Engineered C–N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners

Article abstract of DOI:10.1002/anie.201910704

Aspartic acid derivatives with branched N-alkyl or N-arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N-substituted aspartic acids, including N-(3,3-dimethylbutyl)-l-aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-l-aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine-N,N′-disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C–N lyase (mutant D290M/Y320M) displayed a remarkable 1140-fold increase in activity for the selective hydroamination of fumarate compared to that of the wild-type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step-economic synthesis of an important class of food additives.

Full text of DOI:10.1002/anie.201910704

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Enantioselective Synthesis of Chiral Synthons for Artificial
Dipeptide Sweeteners Catalyzed by an Engineered C-N Lyase
Authors: Jielin Zhang, Eleonora Grandi, Haigen Fu, Thangavelu
Saravanan, Laura Bothof, Pieter G. Tepper, Andy-Mark W. H.
Thunnissen, and Gerrit Jan Poelarends
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201910704
Angew. Chem. 10.1002/ange.201910704
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
Enantioselective Synthesis of Chiral Synthons for Artificial
Dipeptide Sweeteners Catalyzed by an Engineered C-N Lyase
[
a]
[a]
[a]
[a]
[a]
Jielin Zhang, Eleonora Grandi, Haigen Fu, Thangavelu Saravanan, Laura Bothof, Pieter G.
[
a]
[b]
[a]
Tepper, Andy-Mark W.H. Thunnissen, and Gerrit J. Poelarends*
Abstract: Aspartic acid derivatives with branched N-alkyl or N-
arylalkyl substituents are valuable precursors to artificial dipeptide
sweeteners such as neotame and advantame, which have wide-
ranging applications in the food industry. Despite the potential
applications of these amino acid precursors to aspartame-based
sweeteners, the development of a biocatalyst to synthesize these
compounds in a single asymmetric step is an as yet unmet challenge.
Herein we report an enantioselective biocatalytic synthesis of various
difficult N-substituted aspartic acids including N-(3,3-dimethylbutyl)-L-
aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-
aspartic acid, precursors to neotame and advantame respectively,
using an engineered variant of ethylenediamine-N,N'-disuccinic acid
than sucrose. A common synthetic method for neotame and
advantame production is the reductive N-alkylation of aspartame
with the corresponding aldehyde in the presence of hydrogen
using a palladium (Pd/C) or platinum (Pt/C) hydrogenation
catalyst (Scheme 1B, Method 1).[
10-14]
An alternative strategy for
neotame production involves N-(3,3-dimethylbutyl)-L-aspartic
acid (3a) as precursor, which was linked to L-phenylalanine
[
15–
methyl ester by amide bond coupling (Scheme 1B, Method 2).
1
7]
This precursor is chemically synthesized by reductive N-
alkylation of L-aspartic acid (or its ester derivative) using
transition-metal catalysts (Pd/C or Pt/C). Similarly, N-[3-(3-
hydroxy-4-methoxyphenyl)propyl]-L-aspartic acid (3f) could be
chemically prepared by reductive N-alkylation of L-aspartic acid
and serve as precursor to advantame. However, the development
of a biocatalyst for enantioselective synthesis of these difficult N-
substituted aspartic acids 3a and 3f in a single asymmetric step
is an as yet unmet challenge.
Here we report the engineering of an effective C-N lyase,
based on ethylenediamine-N,N'-disuccinic acid (EDDS) lyase
from Chelativorans sp. BNC1,[18-20] for the enantioselective
synthesis of N-(3,3-dimethylbutyl)-L-aspartic acid (3a) and N-[3-
(EDDS) lyase from Chelativorans sp. BNC1. This engineered C-N
lyase (mutant D290M/Y320M) displayed a remarkable 1140-fold
increase in activity for the selective hydroamination of fumarate
compared to that of the wild-type enzyme, which could be rationalized
from the analysis of crystal structures. These results open up new
opportunities to develop practical multienzymatic processes for the
more sustainable and step-economic synthesis of an important class
of food additives.
(
3-hydroxy-4-methoxyphenyl)propyl]-L-aspartic
acid
(3f),
precursors to neotame and advantame respectively, as well as
related chiral synthons for aspartame-based sweeteners starting
from the simple nonchiral compound fumaric acid (1, Scheme 1C).
This newly engineered C-N lyase shows a 1140-fold increase in
activity for the selective hydroamination of fumarate compared to
that of the wild-type enzyme, opening up new opportunities to
design practical multienzymatic processes for the more
sustainable and step-economic synthesis of an important class of
food additives.
Introduction
Artificial low-calorie sweeteners are used as sugar
replacements in the food industry, with the benefits of controlling
energy intake and blood glucose levels, improving dental health
and other health concerns related to sugar overconsumption.[1–6]
The dipeptide aspartame, which is ~200-fold sweeter than
sucrose (Scheme 1A), is one of the most widely used artificial
sweeteners with a substantial production volume each year.[7] The
derivatization of aspartame with branched N-alkyl- or N-arylalkyl-
groups generates even sweeter compounds, such as the more
recently approved food additives neotame and advantame Results and Discussion
Scheme 1A).[6,8-10] Notably, neotame is 7,000-13,000 times
sweeter than sucrose, while advantame is ~20,000 times sweeter
(
Our group has previously reported that an engineered
variant of 3-methylaspartate ammonia lyase (MAL-Q73A) accepts
various amines, including butylamine (2c, Table 1), for
enantioselective hydroamination of fumarate (1).[21,22] Structurally,
amines 2b and 2a have respectively one or two extra methyl
group(s) at the C-3 position compared with 2c. This prompted us
to start our investigations by testing the branched amines 2a and
2b as unnatural substrates in the MAL-Q73A catalyzed
hydroamination of 1. Although amine 2b was accepted by MAL-
Q73A for slow hydroamination of 1 (Figure S1), yielding optically
pure L-3b (e.e. >99%), amine 2a was unfortunately not accepted
as substrate by MAL-Q73A. This suggests that the bulky tert-butyl
group of 2a prevents productive binding in the enzyme active site,
making amine 2a a challenging substrate for C-N lyases.
[
[
a]
b]
J. Zhang, E. Grandi, H. Fu, Dr. T. Saravanan, L. Bothof, P.G.
Tepper, Prof. Dr. G.J. Poelarends
Department of Chemical and Pharmaceutical Biology
Groningen Research Institute of Pharmacy, University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
E-mail: g.j.poelarends@rug.nl
Dr. A.M.W.H. Thunnissen
Molecular Enzymology Group
Groningen Institute of Biomolecular Sciences and Biotechnology,
University of Groningen
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
10.1002/anie.201910704
RESEARCH ARTICLE
Scheme 1. (A) Structures of the low-calorie artificial dipeptide sweeteners aspartame, neotame and advantame; (B) Current synthesis methods for neotame and
advantame involve metal-catalyzed reductive N-alkylation; (C) Biocatalytic asymmetric synthesis of N-substituted aspartic acids 3 as precursors for potential
multienzymatic synthesis of neotame and advantame.
We continued our investigations by testing whether EDDS
lyase, which has previously been shown to possess an
exceptionally broad amine scope,[18-20] can accept the challenging
amine 2a as an unnatural substrate in the hydroamination of 1.
Pleasingly, EDDS lyase accepted 2a for addition to 1, giving rise
to target compound 3a. Under optimized conditions, excellent
conversion (92% after 7 days) and good isolated yield (67%) of
(82-97% after 7 days) and excellent enantioselectivity (>99% e.e.)
were observed (Table 1).
Although EDDS lyase is the first identified biocatalyst to
synthesize target compound 3a in a single asymmetric step, its
catalytic activity for this transformation is quite low resulting in a
rather long reaction time of 7 days when using 0.15 mol%
biocatalyst loading. Therefore,
a
structure-based protein
3
a were achieved using 0.15 mol% biocatalyst loading (Table 1,
engineering strategy was applied to enhance this hydroamination
activity of EDDS lyase. On the basis of the structure of EDDS
lyase in complex with its natural substrate (S,S)-EDDS (Figure
1),[18] two residues (Asp290 and Tyr320) were chosen for site-
saturation mutagenesis (SSM) because of their presumed roles
in positioning of the amine substrate for addition to fumarate.
Specifically, residue Asp290 forms a water-mediated hydrogen
bond with the internal amino group connected to the distal
Figure S2). The enzymatic product 3a was identified as the
desired L-enantiomer with >99% e.e. Amines 2b and 2c were also
readily converted by EDDS lyase to afford the respective optically
pure products L-3b and L-3c (>99% e.e.) with 93-95% conversion
and in 66-74% isolated yield (Table 1). Interestingly, the bulky
arylalkylamines 2d-f were also accepted as substrates by EDDS
lyase, yielding the respective products L-3d-f. High conversion
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
succinyl moiety of (S,S)-EDDS, which appears to be an important
interaction for binding and positioning of ethylenediamine and
other diamine substrates (but not for monoamines such as 2a) for
addition to fumarate. The bulky aromatic ring of Tyr320 may
further preclude optimal positioning of amine 2a.
Table 1. Enantioselective synthesis of neotame and advantame precursors, as well as related compounds, using EDDS lyase or its engineered variant
D290M/Y320M as biocatalyst.
WT[a]
D290M/Y320M[b]
e.e.[d]
Entry
Amine
Product
[
%]
Conv. (yield)[c]
%]
Time
[days]
Conv.(yield)[c]
Time
[hours]
[
[%]
1
2a
3a
92 (67)
7
96 (83)
2.5e
>99
2
3
2b
2c
3b
3c
93 (74)
95 (66)
7
7
93 (81)
92 (68)
2.5e
>99
>99
2.5e
4
5
2d
2e
3d
3e
97 (45)
93 (40)
7
7
96 (49)
90 (40)
6
6
>99
>99
6
2f
3f
82 (34)
7
82 (34)
6
>99
[
2 4
a] Reaction conditions: fumaric acid (1, 10 mM), amine (2a-f, 50 or 100 mM) and EDDS lyase WT (0.05 or 0.15 mol% based on fumaric acid) in NaH PO /NaOH
buffer (pH 8.5) at room temperature. [b] Reaction conditions: fumaric acid (1, 10 mM), amine (2a-f, 50 or 100 mM), glycerol (45%, v/v) and EDDS lyase
1
D290M/Y320M (0.05 mol% based on fumaric acid) in NaH
2
PO
4
/NaOH buffer (pH 8.5) at room temperature. [c] Conversion was determined by H NMR spectroscopy;
isolated yield after ion-exchange chromatography. [d] The e.e. value was determined by high-performance liquid chromatography on a chiral stationary phase using
-
1 -1
m
chemically synthesized authentic standards. [e] The apparent kcat/K values (using 5 mM 1) were estimated to be 5.1 (2c), 12.8 (2b) and 14.5 M s (2a).
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RESEARCH ARTICLE
A
B
Figure 1. (A) Structures of natural substrate (S,S)-EDDS and target compound (S)-3a and (B) a close-up of the active site of EDDS lyase with bound (S,S)-EDDS
PDB: 6G3H). The bound (S,S)-EDDS (green) and side chains of residues forming the amine binding pocket are shown using stick representation. The two target
(
residues for mutagenesis, Asp290 and Tyr320, are shown in yellow.
Accordingly, two focused libraries were constructed by
randomizing positions Asp290 and Tyr320, yielding libraries
D290X and Y320X. The libraries were transformed into
Escherichia coli cells and screened by evaluating ~100
transformants of each library. Initially, we evaluated mutants in
the D290X library by monitoring the depletion of 1 in a
spectrophotometric kinetic assay in multi-well plates using cell
free extracts (CFEs). However, this screening was unsuccessful
because 1 was converted at a similar rate by all CFEs, including
a CFE prepared from E. coli cells not producing EDDS lyase
Using this optimized assay, screening of the D290X and
Y320X libraries resulted in the identification of five mutants
(D290L, D290V, Y320M, Y320V and Y320L) with significantly
improved activity. These mutant enzymes were purified to
homogeneity and assayed for their ability to catalyze the addition
of 2a to 1 to yield target compound 3a. The best mutant from the
D290X library (D290L) showed a 55-fold enhanced activity, while
the best mutant from the Y320X library (Y320M) displayed a
remarkable 620-fold increase in activity compared to that of the
wild-type enzyme (Figure 2, Table S2).
(
Figure S3). We assumed that this relatively high background
To further improve the catalytic activity of EDDS lyase, we
used an iterative saturation mutagenesis (ISM) strategy, using the
best four hits from the single-site libraries as templates and
randomizing the other respective position. Accordingly, the
libraries D290L/Y320X, Y320M/D290X, Y320V/D290X and
Y320L/D290X were constructed. The screening of these libraries
resulted in the identification of four double mutants,
consumption of 1 was caused by indigenous fumarase (FumC)
activity present in the E. coli CFE, resulting in the undesired
hydration of 1 to give L-malic acid, which outcompeted the slower
EDDS lyase mediated hydroamination of 1.[23,24] Considering that
the removal of fumarase by enzyme purification from CFEs is
quite laborious and not suitable for library screening, we tested
whether the addition of fumarase inhibitors (D-malate, citrate and
glycerol) could suppress FumC-dependent hydration of 1. While
D-malate and citrate did not show sufficient inhibition (data not
shown), the addition of glycerol (45%, v/v) to the screening assay
effectively inhibited FumC-catalyzed hydration of 1 (Figures S4A
and S5). It has been reported that glycerol inhibits FumC by
affecting a conformational change, which appears to be the rate-
limiting step, based on its viscogenic effect.[25] Importantly, control
experiments demonstrated that the activity of EDDS lyase,
measured by the addition of ethylene diamine to 1, was not
inhibited by glycerol (Figure S4B). Based on these optimizations,
D290M/Y320M,
D290H/Y320M,
D290L/Y320M
and
D290M/Y320V, which showed activity improvement over the best
single mutant Y320M. Based on assays using purified enzymes,
the mutant D290M/Y320M was shown to be the best mutant
enzyme, with a striking 1140-fold increase in activity compared to
that of the wild-type enzyme (Figure 2, Table S2). Notably, the
mutant enzyme D290L/Y320M, in which the two best single
mutations at each position are combined, displayed a lower
activity compared to that of mutant D290M/Y320M (Figure 2),
illustrating the importance of using an ISM approach to identify
the best mutant.
4
5% (v/v) glycerol was included in the screening assay as additive
To understand how the mutations cause the large increase
in activity we determined crystal structures of EDDS lyase variant
D290M/Y320M. Crystal structures were obtained with fumarate or
formate occupying the active site (Table S3, Figure S6), like
previously shown for the wild-type enzyme.[18] Similar as in the
to suppress the FumC-catalyzed hydration of 1, enabling
hydroamination activity screening of mutant libraries using CFEs
instead of purified proteins.
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RESEARCH ARTICLE
and wild-type structures revealed no significant differences in
overall protein structure, nor in the conformations of the active site
residues responsible for binding and activating fumarate prior to
the amine addition. Mutations D290M and Y320M are located at
opposite sides of the presumed amine binding pocket, and the
main structural consequences of the amino acid substitutions are
a slight reshaping of the pocket and a significant increase of its
hydrophobicity (Figure 3A, B). Further insights were obtained
from docking compound 3a in the EDDS lyase wild-type and
mutant crystal structures (efforts to obtain crystal structures with
bound 3a were unsuccessful). The modeling results show that
while residues Asp290 and Tyr320 in the wild-type structure form
unfavorable polar-apolar contacts with the 3,3-dimethylbutyl
moiety of 3a, residues Met290 and Met320 in the mutant structure
are able to form favorable apolar-apolar contacts (Figure 3C, D).
The increased hydrophobicity of the pocket due to the D290M and
Y320M mutations thus leads to improved binding interactions with
the apolar 3,3-dimethylbutyl moiety of 3a, suggesting that the
large increase in activity of mutant enzyme D290M/Y320M is a
consequence of much stronger, and possibly more productive,
binding of amine 2a.
A
Importantly, we observed that the activity of the mutant
enzyme D290M/Y320M for the addition of 2a (100 mM) to 1 (5
mM) was affected by glycerol. The enzymatic activity decreased
significantly (~2.5-fold) when the glycerol concentration in the
reaction mixture was reduced from 45% to 30% (v/v) and became
almost undetectable when the glycerol concentration was lowered
to ≤20% (Figure S7). This decrease in enzyme activity upon
lowering the glycerol concentration was accompanied by slight
protein precipitation. Since the D290M/Y320M mutant was
observed to be stable and fully active after several hours of
incubation in buffer (without amine substrate) at room
temperature, it appears that in the presence of high
concentrations of amine 2a (100 mM), the D290M/Y320M mutant
is not stable and loses activity. In the reaction mixture with 100
mM amine 2a, the D290M/Y320M mutant was stabilized by
glycerol (45%, v/v), which is a routinely used stabilizing agent for
proteins.[26,27] Note that the addition of 45% (v/v) glycerol did not
effect the hydroamination activity of the wild-type enzyme under
the same conditions (Figure S8).
B
Figure 2. Engineering of EDDS lyase for efficient synthesis of 3a. (A) Activity
improvement of EDDS lyase variants over wild type. Conditions: 1 (5 mM), 2a
100 mM), purified enzyme (0.15 mol% based on fumaric acid), glycerol (45%,
v/v), NaH PO /NaOH buffer (pH 8.5), r.t. Reactions were monitored by following
Interestingly, this implies that glycerol played dual roles in
mutant library screening; it served both as fumarase inhibitor and
as protein stabilizer. The presence of 45% (v/v) glycerol during
library screening was thus essential for the identification of the
D290M/Y320M mutant, suggesting that the incorporation of
cosolvents in screening assays is an appealing strategy to identify
mutants with the desired activity, but having reduced stability, in
enzyme evolution. Our results provide support for the notion that
protein stability is a major constraint in enzyme evolution, and
buffering mechanisms such as the inclusion of stabilizing
(
2
4
the depletion of 1 at 270 nm by UV spectroscopy. Reactions were performed in
triplicate (See Table S2 for details). (B) Progress curves for addition of 2a to 1
catalyzed by EDDS lyase wild type and variants.
wild-type structure, fumarate is bound tightly in the active site of
the mutant through interactions with its two carboxylate groups,
while its Cβ atom faces the hydroxyl oxygen of Ser280, the
catalytic base, at a distance of 3 Å. A second fumarate is bound
somewhat away from the active site, in a region which in the wild-
type structure is responsible for binding the distal succinate group
of (S,S)-EDDS. The formate-bound mutant structure contains
three formate ions, two of which occupy positions in the active site
where the carboxylate groups of fumarate bind, further denoting
the integrity of the active site. A detailed comparison of the mutant
[28]
cosolvents are key in relieving this constraint.
Having generated an EDDS lyase variant with strongly
improved catalytic activity (mutant D290M/Y320M), we tested its
performance as biocatalyst for the synthesis of our target
compound 3a. With 0.05 mol% of biocatalyst loading, starting
substrates 1 and 2a were readily converted to afford optically pure
L-3a (>99% e.e.) with 96% conversion after only 2.5 h (instead of
7
days as observed for the same transformation with the wild-type
enzyme) and in 83% isolated yield (entry 1 in Table 1, Figure S9).
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RESEARCH ARTICLE
Figure 3. Structural analysis of EDDS lyase D290M/Y320M (A) Close-up view of the active site in the crystal structure of fumarate-bound EDDS lyase D290M/Y320M
pdb entry 6RX8). Shown as sticks are bound fumarate (FUM, yellow), the side chain of the catalytic serine Ser280 (orange) and the side chains of Met290 and
(
Met320’ (orange, the apostrophe denotes that the residue is from another subunit). Also shown as an overlay are two formate ions (FMT, green sticks) found in the
crystal structure of formate-bound EDDS lyase D290M/Y320M (pdb entry 6RXA). The second fumarate and third formate found in the crystal structures are omitted
for reasons of clarity. Oxygens are colored red, sulfurs yellow. (B) Binding mode of (S,S)-EDDS (yellow sticks) in the crystal structure of wild-type EDDS lyase
showing the side chains of S280, D290 and Y320’ (cyan, pdb entry 6G3H). Shown as an overlay are the side chains of S280, M290 and M320’ from the formate-
bound crystal structure of the EDDS lyase variant. (C, D) Binding mode of compound 3a (magenta sticks) at the active sites of wild-type EDDS lyase (panel C) and
EDDS lyase D290M/Y320M (panel D), as obtained by docking. Dashed lines indicate unfavorable polar-apolar contacts (yellow, distances ~4 Å) or favorable
hydrophobic apolar-apolar contacts (black, distances 4-5 Å).
To further demonstrate the synthetic usefulness of this newly
engineered C-N lyase, amines 2b-f were tested as substrates in
the hydroamination of 1. The enzymatic reactions proceeded
smoothly to afford enantiomerically pure products L-3b-f (>99%
e.e.) with 82-96% conversion (after a few hours rather than 7
days) and in 34-81% yield (entries 2-6 in Table 1, Figure S10).
These amino acid products (except 3d) are key building blocks for
N-functionalized aspartame derivatives that were reported to be
much sweeter than sucrose.[8] Notably, product 3f is a chiral
precursor for the synthesis of advantame (Scheme 1), which, like
neotame, has already been approved for application in food
products.
laboratory evolution was essential for the identification of this
improved C-N lyase, suggesting that the inclusion of stabilizing
cosolvents is an appealing strategy to reduce the constraining
effects of protein stability during enzyme evolution. The newly
developed biocatalytic methodology offers a useful alternative
route to important chiral synthons for artificial dipeptide
sweeteners. The engineered C-N lyase nicely supplements the
toolbox of biocatalysts for production of unnatural amino acids,
and opens up new opportunities to develop an entirely enzymatic
route for the straightforward synthesis of valuable aspartame-
based sweeteners, starting from the simple non-chiral
dicarboxylic acid 1 (Scheme 1C). Although the engineered EDDS
lyase variant D290M/Y320M exhibits a respectable specific
activity of 1.74 U/mg for the addition of 2a to 1, ongoing enzyme
engineering work in our group is focused on further improving its
stability (in the absence of glycerol) and catalytic efficiency (in
Conclusion
m
terms of kcat/K ) for this reaction. As such, this work sets the stage
In conclusion, we have successfully engineered a C-N lyase
for efficient asymmetric addition of challenging amines to
fumarate to yield optically pure N-(3,3-dimethylbutyl)-L-aspartic
acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-aspartic
acid, which are important precursors to neotame and advantame
respectively. Interestingly, the presence of glycerol during
for further development of practical multienzymatic processes for
the more sustainable and step-economic synthesis of an
important class of food additives.
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
[
[
[
[
22] V. Puthan Veetil, H. Raj, M. De Villiers, P. G. Tepper, F. J. Dekker, W. J.
Quax, G. J. Poelarends, ChemCatChem 2013, 5, 1325–1327.
23] S. A. Woods, S. D. Schwartzbach, J. R. Guest, Biochim. Biophys. Acta,
Protein Struct. Mol. Enzymol. 1988, 954, 14-26.
Experimental Section
Experimental Details. All experimental details can be found in the
Supporting Information.
24] C.-P.
Tseng,
C.-C.
Yu,
H.-H.
Lin,
C.-Y.
Chang,
J.-
T. Kuo, J. Bacteriol. 2001, 183, 461–467.
25] I. A. Rose, T. M. Weaver, Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 3393–
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Acknowledgements
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26] K. Gekko, S. N. Timasheff, Biochemistry 1981, 20, 4667–4676.
27] V. Vagenende, M. G. S. Yap, B. L. Trout, Biochemistry 2009, 48, 11084–
We acknowledge funding from The Netherlands Organization of
Scientific Research (VICI grant 724.016.002) and the European
Research Council (PoC grant 713483). Jielin Zhang and Haigen
Fu acknowledge funding from the China Scholarship Council. The
authors thank Harshwardhan Poddar and Lieuwe Biewenga for
insightful discussions, and Gea Cruiming for assistance with
synthesis and Dr. Robbert H. Cool for assistance with enzyme
purification. Dr. Daniele de Sanctis from the European
Synchrotron Radiation Facility (Grenoble) is acknowledged for
assistance with X-ray data collection at beamline ID29.
1
1096.
[
28] N. Tokuriki, D. S. Tawfik, Nature 2009, 459, 668-673.
Keywords: Artificial sweetener • C-N lyase • protein engineering
•
aspartate derivatives • hydroamination • biocatalysis
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Angewandte Chemie International Edition
10.1002/anie.201910704
RESEARCH ARTICLE
Entry for the Table of Contents
RESEARCH ARTICLE
Jielin Zhang, Eleonora Grandi, Haigen
Fu, Thangavelu Saravanan, Laura
Bothof, Pieter G. Tepper, Andy-Mark
W.H. Thunnissen, and Gerrit J.
Poelarends*
Page No. – Page No.
Engineered carbon-nitrogen lyase working in reverse: The enzyme
ethylenediamine-N,N'-disuccinic acid lyase was optimized by structure-guided
mutagenesis for the enantioselective synthesis of challenging N-substituted aspartic
acids, which are important chiral precursors to artificial dipeptide sweeteners. This
redesigned C-N lyase displayed a remarkable 1140-fold increase in activity for
selective hydroamination of fumarate.
Enantioselective Synthesis of Chiral
Synthons for Artificial Dipeptide
Sweeteners Catalyzed by an
Engineered C-N Lyase
This article is protected by copyright. All rights reserved.