatic nature. In substrate 17, the lone pairs of electrons in the
sulfur atom are more dispersed and less available for bonding,
which possibly results in the lower K
atom in substrates 14 or 15 has lone pairs more available for
bonding, which may result in the very large K values, or lack of
M
. In contrast, the nitrogen
M
activity observed. Substrate 16, with an oxygen atom in the
heterocycle, sits between these substrates in both respects.
In the 3-position (4), the methoxy group has no resonance effect
on the carboxylic acid and so is actually slightly electron
withdrawing by induction, as indicated by the Hammett sigma
constants in Table 2. In many of the CARs, the kcat of 3-
substituted benzoic acids shows a small reduction compared to
Extension of the carboxylate group away from the aryl group in 9
disrupts its influence on the carboxylic acid, and makes a less
sterically rigid substrate. This difference seems to have made the
4-substituted acids, with msCAR showing greatly reduced activity.
M
carboxylic acid group more accessible, as the K is much lower
However, these are still good substrate for most of the CARs. It
is likely that there are further interactions between the substrate
and the active site binding pocket, and that electronic effects
alone cannot account for all differences in activity.
than substrate 1 in most cases. In contrast, the inclusion of a
double bond in substrate 10, should withdraw electrons from the
carboxylic acid group. This would be beneficial for nucleophilic
attack on the carbonyl in steps 2 and 4 of the reaction, but
detrimental to the initial attack by the oxygen of the carboxylate
group on ATP. The result is a significant drop in kcat compared to
substrate 1. The double bond also makes the molecule more rigid,
Very low or no activity was found with the 2-methoxy substituent
of benzoic acid (5). This suggests that there is a steric
interference by the methoxy group on the binding of the nearby
carboxylate group to the relevant area of the active site. This
effect has been reported for other CARs examined to date with
other 2-substituents. However, some cases suggest there is
activity, but at a low level.[22,23] No structure of a CAR enzyme has
yet been described, and this would be highly beneficial in
understanding the effects of groups in the 2-position.
in an apparently favorable conformation, as the K
than for substrate 9. When a triple bond is added to the substrate
11), the molecule is very rigid and flat, with a more electron
M
is even smaller
(
deficient carboxylic acid group. These effects together removed
activity in nearly all the CARs. The presence of a -ketone group
into the -carbon of substrate 9 will have a similar effect to the
inclusion of a double bond in substrate 10, very weakly
withdrawing electrons from the carboxylic acid group. The ketone
All substrates with an electron withdrawing group (6-8) showed
much lower kcat values than benzoic acid, in most cases inhibiting
activity all together. These groups should increase the propensity
of the carbonyl carbon to nucleophilic attack in steps 2 and 4 of
the reaction. Therefore, this strongly suggests again that these
two steps are of limited relevance for substrate specificity. Only
the 3-nitro substituent (7) showed activity with all the CAR
enzymes, likely as in this position the electron withdrawing group
has no resonance effect on the carboxylate group. As is the case
with the methoxy group, it is possible that a 2-nitro substituent (8)
M
group (substrate 12) had mixed effects on the K for the various
CARs, suggesting differing interactions taking place with the
ketone group within the active sites of the enzymes. These
observations agree with the hypothesis that the first step of the
proposed reaction mechanism is rate limiting.
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Fatty acids
efficiency. When choosing an enzyme for industrial use, the
lifespan of the enzyme can be an important consideration. TTN
can be calculated as a measure of how effective an enzyme will
be over its lifetime, which we have demonstrated with three of the
best CAR substrates (Table 5). In this respect the most
thermostable CARs have an obvious advantage in that the total
turnover number of these enzymes will be much greater.[29] We
observed that the lifespan of the enzyme at 30 °C (Table 5)
mirrored the thermostability of the enzymes exactly (Figure 5),
suggesting that a test of thermostability will be a good predictor of
lifespan for CARs.
Fatty acids make interesting substrates, since fatty alcohols can
be used as biofuels, in detergents, surfactants and polymers [7]
.
As was observed for the CAR from Mycobacterium marinum
mmCAR), most of the CARs tested were active against fatty
(
acids between C4 and C18, with similar kinetics to previous work
observed [ . Catalytic efficiency with substrate 18 (C4) was very
10]
M
poor, primarily due to large K ‘s for this substrate suggesting it
might be too small to make the necessary interactions in the
active site of the adenylation domain. However, larger fatty acids
M
showed much lower K ’s, with high turnover numbers resulting in
catalytic efficiencies higher than any of the aromatic substrates
tested in many cases. As the acyl chain length increased past
substrate 19 (C8) kcat decreased, reaching a low residual level for
substrate 21 (C18). Both niCAR and msCAR showed a better
turnover number with substrate 21 than the other CARs,
suggesting these enzymes might be better suited to larger
substrates. Recently two other CARs, in combination with niCAR
and mmCAR have been shown to have activity against ethanoic,
butanoic, 2-methyl butanoic and 2-oxobutanoic acids, highlighting
that CARs can accept small fatty acids and that they can tolerate
the addition of groups such as a methyl or carbonyl group onto
the alpha carbon. However, 2-aminobutanoic acid was also
tested but showed no activity. [11]
The CAR enzymes in this study show only moderate
thermostability. To date no CAR enzymes have been identified in
any thermophilic organisms. A thermostable CAR enzyme would
be attractive for use industrially as this enzyme would likely be
resistant to other denaturing forces such as extremes of pH or
organic solvent and likely offer a higher total turnover number for
use in vitro reactions.
Product inhibition and reaction mechanism
mpCAR was shown to be inhibited by most of its reaction products
and it is assumed that the other CARs share this inhibition. It is
+
unsurprising that NADP acts as a competitive inhibitor of NADPH
+
(
Supplementary Figure 13) as NADP is likely also able to bind to
Effects of pH and temperature
the Rossmann fold of the reductase domain. AMP acts as a
competitive inhibitor against ATP (Supplementary Figure 14),
likely as they are very similar molecules. AMP has also been
shown to be a competitive inhibitor of ATP in long-chain fatty acid
CoA synthetases, in which the adenylation domain shows
significant homology to the CAR adenylation domain.[30]
The operating pH and temperature range of an enzyme is an
important consideration for a potential biocatalyst. Stability at
extremes of pH and in solvents are characteristics often found in
thermostable proteins, as the mechanisms stabilizing these
proteins against high temperature can also be stabilizing against
these other conditions. We observed an optimum pH of 7.5 for
four of the five CARs tested, with a general tolerance to acidic pH,
consistent with previously reported data on the activity of other
CARs.[ In particular, both mpCAR and noCAR were able to
tolerate pH 6 with only a small loss of activity (whilst other CARs
showed a much narrower optimum). In contrast to this, msCAR
is clearly better suited to more alkaline pH values (Figure 4). This
therefore offers a CAR suitable for use in biocatalysis in
conjunction with other enzymes favoring a similarly alkaline pH.
22]
mpCAR showed by far the best thermostability of any
characterized CAR (Figure 5, Table 5). We also observed that it
shows a much lower catalytic efficiency in general than the other
CARs at 30 °C (Tables 2-4). Possibly, there has been a trade-off
between the rigidity of the enzyme (providing thermostability) and
flexibility to allow a broader substrate range. It was notable that
the rate enhancement in mpCAR at its optimum temperature
compared to 30 °C was little greater than that for other CARs
Figure 6 – Model for binding of substrates and inhibitors to the CAR
enzyme. A: binding and release of substrates, products and inhibitors in the
adenlyation domain. The final result is the formation of a thioester intermediate
with the phosphopantetheine arm, represented by CAR-CA.
The
(
(
Figure 5B). In contrast, tpCAR shows very poor thermostability
Figure 5), but is active with many of the substrates that the others
phosphopantetheine arm can then transfer CA to the reduction domain, B,
where it is reduced by NADPH, releasing the aldehyde product
CARs could not turn over (e.g. compounds 5, 6, 11, 14).
A
possible compromise enzyme is msCAR, which shows the next
best thermostability, and also has generally good catalytic
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PP
i
showed mixed inhibition against ATP but competitive
Figure 1).[35] This suggests that the CARs also undergo
characteristic ANL superfamily domain-alteration between steps
1 and 2 (figure 1) to catalytically isolate the adenylation and
inhibition against 4-methylbenzoic acid (Supplementary Figures
15 and 16). This pattern of inhibition is characteristic for ordered
[31]
[18]
sequential bisubstrate reactions.
This indicates that ATP is first
thioester forming reactions.
to bind to the adenylation domain and is then followed by a
carboxylic acid. Long-chain fatty acid CoA synthetases show the
same ordered binding of these substrates.[19] We therefore
propose a model for the ordered binding of substrates and
inhibitors to the CAR enzyme based on these results (Figure 6).
Summary
CARs have been proposed as a useful tool for novel biocatalysis.
This study has demonstrated that, across the entire extant
phylogeny of CARs, similar substrates are preferred by this family
of enzymes, with some enzymes being more promiscuous than
others. In particular, our detailed kinetic analysis of CARs strongly
suggests that the first step in the proposed reaction mechanism,
during which an AMP-carboxylic acid phosphoester intermediate
i
It is also interesting that whilst PP is a product of ATP, its activity
as an inhibitor shows that it preferentially binds to the carboxylic
acid binding site. CARs might therefore need to be combined with
other enzymes such as phosphite dehydrogenase[32] or inorganic
pyrophosphatase[33] to overcome product inhibition in an industrial
process. Indeed the in vitro turnover of niCAR has been shown
to be improved by the addition of an inorganic pyrophosphatase
enzyme.[34]
i
is formed with the release of PP, is critical for determining suitable
substrates. Consequently, the addition of groups that donate
electrons, making the oxygen of the carboxylic acid more
electronegative, will be better substrates; and aliphatic acids are
strongly preferred to aromatic acids. This study also highlighted
that, similarly to other members of the ANL superfamily to which
the CARs belong, this first step is an ordered sequential Bi Bi
reaction, with ATP being bound before the carboxylic acid. Of
CAR phylogeny and insight into the adenylation step
Here we have provided the first glimpse of CAR evolution within
the Actinomycetes. From the phylogeny it can be hypothesized
that the CARs may have propagated through the Nocardia and
Mycobacteria by a series of early horizontal transfer events. This
is most apparent in M. smegmatis, which possesses three CAR
paralogues that cluster in two distinct Mycobacterial clades.
Additionally, it is apparent that a large amount of change has
occurred within the Tsukamurella. This could reflect the slightly
more promiscuous substrate range of tpCAR.
particular relevance to biocatalysis is that all of the by-products of
the reaction (PP
+
i
, AMP and NADP ) appear to be inhibitors: for
the use of CARs in vitro there is a need to remove or regenerate
these. These data further validate CARs as a useful tool for novel
biocatalytic reactions, and highlight their potential when
integrated with other enzymes in vitro for efficient reduction of
carboxylic acids to aldehydes.
We presented evidence that the adenylation domain of the CARs
belongs to the ANL superfamily of enzymes due to the presence
of conserved hallmark motifs. Both nonribosomal peptide
synthetases (NRPSs) and the acyl-CoA synthetases similarly use
an acyl group to form a thioester between a substrate and a
pantetheine thiol, supporting this interpretation. Furthermore, the
Methods
Alignments and Phylogeny Construction
NPRSs mobilize their substrate following thiolation of
a
phosphopantetheine arm bound to a holo-acyl carrier protein
domain. Parallels can be drawn between both above reactions
and the proposed mechanism of CAR activity in Figure 1. This
offers the opportunity to exploit the extensive studies on the ANL
superfamily to gain insight into the finer details of the mechanism
of carboxylic acid reduction employed by CARs.
Unless specified, all algorithms were performed under default settings. 48
sequences were retrieved by homology search in BLAST to the N.
iowensis CAR. Alignments were performed using the MUSCLE plug-in
within Geneious version 9.1 (http://www.geneious.com).[36] Sequence
masking was conducted with the Gblocks algorithm within the Phylogeny.fr
online tool (http://www.phylogeny.fr).[37] ProtTest (version 3.4)[38] analysis
of the aligned dataset was performed in the command line. MrBayes
(
version 3.2.6)[39] was run in the command line as follows: The amino acid
In particular, ANL superfamily members are further partitioned
into two subdomains – a large (~450 aa) N-terminal domain and
a small (~100 aa) C-terminal domain, connected by a flexible
linker. Crystal structures show the substrate-binding pocket is
formed by the N- and C-domain interface. Substrate adenylation
proceeds in a two-step manner, where following the formation of
substitution model was fixed to WAG with a gamma-distributed rate
variation across a proportion of invariable sites and 8 gamma categories.
The analysis was run for 1,000,000 MCMCMC generations, sampling
every 100 generations with two parallel runs and four chains (containing
one heated chain of temperature 0.2), with a burn-in of 25%. Trees were
visualised, midpoint rooted and modified in FigTree version 1.4
(
http://tree.bio.ed.ac.uk/software/figtree/).
i
an acyl-bound intermediate and the release of PP , the active site
undergoes large conformational changes due to a ~140 rotation
of the C-terminal domain. Within the NRPSs and the acyl-CoA
synthetases, the second domain architecture facilitates thiolation
of the phosphopantetheine. Lysines that are required within each
active site are positioned on opposing faces of the C-terminal
domain, and are conserved within the CARs (Supplementary
It must be noted that a more complete list of 124 CAR homologues was
retrieved (Supplementary Figures 17 and 18). However, a reduced set of
sequences was used as this allowed the construction of a more reliable
phylogeny.
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ChemCatChem
10.1002/cctc.201601249
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7.8, 50 mM HEPES pH 7.0 to pH 8.0 and 50 mM Tris pH 7.8 to pH 9.0
Expression and purification
were used. Reactions were carried out as standard with 1 mM ATP, 0.25
mM NADPH, 10 mM MgCl 2-6 g of purified CAR enzyme and 5 mM 4-
2
CAR genes (except niCAR) were cloned into expression vectors pNIC28-
Bsa4 [28] or obtained from Prozomix, cloned into pET28a (Novagen). A pET
methylbenzoic acid. Blanks containing no enzyme were used to subtract
a blank rate at each pH value. Initial rates were calculated as relative
activity against the fastest result at 100 %.
plasmid for the expression of niCAR was obtained from Andrew Hill
(
University of Manchester). All contained a N-terminal 6x histidine tag.[28]
Full sequences for all vectors are supplied as Supplementary information.
Vectors were transformed into BL21 (DE3) E. coli along with a pCDF-
Duet1 vector containing a phosphopantetheine transferase from Bacillus
subtilis for its co-expression with the CARs. Expression was carried out in
LB media with the addition of 50 g/l each kanamycin and spectinomycin.
Cells were grown to approximately 0.6 OD600nm at 37 °C with shaking at
Thermostability
A solution containing 2 g of purified enzyme, 0.25 mM NADPH, 1 mM
ATP, 10 mM MgCl , 100 mM Tris-HCl pH 7.5 was incubated across the
2
temperature gradient of a Biorad thermocycler from 30 °C to 50 °C for 30
minutes. The sample was cooled and assayed for CAR activity against 4-
methylbenzoic acid, in comparison to a control sample that remained on
ice.
225 rpm, at which point IPTG was added to a concentration of 150 M and
temperature was dropped to 20 °C for protein expression overnight. Cells
were harvested by centrifuging and re-suspended in 25 mM Tris-HCl pH
8.0, 0.5 M NaCl. Cell lysate was prepared by sonication on ice followed
by centrifugation to remove the insoluble fraction.
CARs were purified from the cell lysate using a 1 ml His-Trap FF crude
column (GE Healthcare) using an elution gradient from 10 to 250 mM
imidazole in 25 mM Tris-HCl pH 8.0, 0.5 M NaCl. The purified sample was
then applied to a Superdex 200 HiLoad 16/60 gel filtration column (GE
Healthcare) and eluted in 25 mM HEPES, pH 7.5, 0.1 M NaCl at 1.0 ml/min.
Eluted fractions were analyzed by SDS-PAGE before being pooled and
concentrated to approximately 2 mg/ml. To calculate protein concentration
from OD280nm, an extinction coefficient and molecular weight for each
enzyme was calculated using the ExPaSy ProtParam tool, and are shown
in Supplementary Figure 19. Yields of approximately 2 – 10 mg purified
protein per liter of culture were obtained, with 2 – 4 L of culture prepared
per batch. Single use aliquots of protein were stored at -80 °C.
Degradation at 30 °C
2
ml samples at 2 mg/ml in 25 mM HEPES, pH 7.5, 0.1 M NaCl were
incubated at 30 °C over a 120 hour period. At specified time intervals,
samples were taken and assayed for enzyme activity against 4-
methylbenzoic acid. Rates were calculated relative to the first reading at
1
00 %, and fitted to a model of first order thermal deactivation using the
equation Y = Y0 * e- where Y is the relative activity and X is the time in
K*X
hours.
Temperature vs activity
100 mM Tris-HCl pH 7.5 was prepared at assay temperatures between 30
Standard enzyme assay
and 50 °C. Assays were performed as for the thermostability experiment
using the temperature gradient of a Biorad thermocycler from 30 °C to
Unless otherwise specified, assays were carried out in 100 mM Tris-HCl
50 °C over the course of 10 minutes, before rapidly cooling on ice with the
pH 7.5 prepared at 30 °C, 1 mM ATP, 0.25 mM NADPH, 10 mM MgCl
g of purified CAR enzyme and 5 mM carboxylic acid substrate in a total
volume of 200 l. Carboxylic acid substrates were prepared in DMSO at
00 mM. The oxidation of NADPH was used to monitor the reactions by
2
, 2-
addition of 10 mM NaOH. A blank reaction with no substrate was used to
calculate the NADPH used in the reaction. Activity was calculated relative
to the maximum rate at 100 %.
6
5
measuring the absorbance of NADPH at 340 nm. Reactions were
performed in triplicate in a 96-well microtitre plate using a Tecan M200
plate reader at 30 °C over the course of 5 or 10 minutes, after a 5 minute
preincubation at 30 °C. Where convenient, an EpMotion 7050 (Eppendorf)
liquid handling robot was used to set up the assays.
Product inhibition
Potential inhibitors were titrated across a broad range of concentrations to
determine whether inhibition occurred and to give an idea of an
I
approximate K . Kinetic analysis then was performed as described above
using substrates that each inhibitor was likely competitive against, with the
addition of the inhibitors at a range of concentrations based around the
Kinetic analysis of substrate specificity.
I
approximate K . Data were fitted using GraphPad Prism 5.0 by non-linear
Kinetic analysis was performed by picking eight appropriate substrate
least squares regression to different models of enzyme inhibition. The
model with the best fit for the data was used to determine the mode of
inhibition. Where inhibition was not competitive, additional analysis was
carried out with other substrates.
M
concentrations around an approximate K value for each substrate, and
measuring initial rates as described previously. Rates were fitted to the
Michaelis-Menten equation by non-linear least squares regression using
GraphPad Prism 5.0. To calculate constants for ATP and NADPH, 5 mM
(
E)-3-phenylprop-2-enoic acid was used as the carboxylic acid substrate,
except for niCAR where 5 mM 4-methylbenzoic acid was used.
Acknowledgements
pH vs activity.
The authors thank Andrew Hill (University of Manchester) for
providing many of the substrates tested, the pCDF-Sfp plasmid
and the plasmid for the expression of niCAR; and Clive Mountain
Buffers were prepared and titrated to the correct pH using NaOH or HCl
whilst at 30 °C, covering pH values in intervals of 0.2. The buffers 50 mM
MES pH 5.6 to 6.6, 50 mM PIPES pH 6.4 to 7.4, 50 mM MOPS pH 6.6 to
(
GSK), Stacy Clark (GSK), and Alison Hill (University of Exeter)
for advice on the chemistry of the CAR reaction, and Jennifer
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ChemCatChem
10.1002/cctc.201601249
FULL PAPER
Farrar (Georgia Institute of Technology) for providing walltime on
her server to run the Bayesian analyses. Nzomics (Prof. Gary
Black and team) and Prozomix (Simon Charnock and team) are
gratefully acknowledged for cloning msCAR and tpCAR. WF was
funded by BBSRC (grant no. BB/K501001/1) and
GlaxoSmithKline; AT was funded by BBSRC (grant no.
BB/J014400/1). Requests for raw data should be sent to NJH.
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Keywords: carboxylic acid reductase • biocatalysis • enzyme •
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This article is protected by copyright. All rights reserved.