Oxalyl-CoA Decarboxylase Enables Nucleophilic One-Carbon Extension of Aldehydes to Chiral α-Hydroxy Acids

Article abstract of DOI:10.1002/anie.201915155

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine-diphosphate-dependent (ThDP) oxalyl-CoA decarboxylase (OXC)/2-hydroxyacyl-CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl-CoA thioester substrates through the release of the C₁-unit formyl-CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C₁-extensions of various aldehydes to yield the corresponding 2-hydroxyacyl-CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes—a specific oxalyl-CoA synthetase and a 2-hydroxyacyl-CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)-α-hydroxy acids with enantiomeric excess up to 99 %.

Full text of DOI:10.1002/anie.201915155

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Accepted Article
Title: Oxalyl-CoA Decarboxylase Enables Nucleophilic One-Carbon
Extension of
Aldehydes to Chiral α-Hydroxy Acids
Authors: Tobias Jürgen Erb
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201915155
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10.1002/anie.201915155
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Oxalyl-CoA Decarboxylase Enables Nucleophilic One-Carbon Extension of
Aldehydes to Chiral α-Hydroxy Acids
Simon Burgener,[a] Niña Socorro Cortina,[a] Tobias J. Erb*[a,b]
Abstract: The synthesis of complex molecules from simple,
renewable carbon units is the goal of a sustainable economy. Here
we explored the biocatalytic potential of the thiamine diphosphate-
dependent (ThDP) oxalyl-CoA decarboxylase (OXC)/2-hydroxyacyl-
CoA lyase (HACL) superfamily that naturally catalyze the shortening
of acyl-CoA thioester substrates through the release of the C1-unit
formyl-CoA. We show that the OXC/HACL superfamily contains
promiscuous members that can be reversed to perform nucleophilic
C1-extensions of various aldehydes to yield the corresponding 2-
hydroxyacyl-CoA thioesters. We improved the catalytic properties of
Methylorubrum extorquens OXC by rational enzyme engineering and
combined it with two newly described enzymes – a specific oxalyl-
CoA synthetase and a 2-hydroxacyl-CoA thioesterase. This enzyme
cascade enabled continuous conversion of oxalate and aromatic
aldehydes into valuable (S)-α-hydroxy acids with enantiomeric excess
up to 99%. Altogether our study showcases the potential to develop
ThDP-catalyzed nucleophilic C1-extensions as sustainable production
platform for chiral building blocks.
decarboxylase (OXC)/2-hydroxyacyl-CoA lyase (HACL). The
OXC/HACL superfamily comprises of decarboxylating members
(OXCs), as well as non-decarboxylating members (HACLs). Both
catalyze the ThDP-dependent cleavage of formyl-CoA from their
respective acyl-CoA thioester substrates (Scheme 1). OXCs
catalyze the decarboxylation of the C2-compound oxalyl-CoA to
formyl-CoA,[8] whereas HACLs cleave a 2-hydroxyacyl-CoA into a
fatty aldehyde that is shortened by a C1-unit.[9] In OXC and HACL
catalysis has been proposed to proceed through the same
covalent intermediate 1 on the ThDP cofactor (Scheme 1).[9-10]
After release of CO2 or aldehyde, the remaining formyl-CoA
moiety forms 1. Analogous to other ThDP-dependent enzymes
that form similar ThDP carbanion/enamine intermediates, we
speculated that 1 can act as nucleophile in a carboligation
reaction with an electrophilic carbon center, essentially reversing
the native OXC/HACL reactions. This would enable nucleophilic
C1-extension reactions employing formyl-CoA or oxalyl-CoA as
donor, which can in turn be produced from the cheap carbon
sources formate[6b] and oxalate, respectively. Recently, Chou et
al. demonstrated that HACL catalyzes the acyloin condensation
of formyl-CoA with aldehyde acceptor substrates,[11] However, the
study focused on HACL and formyl-CoA, thus the carboligation
potential of OXC with oxalyl-CoA as donor remains unknown.
One of biotechnology’s central goals is the synthesis of
multicarbon compounds under mild and sustainable conditions
from renewable resources. This requires biocatalysts that enable
selective C-C bond formation (‘carboligation’) between two
carbon units. Thiamine diphosphate (ThDP)-dependent enzymes
display high catalytic and substrate promiscuity with respect to
C-C bond breaking and forming reactions and they catalyze
carboligation reactions at high rates and with excellent stereo-
and enantioselectivity.[1] Several biocatalytic applications have
been developed that rely on ThDP-dependent carboligases;
notable examples are pyruvate decarboxylase (PDC),[2]
[4]
benzoylformate decarboxylase (BFD),[3] benzaldehyde lyase
and transketolase (TK).[5] Their broad catalytic repertoire makes
ThDP-dependent enzymes promising starting points for the
development of biocatalysts for C-C bond forming reactions.
In the context of a methanol and/or formate-based economy,
carboligations with one-carbon units are of particular interest.[6]
The potential of ThDP-dependent enzymes for synthetic one-
carbon fixation has been showcased by the computationally
designed enzyme formolase, which condenses three
formaldehyde molecules into dihydroxyacetone.[7] Here, we
focused on the superfamily of ThDP-dependent oxalyl-CoA
Scheme 1. OXC and HACL form the α-hydroxyl-CoA-ThDP carbanion/enamine
intermediate (1) by decarboxylation of oxalyl-CoA and cleavage of a 2-
hydroxyacyl-CoA, respectively. In the second half reaction 1 is protonated and
released as formyl-CoA.
To further explore the carboligase potential within the
OXC/HACL superfamily, we recombinantly produced human
HACL (HACLHs), as well as OXC from Methylorubrum extorquens
(OXCMe), HACLHs expressed very poorly at 25 °C. Lowering
temperature to 15 °C and using the strain ArcticExpress, protein
production was improved, but was still rather low (~2 mg protein
per L culture), especially compared to OXC (~30 mg/L). The low
expression and/or stability limit the use of human HACL for
biocatalytic applications. To test their carboligation activity, the
enzymes were incubated with formyl-CoA and various aldehyde
acceptors. 2-Hydroxyacyl-CoA thioesters were analyzed by LC-
MS and the products verified by MS/MS fragmentation (Figure S1).
Product formation was detected in the presence of formaldehyde,
[a]
[b]
S. Burgener, Dr. N.S. Cortina, Prof. Dr. T. J. Erb
Department of Biochemistry & Synthetic Metabolism
Max-Planck-Institute for terrestrial Microbiology
Karl-von-Frisch-Str. 10, D-35043 Marburg, Germany
E-mail: toerb@mpi-marburg.mpg.de
Prof. Dr. T. J. Erb
LOEWE-Center for Synthetic Microbiology, Philipps-University
Marburg, Karl-von-Frisch-Str. 8, D-35043 Marburg, Germany.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201915155
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acetaldehyde, propionaldehyde, glycolaldehyde, succinic
semialdehyde, benzaldehyde, and phenylacetaldehyde with both
enzymes (Table 1). Glyceraldehyde, glyoxylate and acetone (not
shown) were not accepted by OXCMe and HACLHs. As expected
from its physiological role as fatty acyl-CoA lyase, HACLHs
showed high activity with the aliphatic aldehydes acetaldehyde
and propionaldehyde. On the other hand, OXCMe showed very
high activity with benzaldehyde, indicating a distinct catalytic
spectrum between the two enzymes. In summary, HACLHs and
OXCMe are able to perform nucleophilic C1-extension reactions of
different aldehydes with formyl-CoA as C1-donor.
rapidly decarboxylated oxalyl-CoA into formyl-CoA, followed by
slow carboligation of formyl-CoA with benzaldehyde (Figure S4).
Decarboxylation of oxalyl-CoA proceeds via formation of 1,
which can be either protonated and released as formyl-CoA or
undergo a nucleophilic attack on benzaldehyde to form mandelyl-
CoA. We speculated that mandelyl-CoA formation may be
increased by suppressing the unwanted protonation reaction (and
subsequent release of formyl-CoA) that competes with
carboligation.
Structural, biochemical and theoretical studies on OXC from
Oxalobacter formigenes (OXCOf; 63% sequence identity to
OXCMe) demonstrated that protonation of 1 is mediated by a water
molecule which is coordinated by several polar side chains,
notably a tyrosine and a serine, which are conserved in OXCMe
(Tyr497 and Ser568).[10] In OXCMe variants Y497A and S568A,
formyl-CoA formation was decreased 20- to 50-fold, while the KM
of both variants was largely unaffected (Table 2 & Figure S3).
When starting with oxalyl-CoA and benzaldehyde OXCMe-Y497A
showed 5-fold increased mandelyl-CoA production rate (20 min-1;
Figure S4). The mutation Y497A thus increased the ratio of
carboligation to decarboxylation by a factor of approximately 400
compared to the wild-type, suggesting that we successfully
redirected activity of the enzyme towards carboligation by
suppressing protonation of 1.
Next, we investigated whether both enzymes would also be
able to perform decarboxylating carboligations with oxalyl-CoA as
C1-donor. We argued that the release of CO2 should provide a
Table 1. Comparison of the aldehyde substrate scope of OXCMe and
[a]
HACLHs
.
[c]
[c]
Aldehyde
R
Product name
glycolyl-CoA
lactyl-CoA
OXCMe
100
74
HACLHs
11
2a
2b
2c
2d
2e
2f
H
100
100
100
n.d.[b]
n.d.[b]
100
3
Me
Table 2: Steady-state kinetic parameters of oxalyl-CoA decarboxylation
[a]
2-hydroxybutyryl-
CoA
catalyzed by OXCMe.
5
CH2Me
CH2OH
CHOHCH2OH
COOH
(CH2)2COOH
Ph
Mutation
wild-type
Y497A
kcat (s-1)
kcat/KM (s-1 M-1)
9.3 × 105
KM (µM)
105 ± 11
180 ± 17
23 ± 2
glyceryl-CoA
erythronyl-CoA
tartronyl-CoA
1
98 ± 3
n.d.[b]
n.d.[b]
1
1.32 ± 0.04
5.53 ± 0.11
0.32 ± 0.01
7.3 × 103
1.1 × 105
S568A
2-hydroxyglutaryl-
CoA
2g
2h
2i
103 ± 15
3.1 × 103
Y497A S568A
mandelyl-CoA
100
22
3-phenyllactyl-
CoA
[a] Michaelis-Menten graphs are shown in Figure S3.
100
CH2Ph
Hydrolysis of mandelyl-CoA leads to mandelic acid, which
serves as building block for various drugs as well as a resolving
agent in chiral resolution processes.[16] We set out to identify a
thioesterase capable of hydrolyzing the non-natural metabolite
mandelyl-CoA. We tested PaaI, TesB and YciA from E. coli, which
were shown to hydrolyze a broad range of acyl-CoA thioesters.[17]
YciA showed relatively high mandelyl-CoA thioesterase activity
(kobs ≈ 1.5 s-1), low activity with formyl-CoA (kobs ≈ 0.06 s-1) and no
activity with oxalyl-CoA (Figure S5 & 1B). When used in
combination with OXCMe, the two enzymes formed an enzymatic
cascade for the conversion of oxalyl-CoA and benzaldehyde into
mandelic acid and free CoA (data not shown).
We noticed that subsequent regeneration of free CoA into
oxalyl-CoA would allow us to close a catalytic cycle for the
continuous production of mandelic acid (Figure 1A). To establish
such a catalytic cycle, we obtained an oxalyl-CoA synthetase
AMP-forming (OXS) homologue from M. extorquens,[18] and
[a] The reaction contained 2a-2i (10 mM), formyl-CoA (1 mM), OXCMe
or HACLHs (5 µM). Products were analyzed by LC-MS after 1 h reaction
time. [b] Product not detected. [c] Relative activity in %. Relative activity
refers to the comparison of OXCMe and HACLHs for each aldehyde
substrate.
strong thermodynamic driving force towards carboligation,
analogous to the reactions reported for glyoxylate carboligase,[12]
5b]
PDC,[13] BFD,[14] TK,[5a,
and branched chain ketoacid
decarboxylase.[15] HACLHs possessed only very low oxalyl-CoA
decarboxylation activity (kcat < 1 min-1, see Figure S2), in contrast
to OXCMe (kcat = 98 ± 3 s-1, see Table 2 & Figure S3), which
confirms OXCMe’s physiological function as oxalyl-CoA
decarboxylase.
Given the high activity towards benzaldehyde, we chose it as
model substrate to study the decarboxylating carboligation of
OXCMe in more detail. When started with oxalyl-CoA and
benzaldehyde, OXCMe as expected produced mandelyl-CoA at a
rate of 4 min-1 (Figure S4). However, mandelyl-CoA formation was
preceded by formation of formyl-CoA, suggesting that OXCMe first
determined the enzyme’s steady-state kinetic parameters (kcat
=
1.30 ± 0.02 s-1; KM(oxalate) = 9 ± 1 µM; Figure 1C). We then used
OXS in combination with YciA and OXCMe to continuously
produce mandelic acid from oxalate and benzaldehyde. When we
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replaced OXCMe by the Y497A variant, mandelic acid production
rate increased 5-fold and the conversion increased 4-fold
(Figure 1E). This was likely caused by decreased formation of the
unwanted side product formyl-CoA (and its further hydrolysis by
YciA). Chiral LC-MS revealed that (S)-mandelic acid was
produced with enantiomeric excess of 97% (Figure 1D). Since
YciA showed no stereospecificity in the hydrolysis of mandelyl-
CoA (Figure 1B), the stereochemistry is exclusively determined
The rational engineering of OXCMe-Y497A enabled a three
enzyme cascade comprising of OXS, a newly identified oxalyl-
CoA synthetase, and YciA, an efficient mandelyl-CoA
thioesterase with only minor formyl- and oxalyl-CoA hydrolysis
activities. The OXCMe-mediated production of aromatic (S)-α-
hydroxy acids in high ee from aldehydes and oxalate offers an
alternative to hydrogen cyanide based syntheses catalyzed by
nitrilases.[21] However, the requirements of CoA in catalytic
amounts and an ATP regeneration system may limit the potential
for a synthetic application on the larger scale. To this end,
employing whole-cell catalysts may prove to be advantageous,
providing not only the cascade enzymes but also ATP
regeneration and a CoA pool without addition of purified enzymes
and cofactors.[22]
by OXCMe
.
Next, we tested the substrate scope of the catalytic cycle by
replacing benzaldehyde with substituted variants 2i-2k (Figure
2A). Under limiting ATP concentrations (10 mM), the expected
products 3a-3d were formed at varying yields (57-93%) and ee
(44-99%) (Figure 2B). Notably, also sterically demanding 2k was
converted to 3d with high yield, albeit with moderate ee. The
broad substrate scope of the OXS-OXC-YciA cascade was further
confirmed by an extended screen, in which product formation was
detected for ten other aromatic and three heteroaromatic
aldehydes (3e-3r, Figure 2C). To test if the cascade can be scaled
up, we performed the reaction on a semi-preparative scale (0.625
mmol 2h). We added an ATP regeneration system comprising of
creatine phosphate, creatine kinase and adenylate kinase. With
catalytic amounts of ADP (0.5 mM) this five enzyme one-pot
cascade produced 3a with a yield of 53%. Taken together, these
results indicate that the established enzyme cascade can be
employed to produce various aromatic α-hydroxy acids with
moderate to high (S)-selectivity.
Altogether, our study expands the spectrum of ThDP-
dependent transformations by nucleophilic C1-extensions, which
gives access to α-hydroxy acids that are valuable chiral building
blocks and showcases ways to establish in vitro- and in vivo-
platforms for the continuous production of these compounds in
the future.
Experimental Section
Experimental Details can be found in the Supporting Information.
We demonstrated that members of the OXC/HACL
superfamily are able to catalyze C1-carboligation reactions
between 1 – formed either through decarboxylation of oxalyl-CoA
or deprotonation of formyl-CoA – and several aldehydes to yield
chiral 2-hydroxyacyl-CoA thioesters. These nucleophilic C1-
extension reactions expand the spectrum of ThDP-dependent
enzymes as versatile biocatalysts for C-C bond forming
reactions.[19]
What determines substrate specificity in the OXC/HACL
superfamily? The observed variance in the aldehyde scope of
OXCMe and HACLHs are likely caused by differences in the C-
terminal domain, which forms a ‘lid’ on top of the active site.[10a]
While the bottom part of the active site is virtually identical
between OXC and HACL (with exception of Tyr133 and Glu134
in OXCMe that are replaced by Phe and Gln in HACLHs), the C-
terminal lid-domain shows a high variability between individual
superfamily members. Further characterization of the OXC/HACL
superfamily could reveal more carboligases with aldehyde
preference for a desired application.
We engineered OXCMe towards improved carboligation rate at
the expense of formyl-CoA formation rate by replacing Tyr497
with Ala. Interestingly, the mutation Y497A did not affect the
enantioselectivity for (S)-mandelyl-CoA. This is reminiscent of
engineering efforts on PDC from Zymomonas mobilis, where
Glu473 positions a water molecule that acts as proton donor for
the ThDP carbanion/enamine intermediate. Mutating this amino
acid to glutamine led to a 100-fold preference of carboligation over
aldehyde release, under full retention of enantioselectivity.[13]
Considering the high demand of mandelic acid and its derivatives
in the (R) configuration,[16] it would be interesting to engineer
OXCMe towards inverted enantioselectivity. This has been
achieved for other ThDP-dependent carboligases.[20]
Acknowledgements
We thank L. Schulz for cloning of oxc and B. Heinrich for NMR
measurements. This work was supported by the German Ministry
of Education and Research Grant FormatPlant (part of
BioEconomy 2030, Plant Breeding Research for the Bioeconomy).
A provisional patent application has been filed through the Max-
Planck-Innovation based on the results presented here.
Keywords: oxalyl-CoA decarboxylase • thiamine diphosphate •
C-C coupling • C1 building blocks • biocatalysis
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Figure 1. A) The OXS-OXCMe-YciA cascade converts benzaldehyde and oxalate into mandelic acid and CO2 under consumption of
ATP. B) Thioesterase activity of YciA (2 µM) towards oxalyl-CoA, formyl-CoA and racemic mandelyl-CoA (0.5 mM each). The negative
control without enzyme is shown in Figure S6. Error bars show standard deviation of two replicates. C) Michaelis-Menten graph of OXS
with oxalate as substrate. D) Chromatograms of chiral HPLC analyzing the reaction products of the complete cascade with either wild-
type OXCMe (wt) or Y497A. On top are commercial mandelic acid standards. E) Mandelic acid formation of the cascade over time. The
reaction contained 2h (25 mM), disodium oxalate (10 mM), ATP (10 mM), CoA (0.5 mM), OXS (5 µM), OXC (5 µM), YciA (2 µM). ee of
(S)-mandelic acid is indicated for the last time point (22 h).
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Figure 2. Scope of the OXS-OXCMe-Y497A-YciA cascade for the synthesis of aromatic (S)-α-hydroxy acids. Chromatograms and time
courses of the reactions are shown in Figure S7. A) The reactions contained 2h-2y (25 mM), disodium oxalate (10 mM), ATP (10 mM),
CoA (0.5 mM), OXS (5 µM), OXCMe-Y497A (5 µM), YciA (2 µM). Products were analyzed by chiral LC-MS after 24 h reaction time. ee’s
were estimated based on extracted ion counts. B) For 3a-3d conversion and ee were quantified by comparison to a commercial
standard. For 3b separation of the enantiomers could not be achieved; n.d, not determined. C) 3e-3r were analyzed qualitatively. Where
chromatographic separation allowed, the ee was estimated, assuming identical ionization of the two enantiomers.
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Graphical abstract
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