Arylmalonate Decarboxylase—A Versatile Biocatalyst for the Synthesis of Optically Pure Carboxylic Acids

Abstract

Bacterial arylmalonate decarboxylase (AMDase) is an intriguing cofactor-independent enzyme with a broad substrate spectrum. Particularly, the highly stereoselective transformation of diverse arylmalonic acids into the corresponding chiral α-arylpropionates has contributed to the broad recognition of this biocatalyst. While, more than 30 years after its discovery, the native substrate and function of AMDase still remain undiscovered, contributions from multiple fields have ever since brought forth a powerful collection of AMDase variants to access a wide variety of optically pure α-substituted propionates. This review aims at providing a comprehensive overview of the development of AMDase from an enzyme with unknown function up to a powerful tailored biocatalyst for the synthesis of industrially relevant optically pure α-arylpropionates. Historical perspectives as well as recent achievements in the field will be covered within this work.

Full text

Arylmalonate Decarboxylase—A
Versatile Biocatalyst for the Synthesis
of Optically Pure Carboxylic Acids
Anna K. Schweiger
1
, Kenji Miyamoto
2
and Robert Kourist
1
*
1
Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria,
2
Department of Biosciences and Informatics,
Keio University, Yokohama, Japan
Bacterial arylmalonate decarboxylase (AMDase) is an intriguing cofactor-independent
enzyme with a broad substrate spectrum. Particularly, the highly stereoselective
transformation of diverse arylmalonic acids into the corresponding chiral
α-arylpropionates has contributed to the broad recognition of this biocatalyst. While,
more than 30 years after its discovery, the native substrate and function of AMDase still
remain undiscovered, contributions from multiple fields have ever since brought forth a
powerful collection of AMDase variants to access a wide variety of optically pure
α-substituted propionates. This review aims at providing a comprehensive overview of
the development of AMDase from an enzyme with unknown function up to a powerful
tailored biocatalyst for the synthesis of industrially relevant optically pure α-arylpropionates.
Historical perspectives as well as recent achievements in the field will be covered within
this work.
Keywords: decarboxylase, enantioselectivity, asymmetric synthesis, biocatalysis, optically pure carboxylic acids
INTRODUCTION
Arylmalonate decarboxylase (AMDase, EC 4.1.1.76), originally isolated from the soil bacterium
Bordetella bronchiseptica, catalyzes the decarboxylation of α-aromatic- or α-alkenylmalonic acids to
yield the corresponding optically pure mono-acid without the aid of a cofactor. While the wildtype
enzyme exhibits strict (R)-selectivity, enzyme engineering afforded efficient (S)-selective or
racemizing enzyme variants (Figure 1A). The native function or the natural substrate of
AMDase, however, both remain unknown to date. The unique reactivity and broad substrate
tolerance allows for the production of diverse aryl- or alkenylaliphatic carboxylic acids in
outstanding optical purity, amongst them several α-arylpropionates with non-steroidal anti-
inflammatory activity, the so-called profens (Miyamoto and Kourist, 2016).
DISCOVERY OF ARYLMALONATE DECARBOXYLASE
Miyamoto et al. discovered arylmalonate decarboxylase (AMDase) in the 1990’s in a screening to
identify enzymes for the generation of chiral molecules from prochiral malonates by enzymatic
decarboxylation (Miyamoto Ohta and, Hiromichi, 1990;Miyamoto and Ohta, 1992a)Asthe
decarboxylation of malonyl-ACP is a key step in metabolism, it appeared likely that the bacterial
catabolism might possess malonate decarboxylases. The screening followed the assumption that
decarboxylation of α-phenylmalonic acid 1a could represent the initial step in the metabolism, followed
Edited by:
Frank Hollmann,
Delft University of Technology,
Netherlands
Reviewed by:
Vicente Gotor-Fernández,
University of Oviedo, Spain
Dunming Zhu,
Tianjin Institute of Industrial
Biotechnology (CAS), China
*Correspondence:
Robert Kourist
kourist@tugraz.at
Specialty section:
This article was submitted to
Biocatalysis,
a section of the journal
Frontiers in Catalysis
Received: 15 July 2021
Accepted: 13 September 2021
Published: 12 October 2021
Citation:
Schweiger AK, Miyamoto K and
Kourist R (2021) Arylmalonate
Decarboxylase—A Versatile
Biocatalyst for the Synthesis of
Optically Pure Carboxylic Acids.
Front. Catal. 1:742024.
doi: 10.3389/fctls.2021.742024
Frontiers in Catalysis | www.frontiersin.org October 2021 | Volume 1 | Article 7420241
REVIEW
published: 12 October 2021
doi: 10.3389/fctls.2021.742024

by oxidation to yield 2-oxo-2-phenylacetic acid. Accordingly, it was
proposed that analogous decarboxylation of disubstituted malonic
acids would lead to α-chiral acids, as further oxidation would not
be possible (Figure 1B).
An assay for microorganisms with the ability to grow on
phenylmalonic acid 1a as the sole carbon source led to the
identification of the soil bacterium Alcaligenes bronchisepticus KU
1201 (now: Bordetella bronchiseptica). It could be shown, that whole
cells of this bacterium also converted α-methyl-α-phenylmalonic
acid 2a and analogous substrates with various aromatic residues
(Miyamoto Ohta and, Hiromichi, 1990;Miyamoto and Ohta,
1992b). The decarboxylase was purified from the microorganism
and named arylmalonate decarboxylase (AMDase), due to its
preference towards α-arylmalonates. Characterization of the
purified enzyme revealed that it acts without a cofactor and is
biotin-independent. Yet, the native role and substrate of AMDase
remained unclear, as aryl malonates are not naturally abundant
(Miyamoto and Ohta, 1992b).
Elucidation of Enzyme Mechanism and
Selectivity
Shortly after the enzyme was discovered, Miyamoto et al. aimed
at revealing the enzyme stereoselectivity by means of isotope
FIGURE 1 | (A) Overview on AMDase catalyzed asymmetric decarboxylation by wildtype (variants with Cys188) and (S)-selective variants (Cys74) and AMDase
catalyzed racemization (variants with Cys188/Cys74). (B) Assumed reaction pathway of α-phenylmalonic acid-converting microorganisms (top) and proposed route
towards α-chiral acids via the same decarboxylative activity (bottom).(C) Detected reaction products after decarb oxylation of
13
C-labelled chiral malonic acid substrates
by wildtype AMDase indicates, that the pro-(R) carboxylate is eliminated during the reaction in any case. (D) First postulated mechanism based on the assumption ,
that AMDase acts as a thiol decarboxylase. After thioester formation between the substrate and a cysteine in the active site, the pro-(R) carboxylate is cleaved and the
resulting enolate is enantioselectively protonated from the si-face.
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Schweiger et al. Arylmalonate Decarboxylase

labelling studies (Miyamoto et al., 1992b). For that, both
enantiomers of
13
C-labelled α-methyl-α-phenylmalonic acid 2a
where synthesized. Products of the enzymatic reaction were
analyzed via
13
C NMR spectroscopy, which disclosed that if
the (S)-substrate was used, the (R)-configured product still
contained the
13
C-labelled carboxylate, whereas in case of the
(R)-substrate, enrichment of
13
C was not detected in the
remaining carboxylate of the (R)-configured product
(Figure 1C). These results indicated that, in both cases,
exclusively the pro-(R) carboxylate is cleaved, yielding the final
product via inversion of configuration. Later on, this finding was
confirmed by Okrasa et al. using
18
O labeling of the enantiotopic
carboxylate groups (Okrasa et al., 2009).
The observation that AMDase was inhibited by sulfhydryl
reagents indicated that AMDase is likely to be a thiol
decarboxylase (Miyamoto and Ohta, 1992b). Thus, the initial
step of the reaction was believed to be the formation of a thioester
between the pro-(S) carboxylate of the substrate and a cysteine
residue in the active site (cf. activation by coenzyme A in fatty acid
biosynthesis) (Figure 1D). The observed inversion of
configuration, however, was in discrepancy to other known
decarboxylases, where configuration was strictly retained (Kim
and Kolattukudy, 1980). This prompted the authors to suggest
either a S
E
2-type concerted mechanism with complete inversion,
or the formation of an intermediary enolate, which further gets
protonated in an enantioselective fashion from the si-face
(Figure 1D)(Miyamoto et al., 1992b). Formation of a charged
intermediate was also supported by the finding that substrates
bearing electron-withdrawing groups (EWGs) were converted at
a higher rate, due to their ability to stabilize the supposedly
formed carbanion intermediate (Miyamoto and Ohta, 1992b).
Identification of the AMDase gene and sequence analysis
thereof disclosed that the enzyme contains four cysteine
residues (C101, C148, C171, C188) (Miyamoto and Ohta,
1992a). All were present in a reduced state and only mutation
of C188 to serine (C188S) proved detrimental to enzyme activity
(k
cat
), indicating its critical role in the reaction mechanism
(Miyazaki et al., 1997). Soon thereafter, however, it became
apparent that thioester formation does not play a role in the
mechanism of AMDase. Instead, it was suggested that Cys188
rather acts as a proton donor. This became obvious after the
wildtype enzyme, having the more acidic cysteine residue in
the active center, was inactivated at a pH >9, whereas the
corresponding C188S variant was not. Interesting evidence was
also found among quite remotely related (30% homology)
enzymes from the racemase and isomerase family (Matoishi
et al., 2004). Those enzymes, which were well-studied with
regards to their mechanism, also exhibit a highly conserved
cysteine residue in this region, which functions as a proton
donating residue (Glavas and Tanner, 1999).
INVERSION OF ENANTIOSELECTIVITY AND
INTRODUCTION OF RACEMASE ACTIVITY
Some enzymes with about 30% homology to AMDase were
identified via PSI-BLAST (position-specific iterative basic local
alignment search tool), all belonging to the class of isomerases
(EC 5). Glutamate racemase (Lactobacillus fermenti), aspartate
racemase (Streptococcus thermophilus), hydantoine racemase
(Pseudomonas sp. strain NS671) and maleate isomerase
(Alcaligenes faecalis) all share the conserved cysteine at
position 188 with AMDase, while enzymes from the isomerase
class have an additional cysteine at around residue 74 (Figure 2A)
(Ijima et al., 2005). As the reaction mechanism and crystal
structure of glutamate racemase were already well-studied, two
cysteine residues on both sides of the substrate were proposed to
be essential for the racemizing activity, by either abstracting or
donating protons from opposite sites in a so-called two-base
mechanism (Glavas and Tanner, 1999).
Inspired by these findings, Ijima et al. decided to introduce a
cysteine at position 74 instead of the glycine residue present in
AMDase (Ijima et al., 2005). Additionally, previous studies have
shown that the mutation G188S led to a drastic decrease of native
AMDase activity (Miyazaki et al., 1997). While the proton-
donating ability of serine is already low compared to cysteine,
the authors proposed that an amino acid without any acidic
proton would be beneficial for the enantiomeric excess of the
obtained product. Surprisingly, the mutant G188A proved to be
completely inactive. Hence, the double-variant G74C/C188S was
prepared, and was found to produce the opposite enantiomers in
94–96 %ee as compared to wildtype AMDase. Yet, activity of the
G74C/C188S variant even fell below the tremendously reduced
activity of C188S (Ijima et al., 2005). Interestingly, also for the (S)-
selective variant S36N/G74C/C188S, preference for cleavage of
the pro-(R) carboxylate was observed by isotope labeling studies
(Terao et al., 2006a), thus confirming that enantioselective
reprotonation of the enolate intermediate is decisive for the
final configuration of the product (Miyamoto et al., 2007a). As
already observed by Ijima et al., the single amino acid exchange
G74C rendered arylmalonate decarboxylase a racemase (Terao
et al., 2006b). Notably, the decarboxylase activity of AMDase was
preserved in the G74C variant, yielding racemic arylpropionates
from arylmalonates. Kinetic analysis disclosed that, in terms of
catalytic efficiency (k
cat
/K
M
), decarboxylation (0.96 s
−1
mM
−1
)
exceeded racemization (0.56 s
−1
mM
−1
), which was also
reflected by the racemic product already detected at an early
stage of the reaction (Terao et al., 2006b).
CRYSTAL STRUCTURE AND MECHANISM
Despite the crystal structure of AMDase remained unsolved until
2008, considerable insights were already acquired by that time,
including suggestions on the enzyme mechanism (Miyamoto
et al., 1992b;Miyamoto et al., 2007a;Matoishi et al., 2004),
identification of key residues (Matoishi et al., 2004;Terao
et al., 2007), switch in enantioselectivity (Ijima et al., 2005;
Terao et al., 2006a) and introduction of a racemase activity
(Terao et al., 2006b). Yet, the exact mechanism for
stabilization of the highly unstable enediolate intermediate
remained elusive. While other enzymes with related enolate
intermediates use Mg
2+
for stabilization (Gerlt et al., 2005),
activity of AMDase is not dependent on metal ions or other
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Schweiger et al. Arylmalonate Decarboxylase

cofactors (Miyamoto and Ohta, 1992b). Further, delocalization of
electron density into the aromatic ring system might partly
account for stabilization of the enediolate but would be
insufficient to explain the observed highly efficient enzymatic
decarboxylation (Okrasa et al., 2008).
By solving the first crystal structure of wildtype AMDase
(PDB: 3DG9), Okrasa et al. were able to unravel key features
of enzyme activity and selectivity in more detail. Interestingly, in
the core structure consisting of two four-stranded parallel
β-sheets surrounded by several α-helices, a tightly bound
phosphate ion was found near the active site cysteine 188. A
total of six hydrogen bonds were established by side-chain and
backbone interactions to Thr75, Ser76, Tyr126, Cys188 and
Gly189, belonging to two adjacent oxyanion holes (Okrasa
et al., 2008). The authors suggested that the phosphate might
resemble the position of the enediolate intermediate. In
retrospective, this structural motif termed “dioxyanion hole”
was also found in related enzymes of the isomerase family and
mutational studies on Thr75 and Ser76 or Tyr126 of AMDase
have already demonstrated the essential role of this region for
enzyme activity (Terao et al., 2007;Okrasa et al., 2008).
After reconfirming loss of the pro-(R) carboxylate by a
18
O-labelling strategy, the enediolate intermediate resulting from
decarboxylation of α-methyl-α-phenylmalonic acid 2a was placed
in the active site. It became obvious, that the phenyl moiety occupies a
large solvent accessible pocket, whereas the small methyl substituent
is left at an orientation, where only little space is available.
Considering these restrictions and the fixed position of the pro-(S)
carboxylate entrapped in the dioxyanion hole, it was suggested that
the pro-(R) carboxylate must point towards a small hydrophobic
pocket (Leu40, Val43, Val156, Tyr48), which could act as a driving
force for decarboxylation by hydrophobic destabilization
(Figure 2B). Thus, the key for cofactor-free decarboxylation by
AMDase was proposed to be the extensive stabilization of one
carboxylate, while the other one is destabilized by unfavorable
electrostatic interactions (Okrasa et al., 2008).
This proposed binding mode was confirmed when AMDase
was co-crystallized with the mechanism-based inhibitor
benzylphosphonate (K
i
5.2 mM) (PDB: 3IP8) (Okrasa et al.,
2009). Similarly, the phosphonate dianion was tightly engaged by
six hydrogen bonds to the residues of the dioxyanion hole and the
phenyl residue was positioned in the large pocket stacked within
the Gly189-Gly190 amide bond and Pro14 through van der Waals
interactions.
Obata et al. solved the crystal structure of the G74C/C188S variant
containing the ligand 2-phenyl acetate (PDB: 3IXL, Figure 3A)andof
the G74C variant (PDB: 3IXM, Figure 3C)andWTenzyme(PDB:
3DTV, Figure 3D) in a sulfate associated form (Obata and Nakasako,
2010). While the overall structure of AMDase G74C/C188S was
mostly preserved when compared to wildtype structures either
containing BnzPO (PDB: 3IP8, Figure 3A)orPO
43−
(PDB:
3DG9) in the active site (RMSD of C
α
atoms approx. 0.25 Å),
considerable conformational differences became obvious by
comparingtotheSO
42−
-associated structures of the wildtype and
FIGURE 2 | (A) Amino acid sequence homology between AMDase and selected racemases identified via PSI-BLAST. Glutamate racemase from Lactobacillus
fermenti, aspartate racemase from Streptococcus thermophilus, hydantoine racemase from Pseudomonas sp. strain NS671, maleate isomerase from Alcaligenes
faecalis and AMDase from Bordetella bronchiseptica.(B) Proposed binding mode and interaction network of α-methyl-α-phenylmalonic acid 2a (left) and the resulting
enediolate intermediate (right) in the active site of AMDase WT. The pro-(R) carboxylate is shown in blue and the dioxyanion hole in gray.
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Schweiger et al. Arylmalonate Decarboxylase

G74C variant (PDB: 3DTV and 3IXM). It was suggested that a
positional shift of Cys188-Gly189-Gly190 towards Gly74-Thr75 was
induced upon ligand binding, thereby triggering the formation of a
hydrophobicnetworkcoveringtheactivesite(Val43,Thr154,Val156).
In the unliganded (SO
42−
-associated) crystal structures, such cluster
was not observed, as the corresponding loop regions are not
contacting each other. Additionally, in this conformation, the key
residue 188 is rotated away from the ligand towards the hydrophobic
pocket in a rather “non-productive”orientation (Figures 3C,D).
These observations indicated the presence of either an “open”or
“closed”conformation, regulated by ligand binding (Figure 3)(Obata
and Nakasako, 2010). A similar behavior between structures of the
empty and the ligand-bound enzyme was found for the related
glutamate racemase (Puig et al., 2009).
Most important, however, was the observation that the position
of Cys74 of the G74C/C188S variant was indeed in mirror symmetry
to C188 of AMDase WT with regards to the C
α
atom of the
enediolate intermediate, which gets protonated during the
reaction, thereby confirming the early proposed rationale for
inversion of enantioselectivity (Ijima et al., 2005;Terao et al.,
2007). In this binding mode, configuration of the final product is
only dependent on the enantioface-selective protonation, which
occurs from si-face in case of Cys188 or from re-faceincaseof
Cys74, resulting in (R)- or (S)-2-phenylpropionate, respectively in
accordance to experimental data (Miyauchi et al., 2011).
COMPUTATIONAL MODELLING OF THE
ENZYME MECHANISM
The reaction mechanism of AMDase was also subject of
computational studies. Lind and Himo studied the mechanism
FIGURE 3 | Representation of the active site of (A) AMDase G74C/C188S in association with 2-phenyl acetate (PDB: 3IXL) and superimposed with (B) AMDase WT
in complex with benzylphosphonate (BnzPO) (PDB: 3IP8), (C) AMDase G74C in complex with SO
42-
(PDB: 3IXM) and (D) AMDase WT in complex with SO
42-
(PDB:
3DTV). Active site residues of AMDase G74C/C188S are shown in color and the respective superimposed structure in gray. Dotted lines represent H-bonds formed
between the carboxylate and residue side-chains (red) or backbone NH groups (blue). Figure created with PyMOL.
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Schweiger et al. Arylmalonate Decarboxylase

by means of density functional theory (DFT) calculations (Lind
and Himo, 2014). By employing the quantum chemical cluster
approach, the mechanism, stationary points thereof and
enantioselectivity were investigated with the aid of two different
truncated active site models. The smaller model only partly
contained the residues of the dioxyanion hole and the catalytic
Cys188 (81 atoms), whereas the second model additionally
included residues from the small and large binding pockets (223
atoms). Results obtained from using the small model indicated that
a two-step mechanism proceeding via aplanarenediolate
intermediate is plausible, with the decarboxylation step being
rate-limiting. However, binding modes leading either to cleavage
of the pro-(R)orpro-(S) carboxylate were very similar in energy
(1.9 kcal mol
−1
for initial binding pose; 1.5 kcal mol
−1
for transition
state) probably due to the absence of the binding pockets
determining the orientation of the remaining substituents,
therefore being unable to explain the observed enantioselectivity
with this small model (>99 %ee corresponds to at least
3kcalmol
−1
). By using the larger model, the calculated energies
for binding and decarboxylation were 14.1 and 18.3kcal mol
−1
higher for the formation of the (S)-enantiomer than for the (R)-
product, mostly caused by unfavorable steric clashes of the large
aryl substituent and the residues of the small binding pocket. When
the much smaller α-methyl-α-vinylmalonic acid was studied,
ambiguous results were obtained concerning absolute transition
state energies, probably being a result of the too small and rigid
models of the binding pockets. Both binding modes and transition
states, however, were much closer in energy as compared to
α-methyl-α-phenylmalonic acid 2a. This would indicate that
stereoselectivity is exclusively determined by substrate binding
in case of bulky aromatic compounds, whereas in case of
smaller substrates, the decarboxylation transition state could
also contribute to enantiodiscrimination (Lind and Himo,
2014). Just recently, Dasgupta et al. emphasized that geometric
constraints such as introduced in quantum-chemical studies can
lead to artifacts like imaginary vibrational frequencies and
impaired efficiency of the overall optimization process
(Dasgupta and Herbert, 2020). The authors thus introduced soft
harmonic confining potentials to the terminal atoms of the model,
thereby avoiding the artificial strain and rigidity of fixed-atom
truncated active site models. By employing this system, they were
able to reproduce the results from previous studies (Lind and
Himo, 2014) and further claimed that this methodology is easy to
implement and can dramatically reduce optimization efforts
(Dasgupta and Herbert, 2020).
In the previously described modeling approaches, results were
substantially dependent on the size of the truncated enzyme
models used (Lind and Himo, 2014;Dasgupta and Herbert,
2020). In order to obtain a more comprehensive overview of
the molecular level origin of AMDase selectivity, a full enzyme
model would be highly preferable. Particularly, counterintuitive
effects observed after iterative saturation mutagenesis within the
AMDase active site cavity (Okrasa et al., 2009;Miyauchi et al.,
2011;Yoshida et al., 2015) require consideration of the entire first
coordination sphere. Thus, the empirical valence bond (EVB)
approach was the methodology of our choice, which was further
complemented by metadynamics simulations (Warshel and
Weiss, 1980;Barducci et al., 2008;Biler et al., 2020). EVB
simulations were able to reproduce experimentally observed
activation energy barriers (within 3 kcal mol
−1
)andthe
preferential cleavage of the pro-(R) carboxylate group which was
already determined during substrate positioning in the Michaelis
complex. Yet curiously, the (S)-selective CLGIPL variant showed
preferential cleavage of the pro-(S)groupwhichstillledtothe
expected formation of (S)-enantiomers due to reacting via adifferent
substrate binding pose. For compounds, which were not or only
poorly converted by AMDase and variants thereof, an increased
flexibility and motility within the active site was observed during
simulations. The resulting inadequate substrate positioning
furthermore allowed water to enter the active center, which is
likely to destroy the vulnerable interaction network vital for
catalysis (Biler et al., 2020). By consulting the grid
inhomogeneous solvation theory (GIST) (Nguyen et al., 2012;
Nguyen et al., 2014) to analyze the local hydrophobicity within
the AMDase active site, we found clear evidence for a mechanism
driven by ground-state destabilization by entrapment of the
carboxylate to-be-cleaved in a hydrophobic environment. These
analyses also revealed the emergence of an additional
hydrophobic cavity in the CLGIPL variant, which allows this
variant to react via an alternative binding pose and unexpected
cleavage of the pro-(S) carboxylate. While the alteration of
hydrophobic pockets in the CLGIPL variant was not by design,
the targeted engineering of the active site hydrophobicity presents a
seminal approach for future engineering of AMDase. WT-MetaD
simulations further confirmed that the reactive binding pose
(according to EVB simulations) also coincides with the most
populated binding pose observed at the Michaelis complex in
most of the studied cases. Thus, AMDase selectivity is partly
already determined at the stage of substrate binding, but selective
destabilization of a distinct carboxylate group (leading to ground-
state destabilization) seems to be the true determinant of the eventual
reactive transition state (Biler et al., 2020).
Busch et al. used semiempirical QM/MM calculations to study
the mechanism of the AMDase racemase variant G74C (Busch
et al., 2016). MD simulations revealed that both catalytic cysteines
need to be in their deprotonated state for successful catalysis. In
contrast to glutamate racemase, where co-catalytic residues
activate the cysteines (Glavas and Tanner, 2001), water is
suggested to deprotonate the corresponding residues of
AMDase G74C prior to substrate binding, which are then
further stabilized by thiolate holes. This might also explain the
pronounced pH-dependency of the reaction. Further
semiempirical calculations indicated a stepwise mechanism
similar to decarboxylation, where a delocalized π-electron
system within the substrate is necessary to stabilize the
enediolate intermediate (Busch et al., 2016). Likewise to
previous studies (Lind and Himo, 2014), there was no
indication for a concerted mechanism as described for
glutamate racemase (Puig et al., 2009).
Another study conducted by Karmakar et al. aimed at the
simulation of CO
2
and product release from the active site
(Karmakar and Balasubramanian, 2016). By performing
(steered) MD simulations it was demonstrated that release of
the decarboxylated product is energetically unfavorable and
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Schweiger et al. Arylmalonate Decarboxylase

might even surpass the decarboxylation step, thus potentially
rendering product release the true rate-limiting step (WT:
∼14 kcal mol
−1
for decarboxylation vs. ∼23 kcal mol
−1
for
product release). The authors claimed that this might also
partly account for the reduced activity of the G188S/G74C
variant (20,000-fold) (Miyauchi et al., 2011), as the calculated
energy barrier for product release was even higher in this case
(∼20 kcal mol
−1
for decarboxylation vs. ∼37 kcal mol
−1
for
product release) (Karmakar and Balasubramanian, 2016).
SUBSTRATE SCOPE
Already after the discovery of AMDase, Miyamoto and Ohta
thoroughly characterized the enzyme and its substrate scope
(Miyamoto and Ohta, 1992b). These fundamental findings,
obtained in absence of any structural and mechanistic knowledge
on the enzyme, provided the basis for studies up to the present by
demonstrating the essential limitations of AMDase substrates. 1)
Steric effects play a crucial role for the small substituent, as mono-
substituted malonates were converted faster than the corresponding
methylated substrates. Further, the ethyl analogue was not
converted. 2) The aromatic substituent is essential for activity, as
substrates with a benzyl-, phenoxy- or phenylthio- instead of an
phenyl-group were inert (Miyamoto Ohta and, Hiromichi, 1990).
This was also true for malonic acid (R
L
H) or methylmalonic acid
(R
L
CH
3
). 3) Only free malonic acids can act as a substrate, as the
corresponding mono- and diesters were not accepted. 4) Electron-
withdrawing substituents on the aromatic moiety enhance the
reaction rate by better stabilizing the proposed carbanion
intermediate. Table 1 summarizes substrates studied in AMDase
catalyzed decarboxylation and racemization.
The large substituent R
L
offers major variability within the
substrate scaffold. Phenyl- (Miyamoto Ohta and, Hiromichi, 1990;
TABLE 1 | Overview on the AMDase substrate scope.
Substituent Generally accepted Accepted by
racemase
Not accepted
R
L
RH, p-Me, p-iso-bu, p-NO
2
,p/m/o-OMe, p/m/o-F, p/m/o-Cl, p/m/o-CF
3
,
p-Ph-m-F, m-COPh
XCH
2
,O,S
XS; R HorXO; R Me, OMe, 2 Me
RH, OMe
R
1
H; R
2
H, Me, Et, Ph or R
1
Me; R
2
Me
R
S
H, D, Me, OH, F, NH
2
Et iPr, nPr
EWG CO
2
HNO
2
COMe, COH, CO
2
Me, CO
2
Et, CONH
2
,CH
2
OH,
COSEt, CN
R
L
: large (unsaturated) substituent; R
S
: small substituent; EWG: electron withdrawing group.
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Schweiger et al. Arylmalonate Decarboxylase

Miyamoto and Ohta, 1992b;Miyamoto et al., 1992a;Miyamoto
et al., 1994;Miyazaki et al., 1997;Fukuyama et al., 1999;Matoishi
et al., 2000;Terao et al., 2006b;Terao et al., 2007;Okrasa et al.,
2008;Okrasa et al., 2009;Tamura et al., 2008;Kourist et al., 2011b;
Miyauchi et al., 2011;Yoshida et al., 2015;Lewin et al., 2015), 2-
naphthyl- (Miyamoto and Ohta, 1992b;Ijima et al., 2005;Terao
et al., 2006a;Terao et al., 2006b;Terao et al., 2007;Miyauchi et al.,
2011) and thienyl- (Miyamoto Ohta and, Hiromichi, 1990;
Miyamoto and Ohta, 1992b;Ijima et al., 2005;Terao et al.,
2006a;Terao et al., 2006b;Terao et al., 2007;Lewin et al., 2015)
malonic acids are typical substrates of AMDase and omnipresent in
literature. Over time, the substrate scope was extended to a vast
number of variously substituted phenyl derivatives (o-Me
(Miyamoto Ohta and, Hiromichi, 1990), p-Me (Miyamoto and
Ohta, 1992b;Kourist et al., 2011b), p-iso-butyl (Yoshida et al.,
2015), p-NO
2
(Kourist et al., 2011b), OMe (Miyamoto Ohta and,
Hiromichi, 1990;Miyamoto and Ohta, 1992b;Kourist et al.,
2011b), F (Miyamoto et al., 1992a;Miyamoto and Ohta,
1992b), Cl (Miyamoto Ohta and, Hiromichi, 1990;
Miyamoto and Ohta, 1992b;Miyamoto et al., 1994), CF
3
(Miyamoto et al., 1992a), p-Ph-m-F (Kouristetal.,2011b;
Gaßmeyer et al., 2016), m-COPh (Kourist et al., 2011b)),
naphthyl derivatives (1-naphthyl (Miyamoto and Ohta,
1992b), 6-methoxynaphthalen-2-yl (Miyamoto Ohta and,
Hiromichi, 1990;Kouristetal.,2011b;Miyauchi et al.,
2011;Yoshida et al., 2015;Gaßmeyer et al., 2016))
heterocyclic (pyridinyl (Kouristetal.,2011b;Lewin et al.,
2015), furanyl and bicyclic systems (Lewin et al., 2015)) and
also alkenyl (Okrasa et al., 2009;Kouristetal.,2011b)
substrates.
With regards to the small substituent R
S
, it was soon
recognized that, besides H and CH
3
, other small groups like D
(Matoishi et al., 2000), F (Miyamoto et al., 1992a), OH or NH
2
(Tamura et al., 2008) were generally tolerated, while larger alkyl
groups (ethyl (Miyamoto and Ohta, 1992b) or propyl (Terao
et al., 2006b;Kourist et al., 2011b)) were not or only converted at a
very low rate (Terao et al., 2006b;Kourist et al., 2011b). This was
later on explained by structural analysis, as only limited space is
available at the position left for this group (Okrasa et al., 2008;
Obata and Nakasako, 2010).
As already exposed by Miyamoto et al. in 1992, presence of the
free di-acid is indispensable for decarboxylation to proceed
(Miyamoto and Ohta, 1992b). This was also proven valid for
the racemization activity of AMDase, as carboxylate derivatives
such as alcohols, amides, nitriles (Terao et al., 2006b), esters
(Terao et al., 2006b;Kourist et al., 2011b), thioesters, ketones and
FIGURE 4 | (A) Coplanar arrangement of aromatic ring and α-substituent R in a syn-oranti-periplanar conformation with respect to the ortho-substituent X (bold
gray bonds). Indane-1,1-dicarboxylic acid (IDA) serves as conformationally restricted model substrate to study entropic contributions for substrate acceptance. (B)
Potential energy diagram for the C-C bond rotation of α-(o-chlorophenylmalonic) acid (black squares) and α-(o-chlorophenyl)-α-methylmalonic acid (gray circles)
calculated with HF/3-21G*. The phenyl ring is represented as rectangle in the Newman projection of the respective rotamers. Figure created based on data
presented in Miyamoto et al. (1994).
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Schweiger et al. Arylmalonate Decarboxylase

aldehydes (Kourist et al., 2011b) were completely inactive. Thus,
stabilization of the free carboxylate via the complex network
within the dioxyanion hole seems crucial, regardless of
decarboxylation or racemization. The only exceptional case
known so far is (nitromethyl)benzene, which was accepted as
a substrate by AMDase racemase G74C and G74C/V43A (Kourist
et al., 2011b). It was generally concluded that the substrate
structure seems more decisive for successful conversion, than
α-proton acidity of the α-aryl propionates (Kourist et al., 2011b).
In terms of spatial arrangement, Miyamoto et al. proposed that
a co-planar alignment of the phenyl group and α-substituent
would allow optimal overlap of the π-orbitals in order to stabilize
the formed charge on the enolate intermediate (Miyamoto et al.,
1994). In case of ortho-substituted substrates like
α-(o-chlorophenyl)malonic acid, two such conformations come
into consideration, namely a syn- and anti-periplanar
arrangement (Figures 4A,B).
Kinetic studies revealed that, while the activity of AMDase towards
α-(o-chlorophenyl)malonic acid even surpassed α-phenylmalonic acid
1a,thecorrespondingα-methyl-substituted compound was not
accepted at all. This led to the assumption, that a syn-periplanar
arrangement is necessary for AMDase-mediated decarboxylation,
which might in turn disfavor substrates with an additional
α-substituent, due to steric clashes with the ortho-substituent
(Miyamoto et al., 1994). Ab initio calculations were thus
performed to assess the energy barriers encountered during
rotation along the C
α
-C
Ar
bond. Results obtained for
α-(o-chlorophenyl)malonic acid indicated two stable conformations
corresponding to a syn-oranti-periplanar orientation (approx. 0°or
360°and 180°respectively). The small energy difference between both
structures (<1kcalmol
−1
) implies, that steric repulsion between o-Cl
and α-H is negligible. Repulsive contributions were mainly caused by
interaction of the chlorine residue with the carboxylates (Figure 4B).
In case of α-(o-chlorophenyl)-α-methylmalonic acid, an anti-
periplanar or perpendicular arrangement of substituents was
energetically favored, whereas syn-periplanar-like conformations
werefoundtobeunstable(Figure 4B).
It was further proposed that by using the conformationally
restricted indane-1,1-dicarboxylic acid (IDA), a syn-periplanar
conformation would be mimicked, thereby lowering the activation
entropy ΔS
‡
(Figure 4A). Indeed, the model substrate IDA was
accepted by AMDase in contrast to the corresponding α-methyl-
α-(o-tolyl)malonic acid, thus underlining the inability of the latter to
overcome rotational energy barriers towards a suitable syn-periplanar
arrangement for conversion (Miyamoto et al., 1994). The remarkably
low K
M
value observed for IDA in comparison to other studied
substrates further affirmed the suitability of such spatial arrangement
for AMDase-catalyzed decarboxylation (Miyamoto et al., 1994;
Kawasaki et al., 1996). In this context it was also proposed that a
hydrophobic pocket within the active site might be responsible for
correct positioning of unrestricted substrates via CH-πinteractions
(Nishio et al., 1995), thereby reducing the activation entropy in a
similarmanner(Kawasaki et al., 1996).
ENZYME ENGINEERING
In view of the dramatically reduced efficiency of the initially
created variants with inversed (Ijima et al., 2005) or racemizing
activity (Terao et al., 2006b) in comparison to the native enzyme,
considerable efforts to recover or even to increase the activity by
enzyme engineering have been made until today.
Enzyme Engineering of (S)-Selective C188X/
G74C-Based Variants
At the time, when Ijima et al. first described the AMDase G74C/
C188S variant, the obtained inversion of enantioselectivity by merely
TABLE 2 | Kinetic parameters of AMDase WT and (S)-selective variants towards α-methyl-α-phenylmalonic acid 2a according to Yoshida et al. (2015). Relative activity was
calculated by dividing the catalytic efficiency (k
cat
/K
M
) of each variant by the catalytic efficiency of G74C/C188S variant.
AMDase variant k
cat
/K
M
(s
−1
mM
−1
) Relative activity Ref.
WT 279/26.9 28,090 Okrasa et al. (2008)
G74C/C188S (CS) 0.0048/13 1 Yoshida et al. (2015)
G74C/M159L/C188G (CLG) 1.1/1.8 1,655 Yoshida et al. (2015)
G74C/V156L/M159L/C188G (CLGL) 1.7/1 4,604 Yoshida et al. (2015)
V43I/G74C/A125P/V156L/M159L/C188G (CLGIPL) 3.8/1.1 9,356 Yoshida et al. (2015)
FIGURE 5 | Enzyme variants evolved from AMDase G74C/C188S in
three rounds of directed evolution via iterative saturation mutagenesis (ISM)
and their improvement of specific activity towards α-phenylmalonic acid 1a.
First (gray), second (green) and third (blue) screening are shown as
arrows. Reproduced from Miyauchi et al. Miyauchi et al. (2011) with
permission from the Royal Society of Chemistry.
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Schweiger et al. Arylmalonate Decarboxylase

employing the yet uncovered spatial mirror symmetry of
residues 188 and 74 was intriguing. Yet, the variant
suffered from an incisive loss of activity due to diminished
k
cat
(see Table 2)(Ijima et al., 2005). Early attempts on
recovering the activity by random mutagenesis led to a
variant (S36N/G74C/C188S) with increased (10-fold)
activity among a total of 50,000 screened clones (Terao
et al., 2006a). This reflected the difficulty of enzyme
evolution purely relying on randomization in the absence
of rational guidance.
After structural data of AMDase became available and,
inspired by previous findings, that variations of the
hydrophobic pocket residues can strongly enhance enzyme
activity (Okrasa et al., 2009) Miyauchi et al. also attempted
directed evolution of the G74C/C188S variant. This time,
structural information provided the basis for three rounds of
directed evolution via iterative saturation mutagenesis (ISM)
(Reetz et al., 2008) of Ser188 in the first place, and residues of
the hydrophobic pocket (Leu40, Val43, Tyr48, Leu77, Val156,
Met159) in the following rounds (NNK codon for introduction
of all 20 amino acids) (Figure 5)(Miyauchi et al., 2011). The critical
role of the Cys188-substituting residue on enzyme activity was
demonstrated earlier by the G74C/C188A variant being completely
inactive (Ijima et al., 2005). From the first round of screening,
variant G74C/C188G was identified to exhibit 5.6-fold increased
activity towards α-phenylmalonic acid 1a.Byusingthisvariantasa
template for the second generation, a triple mutant (G74C/M159L/
C188G) with 210-fold higher activity evolved. Interestingly, the
mutant selected from the last screening round (Y48F/G74C/
M159L/C188G) with 920-fold increased activity carried the
mutation Y48F, which was already found beneficial in the
second-generation mutants. All identified beneficial mutations
within the hydrophobic pocket were due to hydrophobic
substitutions, thus underlining the high importance of this
destabilizing environment for decarboxylation.
As selection was based on conversion of α-phenylmalonic
acid 1a, activity towards α-aryl-α-methylmalonic acids
differed considerably. While the G74C/M159L/C188G
variant showed a comparably increased activity in the
synthesis of Naproxen 8b from its corresponding malonate
(220-fold higher specific activity), the quadruple variant Y48F/
G74C/M159L/C188G, superior for conversion of
α-phenylmalonic acid 1a, exhibited only reduced activity.
Additionally, the slightly impaired enantioselectivity of the
G74C/C188S variant was completely abolished in the C188G-
based variants (>99%ee), indicating potential disadvantageous
interactions of the serine moiety with the enolate intermediate
(Miyauchi et al., 2011).
Due to previously created variants, synergistic effects through
multiple amino acid exchanges within the hydrophobic pocket
were strongly indicated. Yoshida et al. conducted simultaneous
saturation mutagenesis (SSM) on the previously evolved G74C/
M159L/C188G (CLG) variant (Miyauchi et al., 2011), which
allowed for simultaneous variation of multiple sites (Yoshida
et al., 2015). Further, the authors argued, that evolution towards
methyl substituted α-arylmalonic acids would be advantageous
compared to simple α-phenylmalonic acid 1a, as the
corresponding optical pure products are of major interest
(Kourist et al., 2011a). Again, residues from the hydrophobic
pocket were chosen as mutagenesis targets, due to their vicinity to
the aryl residue (Leu40, Val156) or the proton-donating Cys74
(Met73). AMDase CLG was used as the starting variant, due to its
superior activity in the synthesis of Naproxen 8b (Miyauchi et al.,
2011) and a limited set of amino acids was introduced at the
selected sites via the VTK codon (Leu, Ile, Val and Met).
Surprisingly, only variants with mutations at V156 were
selected from the first-generation library, thus indicating its
crucial role in the decarboxylation mechanism. The best
variant CLG-L (G74C/V156L/M159L/C188G) was chosen for
the second round of SSM (see Table 2), where the region
around the important residues Val156 and Met159 was
targeted (Val43 and Ala125). Interestingly, from the three
selected variants with improved activity, two included the
mutation A125P, which seemed surprising, as A125 was part
of an α-helix. The most active variant identified in this screening
was CLGL-IP (V43I/G74C/A125P/V156L/M159L/C188G),
where the catalytic efficiency (k
cat
/K
M
) was improved 2.1-fold
compared to CLG-L and over 9,000-fold compared to the initial
G74C/C188S variant (see Table 2).
The CLGIPL variant turned out to be highly (S)-selective
and most active amongst the (S)-selective enzyme variants
towards all tested substrates. Notably, malonate 7a was
converted by AMDase CLGIPL with 1.3 U mg
−1
(not
converted by G74C/C188S) which was also more than
twofold faster than the wildtype enzyme (0.53 U mg
−1
). Also
Naproxen 8b was formed by CLGIPL (17 U mg
−1
) with high
activity, which was yet surpassed by the (R)-selective wildtype
(88 U mg
−1
)(Yoshida et al., 2015).TheAMDaseCLGIPL
variant also turned out highly efficient in the conversion of
malonate 6a (55 U mg
−1
) when compared to the wildtype
(33.1 U mg
−1
)orthe(S)-selective CLG variant (15.8 U mg
−1
)
(Gaßmeyer et al., 2016). Interestingly, AMDase wildtype
possesses higher activity towards the formation of Naproxen
8b compared to Flurbiprofen 6b, while AMDase CLGIPL
shows an inversed preference. This emphasizes once more
that the inherent reactivity of a substrate is less decisive for
AMDase activity compared to the ability of productive binding
in the active site and the sterical and electronical interactions
resulting thereof.
Enzyme Engineering of (R)-Selective
WT-Based Variants
By solving the AMDase WT crystal structure in complex with the
mechanism-based inhibitor benzyl phosphonate, Okrasa et al. were
able to further rationalize interactions of the enzyme active site
residues and the substrate scaffold (Okrasa et al., 2009). They
further proposed that alkenyl moieties might also afford suitable
delocalization of electron density during the reaction, similarly to
the previously studied substrates with aryl residues. Indeed, the
tested alkenyl substrates proved to be suitable for AMDase-
catalyzed decarboxylation, yet at lower efficiencies as their
aromatic counterparts. Despite their relatively low turnover
numbers (k
cat
), most of the alkenyl substrates often showed a
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Schweiger et al. Arylmalonate Decarboxylase

higher affinity (K
M
) for AMDase than the respective phenyl
derivatives. To prove the hypothesized interaction patterns in
the active center, two sets of residues (Pro14, Pro15 and Gly190
from the large binding pocket and Val43 and Met159 from the
hydrophobic pocket) were selected for three rounds of iterative
saturation mutagenesis (NNK coding for all 20 amino acids). Due
to the established setup, mutants were screened for
α-phenylmalonic acid 1a, but were later on also tested towards
the newly introduced alkenyl substrates (Okrasa et al., 2009).
By introducing the single point mutation M159V, the relative
activity towards α-phenylmalonic acid 1a was raised 51-fold, thus
implying at the same time, that it is not the native substrate of
AMDase (Table 3, entry 1). The double mutant P14V/P15G
exhibited enhanced activity towards several different substrates
(Table 3,entry2–4), which could be attributed to a generally
increased flexibility within the larger binding pocket. The
applicability of the M159V and P14V/P15G variants was later
on also exemplified with a series of α-heterocyclic prochiral
malonic acids (Lewin et al., 2015). The importance of substrate-
fit was also demonstrated by the G190A variant, which converted
α-methyl-α-vinylmalonic acid 4a, the smallest of all tested
substrates, with 4.6-fold increased activity (Table 3, entry 5).
The authors proposed that the slightly decreased space within
the pocket might be beneficial for binding of the small vinyl group,
which was also reflected in the lowered K
M
value (0.8 mM) as
compared to the wildtype (7.8 mM) (Okrasa et al., 2009).
Gaßmeyer et al. showed that by transferring the set of amino
acid substitutions previously found beneficial in the (S)-selective
CLGIPL variant (V43I/A125P/ V156L/M159L excluding C188G/
G74C), a potent (R)-selective variant (IPLL) for production of
(R)-Flurbiprofen 6b is created. The quadruple variant converted
the respective malonate with 209 U mg
−1
to yield (R)-
Flurbiprofen 6b in 98%ee, which represents a six-fold increase
of specific activity compared to the native AMDase (Table 3,
entry 6) (Gaßmeyer et al., 2016).
Enzyme Engineering of G74C-Based
Variants With Racemising Activity
The limited activity of the AMDase G74C racemase towards
bulkier and industrially relevant α-aryl propionic acid derivatives
(also referred to as profens) prompted Kourist et al. to create
more suitable racemase variants by rational enzyme engineering
(Kourist et al., 2011b). While attempts to introduce co-catalytic
residues (Asp, Glu, His) as present in the related glutamate
racemase GluR (Glavas and Tanner, 2001) at the
corresponding positions of AMDase (Val13, Pro14, Gly190)
only led to inactive mutants, variation of the hydrophobic
pocket residues proved more successful. Destabilization within
the hydrophobic pocket is crucial for decarboxylation, however,
only plays a minor role in racemization. Yet, most of its
constituting residues are in close proximity of the reaction
center, thereby indirectly affecting the activity of the racemase.
In the case of AMDase G74C, MD simulations revealed, that
Val43 and Met159 are potent engineering targets, due to their
vicinity to the small substituent. Two of the designed variants,
AMDase G74C/V43A and G74C/M159L, were found to exhibit
enhanced racemization activity, while efficiency towards
decarboxylation was decreased at the same time, thus
underlining the different determinants for both promiscuous
activities. In total, activity of the G74C/V43A variant was 20-
fold shifted from decarboxylation towards racemization.
Remarkably, activity towards racemization of ketoprofen 9b
was also enhanced 30-fold (Kourist et al., 2011b). In a later
TABLE 3 | Kinetic parameters of AMDase wildtype-derived variants towards different substrates. Relative activities were taken from literature and were either calculated by
dividing the catalytic efficiency (k
cat
/K
M
) of each variant by the catalytic efficiency of wildtype AMDase (entry 1–5) or the corresponding specific activities (entry 6).
Entry Substrate AMDase variant k
cat
/K
M
(s
−1
mM
−1
)
Relative activity Ref.
1
1a
M159V 450/0.3 51 Okrasa et al. (2009)
2 P14V/P15G 1,143/3.5 11 Okrasa et al. (2009)
33a P14V/P15G 99.8/15.3 1.9 Okrasa et al. (2009)
44a G190A 20.8/0.8 4.6 Okrasa et al. (2009)
55a P14V/P15G 34.4/8.0 1.5 Okrasa et al. (2009)
66a IPLL n.d. 6 Gaßmeyer et al. (2016)
IPLL: AMDase V43I/A125P/V156L/M159L; n.d.: not determined.
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Schweiger et al. Arylmalonate Decarboxylase

TABLE 4 | Overview on AMDase substrates with relevance for research and industry.
AMDase substrate Research interests Ref.
6a Flurbiprofen 6b is sold as racemate; (S)-enantiomer acts as
NSAID;
Kourist et al. (2011b);Gaßmeyer et al. (2016)
(R)-enantiomer shows other activities (Geerts, 2007;Jin et al.,
2010;Liu et al., 2012);
Best variant: IPLL (R) or CLGIPL (S)(Gaßmeyer et al., 2016).
7a Ibuprofen 7b is sold as racemate; (S)-enantiomer acts as NSAID; Kourist et al. (2011b);Yoshida et al. (2015)
Best variant: WT (R) or CLGIPL (S)(Yoshida et al., 2015).
8a Naproxen 8b is sold as pure (S)-enantiomer; Kourist et al. (2011b);Miyauchi et al. (2011);Yoshida et al. (2015);
Gaßmeyer et al. (2016)(S)-enantiomer acts as NSAID;
Best variant: WT (R) or CLGIPL (S)(Yoshida et al., 2015).
9a Ketroprofen 9b is sold as racemate; (S)-enantiomer acts as
NSAID;
Kourist et al. (2011b)
Not well accepted by AMDase to date.
AMDase CLGIPL, V43I/G74C/A125P/V156L/M159L/C188G and IPLL,V43I/A125P/V156L/M159L. NSAID, non-steroidal anti-inflammatory drug.
FIGURE 6 | (A) Chemo-enzymatic synthesis routes towards optically pure Flurbiprofen 6b. The upper route was inspired and adapted from an industrial chemical
synthesis and the lower route was adapted to the aspired conditions of the chemo-enzymatic process. (B) Chemo-enzymatic synthesis of electron-deficient
N-heteroaromatic propionic acids.
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Schweiger et al. Arylmalonate Decarboxylase

study, structure-guided protein engineering of the racemase was
performed based on STD-NMR (saturation-transfer-difference
NMR) analysis, which eventually led to the quadruple variant
V43A/G74C/A125P/V156L with generally increased activity
towards all tested profen derivatives and a maximum of 40-
fold activity-increase towards Naproxen 8b (Gaßmeyer et al.,
2015).
RECENT APPLICATIONS
Production of Non-Steroidal
Anti-Inflammatory Drugs
Chiral α-aryl propionates are members of the group of non-
steroidal anti-inflammatory drugs (NSAIDs) and possess anti-
inflammatory as well as analgesic properties (Brogden, 1986).
Members of this drug class belong to top-selling over-the-counter
drugs and are used for the treatment of rheumatic disease
(muscoskeletal pain) and acute or chronic pain (Brooks, 1998).
NSAIDs inhibit prostaglandin biosynthesis by acting on the
cyclo-oxygenase (COX) enzyme system, which at the same
time causes common adverse effects on gastrointestinal
functions by non-specific inhibition of a different COX
isoform (Brogden, 1986;Brooks, 1998). Amongst the
commonly known profens, only Naproxen 8b is marketed as
pure (S)-enantiomer, whereas Ibuprofen 7b, Ketoprofen 9b or
Flurbiprofen 6b are administered in a mixture together with their
inactive (R)-enantiomers (Brooks, 1998). However,
unidirectional in vivo interconversion of the inactive (R)- to
the pharmacologically active (S)-enantiomer can occur to a
varying extent (Tracy and Hall, 1992;Brooks, 1998). Even
though Flurbiprofen 6b is mostly sold as a racemate, the (S)-
enantiomer primarily accounts for the anti-inflammatory
effect (Abdel-Aziz et al., 2012). Yet, other biological effects
of (R)-Flurbiprofen 6b had been described (Geerts, 2007;Jin
et al., 2010;Liu et al., 2012), thus making the accessibility of
both enantiomers in pure form a desirable goal. The ability of
AMDase to convert prochiral malonates into optically pure
arylpropionic acids was early recognized in the context of
profen synthesis, yet hampered by the availability of efficient
enzyme variants or limited stability of the malonate
precursors (Terao et al., 2003). Table 4 should give an
overview on NSAID precursors studied in AMDase
catalyzed decarboxylation and the corresponding best-
performing enzyme variants to date.
Interestingly, several industrially applied routes for profen
synthesis employ chemical malonate decarboxylation (Hylton
and Walker, 1981;Mizushima et al., 2014). Due to the
inherent instability of the intermediately formed malonic acid,
ester hydrolysis and decarboxylation are often carried out
simultaneously, thus yielding racemic product mixtures
(Hylton and Walker, 1981;Lu et al., 2006;Mizushima et al.,
2014). Yet, efforts to obtain pure enantiomers, like
recrystallization of diastereomeric mixtures or enzymatic
kinetic resolution, are often tedious and suffer from low yields
or limited enantiomeric excess (Terao et al., 2003). In this respect,
also enzymatic racemization under mild conditions could
contribute to higher overall yields by recycling the undesired
enantiomer for resolution processes (Kourist et al., 2011b).
In the synthesis of Flurbiprofen 6b, the low stability of the
corresponding malonic acid substrate with its electron-
withdrawing fluorine substituent represents a serious problem
for its work-up and isolation after the saponification of
α-arylmalonic acid esters. Inspired by a protection group
strategy used by Miyamoto and Ohta for the preparation of
isotope-labelled pseudochiral malonates (Miyamoto et al.,
1992b), we implemented hydrogenolysis of benzyl esters
instead of alkyl ester hydrolysis to avoid problems associated
TABLE 5 | Sequential chemo-enzymatic cascade of AMDase catalyzed decarboxylation of α-alkenyl-α-methylmalonic acids followed by CC bond reduction with in situ
generated diimide. After completion of the biocatalytic reaction step, hydrazine (20 eq) and CuCl
2
(0.01 eq) were added directly to the reaction mixture. The stated
conversion refers to the final reaction step and was detected after 23 h.
Entry Substrate AMDase variant Conversion (%) ee of c (%)
14a IPLL >99 98 (R)
24a CLGIPL >99 66 (S)
35a IPLL 20 >99 (R)
45a CLGIPL 11 >99 (S)
511a IPLL 80 >99 (R)
611a CLGIPL 78 >99 (S)
712a IPLL 86 >99 (R)
812a CLGIPL 89 >99 (S)
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Schweiger et al. Arylmalonate Decarboxylase

to spontaneous decarboxylation (Gaßmeyer et al., 2016). This
strategy is particularly suitable for all malonates with electron-
poor larger substituents.
We then developed an integrated chemo-enzymatic pathway
towards Flurbiprofen 6b including AMDase-mediated
decarboxylation and the previously introduced protecting
group strategy (Gaßmeyer et al., 2016;Enoki et al., 2019a).
We envisioned two potential chemo-enzymatic routes similarly
to previously described chemical synthesis strategies, yet adapted
to the corresponding dibenzyl malonic acid esters (Figure 6A)
(Terao et al., 2003;Lu et al., 2006). Interestingly, the AMDase-
catalyzed reaction step (Figure 6A, step (viii)) was planned at two
different stages of the process. Either after Suzuki coupling,
thereby using the established malonate 6a as substrate
(Figure 6A, step (viii-a)), of before Suzuki coupling, which
would require that AMDase exhibits activity towards α-(4-
bromo-3-fluorophenyl)-α-methylmalonic acid (Figure 6A, step
(viii-b)) (Enoki et al., 2019a).
After necessary adaptions were established for the initial
chemical steps towards the central intermediate dibenzyl α-(4-
bromo-3-fluorophenyl)-α-methylmalonic acid ester (Figure 6A,
steps (i)-(v)), the authors focused on the second half of the
cascade, including Pd-catalyzed Suzuki coupling (step (vi)),
deprotection of the dibenzyl-protected malonates (step (vii))
and eventually AMDase catalyzed decarboxylation (step (viii)).
It was suggested that palladium on charcoal (Pd/C) should be a
suitable catalyst for both Suzuki coupling and cleavage of benzyl
esters. Notably, during hydrogenolysis, the central intermediate
was less reactive (step (vii-b)) compared to dibenzyl flurbiprofen
malonic acid ester (step (vii-a)), yet more stable towards
spontaneous decarboxylation, which proved advantageous for
obtaining a high final ee of the product (Enoki et al., 2019a).
While Suzuki coupling under mild conditions in water, using Pd/
C as a catalyst and Ph
4
BNa as phenyl group donor, was reported
(step (vi-b)) (Lu et al., 2006), those particular conditions proved
impractical for the corresponding reaction of the alternative route
(step (vi-a)), due to solvent incompatibilities of the reagents.
Alternative Suzuki coupling strategies in organic solvents were
not considered at this point, by reasons of the aspired
environmentally benign reaction conditions. Eventually,
AMDase variants also proved active towards the yet
uncharacterized substrate in step (viii-b) with 114.3 U mg
−1
(WT), 480.3 U mg
−1
(IPLL), 41.7 U mg
−1
(CLG) or
40.7 U mg
−1
(CLGIPL), which was comparable to the
activities towards malonate 6a. Both enantiomers of
Flurbiprofen 6b could thus be produced in high yields and
>99%ee (Enoki et al., 2019a).
Synthesis of Decarboxylation of
Heteroaromatic Malonic Acids
The deprotection strategy was also successfully applied for the
preparation of electron-deficient N-heteroaromatic malonic
acids (Blakemore et al., 2020). The inherent instability of this
compound class even surpasses the one of their common
aromatic counterparts, thus fully preventing successful
isolation without undesired spontaneous decarboxylation
even under the typically mild hydrogenolysis conditions.
After optimization, hydrogenolysis was performed at carefully
balanced basic conditions in a biphasic system (5 wt% Pd/C;
10 ml g
−1
0.5 M Tris-HCl, pH 8.5; 2 eq. NaOH; 2 ml g
−1
toluene,
20°C, 4 h), which furnished the respective malonate dianions in
the buffered aqueous phase. This layer was separated and
directly used for enzymatic decarboxylation (3.5 wt%
lyophilized CFE, 20°C, 18 h), thus circumventing intermediary
isolation of the unstable malonic acid derivatives. The usability
of this telescoped hydrogenation-AMDase process was
demonstrated on a 120 g scale, producing optically pure 10b
in 76% yield and 98%ee (R)(Figure 6B). To prevent undesired
spontaneous decarboxylation of the AMDase substrates, the
importance of careful pH and temperature control
throughout the process was highlighted.
TABLE 6 | Overview on immobilization techniques studied in AMDase catalyzed decarboxylation.
Entry Carrier Interaction Enzyme (formulation)
1 Polystyrene nanoparticle, CoA functionalized Covalent AMDase WT (purif.) (Wong et al., 2010)
2 LentiKats
®
(PVA gel) Entrapment AMDase WT (CFE) (Markošová et al., 2018)
3 Activated MMP (magnetic microparticles) Covalent AMDase WT (CFE) (Markošová et al., 2018)
4 MMP-LentiKats
®
Combined AMDase WT (CFE) (Markošová et al., 2018)
5 Amino C2 acrylate Covalent AMDase WT (purif.) (Wong et al., 2010) AMDase CLGIPL (purif.)
(Gaßmeyer et al., 2016) AMDase CLGIPL (CFE) (Aßmann et al.,
2017a;Aßmann et al., 2017b)
6 Sepabeads EC-EP (polymethacrylate) Covalent AMDase WT (purif.) (Aßmann et al., 2017a)
7 Sepabeads EC-HA (polymethacrylate) Covalent AMDase WT (purif.) (Aßmann et al., 2017a)
8 Trisoperl
®
(porous glass) Adsorption AMDase WT (purif.) (Aßmann et al., 2017a)
9 Trisoperl
®
amino (porous glass) Adsorption AMDase WT (purif.) (Aßmann et al., 2017a)
10 EziG1™(Fe
III
or Co
II
) (porous glass with longchain aminoalkyl coating) Complex AMDase WT (purif.) (Aßmann et al., 2017a) AMDase CLGIPL
(purif.) (Aßmann et al., 2017a)
11 EziG2™(Fe
III
or Co
II
) (porous glass with vinylbenzyl-chloride coating) Complex AMDase WT (purif.) (Aßmann et al., 2017a) AMDase CLGIPL
(purif.) (Aßmann et al., 2017a)
12 EziG3™(Fe
III
or Co
II
) (porous glass with styrol/ acrylonitrile copolymer coating) Complex AMDase CLGIPL (purif.) (Aßmann et al., 2017a)
Purif, purified enzyme; CFE, Cell-free extract.
Frontiers in Catalysis | www.frontiersin.org October 2021 | Volume 1 | Article 74202414
Schweiger et al. Arylmalonate Decarboxylase

Synthesis of Optically Pure Alkanoic Acids
AMDase requires a substituent with a π-electronsystemonthe
substrate and does not convert aliphatic malonic acids.
Nevertheless, enantiopure alkanoic acids could be accessed in
two steps by combining AMDase-mediated decarboxylation
and reduction of the non-activated double bond (see
Table 5). With other catalytic methods, such molecules are
not easily accessible, particularly due to the difficulty of
chemical catalysts to distinguish the structurally similar
substituents (Enokietal.,2019b). While the activity of
wildtype AMDase towards alkenyl malonic acids was already
reported (Okrasa et al., 2009), reactivity of other (R)- and (S)-
selective enzyme variants was not yet studied with this class of
substrates.
As already demonstrated with other substrate types, AMDase
IPLL and CLGIPL outperformed the wildtype enzyme and other
(S)-selective variants, yet with a 19–170 and 190–2,200 times
reduced activity, respectively, as compared to aromatic substrates.
This reflected the lower capacity of an alkene to stabilize the
enediolate intermediate (Enoki et al., 2019b).
Interestingly, all tested enzyme variants proved to be highly
stereoselective, with the conversion of α-methyl-α-vinylmalonic
acid 4a by AMDase CLGIPL as only exception. This reaction
produced (S)-2-methylbut-3-enoic acid with surprisingly low
66%ee (S)(Table 5, entry 2). In this case, either an alternative
proton donor (e.g. water) or binding mode was discussed to cause
such impaired enantioselectivity, as control reactions with other,
highly selective AMDase variants ruled out a racemizing side-
reaction as source of this low enantiomeric excess (Lind and
Himo, 2014). Moreover, AMDase CLGIPL decarboxylated
α-vinyl malonates with additional substituents present on the
alkene (11–12a) with outstanding enantioselectivity, thus
reflecting the fragile interaction network responsible for ligand
recognition and binding.
Regarding chemical CC bond reduction, the potential risk of
double bond isomerization during transition-metal catalyzed
reduction, which would cause product racemization, could be
eventually eliminated by using in situ generated diimide as
reductant. The high enantiomeric excess observed after AMDase-
catalyzed decarboxylation could be thus retained until after the final
chemical reduction step (Table 5)(Enoki et al., 2019b).
Immobilization of Arylmalonate
Decarboxylase
Enzyme immobilization can significantly contribute to an
economical use of a biocatalyst (e.g.: stability, reuse) and
facilitate downstream processing (Cantone et al., 2013).
Considering the limited stability of purified AMDase under
process conditions (t
1/2
1.2 h) (Aßmann et al., 2017a),
differing immobilization strategies were tested recently,
including site-specific(Wong et al., 2010) or conservative
(Gaßmeyer et al., 2016;Aßmann et al., 2017b;Aßmann et al.,
2017a) covalent immobilization, adsorption, complexation
(Aßmann et al., 2017a), entrapment or combined approaches
(Table 6)(Markošová et al., 2018). Wong et al. studied the
phosphopantetheinyl transferase (Sfp)-catalyzed
immobilization of ybbR-tagged proteins (12-mer N-terminal
tag) on CoA-functionalized polystyrene nanoparticles
TABLE 7 | Overview on AMDases identified from different organisms.
Organism Maximum
activity
Optimum
pH
Ref.
Bordetella bronchiseptica KU1201 (formerly: Alcaligenes bronchisepticus
KU1201)
45°C8.5Miyamoto and Ohta (1992b);Miyamoto and Ohta
(1992a)
Achromobacter sp. KU1311 40°C8.5Miyamoto et al. (2007b)
Enterobacter cloacae KU1313 35°C5.5Yatake et al. (2008)
Chelativorans sp. BNC1 (formerly: Mesorhizobium sp. BNC1) n.d. n.d. Okrasa et al. (2008)
Variovorax sp. HH01 34°C6.0Maimanakos et al. (2016)
Variovorax sp. HH02 30°C7.0Maimanakos et al. (2016)
Polymorphum gilvum SL003B-26A1 37°C7.0Maimanakos et al. (2016)
n.d, not determined.
TABLE 8 | Identified conserved sequence motifs and residues involved in the catalytic mechanism of AMDase (in bold). Unconserved residues are denoted with x. Residue
numbering according to B. bronchiseptica AMDase (Maimanakos et al., 2016).
No. Sequence pattern Residues Description
1 GLIVPPAxGxVPxE (res.10-23) P14, P15 Part of large pocket, aryl binding
2 GLGLxxVxxxGY (res. 37-48) L40, V43, Y48 Part of hydrophobic pocket
3 GAxxVxLMGTSLSFYRG (res. 66-82) G74; C74 characteristic for racemases; part of dioxyanion hole
T75, S76
4 RVAVxTAY (res. 119-126) Y126 Part of dioxyanion hole
5 LxIxxVxxM (res. 151-159) V156, M159 Part of hydrophobic pocket
6 DALLISCGxL (res. 182-191) G189; Part of dioxyanion hole; catalytic active residue
C188
Frontiers in Catalysis | www.frontiersin.org October 2021 | Volume 1 | Article 74202415
Schweiger et al. Arylmalonate Decarboxylase

(Table 6, entry 1) (Wong et al., 2010). The covalent and site-
specific linkage was efficiently achieved under mild conditions,
and the obtained biocatalyst revealed high operational stability
over four cycles (approx. 7% loss of activity). Yet, the observed K
M
values were approximately three times higher compared to the
free enzyme, whereas K
M
values for AMDase and ybbR-AMDase
were similar, thus indicating that unfavorable interactions
between the nanoparticle and substrate are causing this
observed loss in efficiency rather than the immobilization itself
(Wong et al., 2010).
Recently, Markošová et al. attempted entrapment of AMDase
in polyvinyl alcohol (PVA) gel (Table 6, entry 2) (Markošová
et al., 2018). While AMDase showed enhanced stability, the
formulation suffered from severe activity loss after repeated
use, probably due to leaching of the enzyme. In a combined
approach (Table 6, entry 4), the combination of the operational
stability of covalently bound AMDase on magnetic microparticles
(MMP) and easy handling of PVA gel beads afforded a
biocatalyst, which was stable over eight biocatalytic reactions
and upon storage at 4°C (64% retained activity after 3 months)
(Markošová et al., 2018).
Covalent immobilization on well-established amino C2
acrylate carrier proved practical to obtain a stable biocatalyst
(approx. 20% activity yield) with a half-life of 16.5 h and a total
turnover number (TTN) of over 20,000 (Table 6, entry 5)
(Gaßmeyer et al., 2016). The same strategy was further studied
and applied by Aßmann et al. in an upscaled process for (S)-
naproxen 8b synthesis via decarboxylation of 8a (Aßmann et al.,
2017a;Aßmann et al., 2017b). This example demonstrates the
complementarity of enzyme engineering and process
engineering. While enzyme engineering could recover the
initially reduced activity of the (S)-selective variants by several
hundred folds to levels comparable to the wildtype, enzyme
immobilization achieved a substantial improvement of the
stability. Compared to the (S)-selective variant G74C/C188S in
solution, both protein and reaction engineering achieved a
tremendously improved productivity of the enzyme variants
with TTNs of 83,000–107,000 over five batches (Aßmann et al.,
2017a;Aßmann et al., 2017b). Also in this case, immobilization
increased the K
M
-value towards 8a from 0.08 to 22 mM,
underlining the strong effect of mass-transfer limitation. At the
same time, different other support materials and binding strategies
for immobilization were evaluated (Table 6,entry5–12). Overall,
covalently attached enzyme preparations were clearly preferred in
terms of enzyme loading and long-term stability in repeated
batches. For example, activity of AMDase immobilized on
EziG1 was formidable, yet the coordinative binding between the
enzyme’s 6xHis-tag and Fe(III) or Co(II) on the carrier is
considerably weaker and thus led to extensive leaching during
repeated experiments. Immobilization on amino C2 acrylate was
further optimized by using CFE instead of purified protein, which
certainly led to a decreased catalytic activity (30% compared to
purified AMDase), yet to an enhanced long-term operational
stability (t
1/2
8.6 d) (Aßmann et al., 2017a).
Immobilized AMDase was used by Aßmann et al. for an
intensification and up-scale of the synthesis of (S)-Naproxen 8b.
A careful analysis of kinetic parameters was greatly facilitated by
an in-line reaction monitoring via Raman spectroscopy.
Importantly, AMDase CLGIPL was inhibited by the product,
but only to an extent, where in situ product removal was not
necessary. A productivity of 140 kg
product
kg
enzyme
−1
was
calculated for the implemented process, which exceeds the
minimum specified value for pharmaceutical processes of
50–100 kg
product
kg
enzyme
−1
. Further, product isolation via
precipitation was optimized and (S)-naproxen 8b was isolated
in 92% yield and 99%ee (Aßmann et al., 2017b).
ARYLMALONATE DECARBOXYLASES
FROM OTHER ORGANISMS
In search of organisms with similar reactivities and characteristics
as AMDase from Alcaligenes bronchisepticus (now: Bordetella
bronchiseptica) KU1201, Ohta and co-workers were able to
identify two further strains from soil samples by their ability
to degrade α-phenylmalonic acid 1a (Miyamoto et al., 2007b;
Yatake et al., 2008). The strains KU1311 (Miyamoto et al., 2007b)
and KU1313 (Yatake et al., 2008) were the most active ones and
identified as Achromobacter sp. and Enterobacter cloacae,
respectively. Respective genes were amplified from the
genomic DNA and heterologously produced in E. coli for
characterization. Both genes encoded a protein of 240 amino
acids length, consistent with the originally identified AMDase
from strain KU1201 (Miyamoto and Ohta, 1992a) and both
shared a high sequence homology of 94% (KU1311) and 85%
(KU1313). Accordingly, the observed substrate selectivity and
activity were comparable to the original AMDase and all shared
the same strict enantiopreference (Miyamoto and Ohta, 1992a,
1992b;Miyamoto et al., 2007b;Yatake et al., 2008).
While those three AMDases show considerable similarity, the
shifted pH optimum (pH 5.5) towards acidic conditions of
AMDase from strain KU1313 was intriguing. It was the only
microorganism, which showed AMDase activity when soil
samples were screened at acidic pH, others were rather active
at neutral to slight basic pH (pH 7–8.5) (Table 7).
Okrasa et al. tried to identify novel AMDase enzymes from
protein sequences with a similarity of 30-52% and a requisite
cysteine residue at the same relative position as in the enzyme
from B. bronchiseptica. The putative AMDases were
characterized, however only the enzyme from Mesorhizobium
sp. (now: Chelativorans sp.) BNC1, exhibiting the highest
similarity, showed satisfactory decarboxylase activity, while the
other candidates rather acted as racemases (Okrasa et al., 2008).
The difficulty of identifying novel AMDases accompanied by
the issue of inappropriate gene annotation was addressed by the
work of Maimanakos et al., who developed a sequence-based
search algorithm for a more reliable prediction of enzymes
possessing AMDase activity (Maimanakos et al., 2016).
Sequence information of confirmed AMDases was used to
specify 12 conserved sequence patterns (e.g. residues of the
hydrophobic pocket, dioxyanion hole, aryl binding pocket and
residues C188 and G74 including their surroundings), which, in
turn, were used for concise database screening. In this way, 58
additional ORFs encoding putative AMDase-like enzymes were
Frontiers in Catalysis | www.frontiersin.org October 2021 | Volume 1 | Article 74202416
Schweiger et al. Arylmalonate Decarboxylase

found and used for generating a Hidden Markov Model (HMM),
which furnished six specific motifs (Table 8) and was
implemented in the applied search algorithm. To prove the
reliability and applicability of these search criteria, the enzyme
from Polymorphum gilvum SL003B-26A1 identified via data base
search was characterized together with two enzymes from
Variovorax sp. HH01 and HH02 identified via screening of
soil samples. All three enzymes showed the desired AMDase
activity within common mesophilic temperature and around
neutral to slightly acidic pH (Table 7)(Maimanakos et al., 2016).
Interestingly, all putative AMDase-encoding ORFs identified
by Maimanakos et al. belonged to the class of α-β- and
γ-proteobacteria. Interestingly, when AMDase flanking regions
were analyzed, genes encoding mandelate racemase/muconate
lactonizing enzyme or several transporters (e.g. tripartite
tricarboxylate transporter TTT, TRAP or ABC transporters)
were commonly found. According to these flanking regions
and sequence similarities, AMDases were classified into eight
enzyme clusters. While the natural role of this enzyme still
remained elusive, the fact, that close relatives of AMDase-
producing bacteria often do not encode this enzyme, strongly
indicated, that this gene was frequently lost in evolution and
might be therefore only temporarily advantageous for organisms
under certain physiologic conditions (Maimanakos et al., 2016).
CONCLUSION
30 years after its discovery, AMDase has arrived at the stage of
industrial applications. The history of research on this unique
decarboxylase is exemplary for the technological progress in
biocatalysis within the last decades. After AMDase discovery,
based on a purely functional screen, the first important milestone
achieved by the Ohta group was the identification of the
corresponding gene, which allowed recombinant production of
the enzyme. Enzymological characterization and studies with
isotope-labelled pseudochiral malonic acids allowed initial
mechanistic insights: During the reaction, only one carboxylate
group is cleaved, and this step is not directly determining the
stereooutcome of the reaction. Based on homology models, the
enantioselectivity was completely inverted, and a promiscuous,
unique profen racemase was generated. Yet, elucidation of the
crystal structure of AMDase in its liganded form in the early
2010s was groundbreaking for the formulation of a hypothetical
decarboxylation mechanism, which was confirmed by several
computational studies in recent years. Despite the availability
of several crystal structures, it remains exceedingly difficult to
accurately predict the influence of specific amino acid
substitutions in the hydrophobic pocket on enzyme activity. In
lieu of predictability, site-saturation mutagenesis and
simultaneous saturation mutagenesis proved to be efficient
methods to increase the activity of enzyme variants in the
synthesis of both enantiomers of α-aryl propionic acids, and
to identify optimal variants for different substrates.
The tendency of α-aryl α-methylmalonic acids to undergo
spontaneous decarboxylation proved to be an obstacle for
industrial application, particularly regarding the
decarboxylation of substrates with large electron-poor
substituents. By developing a carefully balanced deprotection
strategy for the corresponding malonic acid esters, conditions
leading to spontaneous decarboxylation could be avoided. A
telescoped deprotection/decarboxylation approach resulted in a
robust, practicable reaction that allows to obtain α-aryl
propionates in either their (S)- or (R)-form in outstanding
optical purity. While the activity of the enzyme is generally
high, immobilization greatly increased the stability, resulting in
an excellent productivity.
Starting with a completely unknown enzyme with unknown
mechanism that produced the “wrong”enantiomer of profens, a
combination of biocatalysis, enzymology, structure elucidation,
molecular modeling, protein engineering, organic chemistry and
process intensification succeeded to create a powerful toolbox of
AMDase variants that provide access to a large diversity of
α-substituted propionates in their enantiopure form.
AUTHOR CONTRIBUTIONS
AS devised the article and prepared figures, RK and KM
participated in planning of the article and contributed to the text.
FUNDING
All sources of funding have been submitted. RK is funded by the
Austrian Science Fund (FWF) P34820.
ACKNOWLEDGMENTS
RK would like to thank the Austrian Science Funds (FWF,
P34280) for financial support.
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The handling editor declared a past co-authorship with one of the authors RK
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