Modular Enzymatic Cascade Synthesis of Vitamin B5 and Its Derivatives

Article abstract of DOI:10.1002/chem.201804151

Access to vitamin B₅ [(R)-pantothenic acid] and both diastereoisomers of α-methyl-substituted vitamin B₅ [(R)- and (S)-3-((R)-2,4-dihydroxy-3,3-dimethylbutanamido)-2-methylpropanoic acid] was achieved using a modular three-step biocatalytic cascade involving 3-methylaspartate ammonia lyase (MAL), aspartate-α-decarboxylase (ADC), β-methylaspartate-α-decarboxylase (CrpG) or glutamate decarboxylase (GAD), and pantothenate synthetase (PS) enzymes. Starting from simple non-chiral dicarboxylic acids (either fumaric acid or mesaconic acid), vitamin B₅ and both diastereoisomers of α-methyl-substituted vitamin B₅, which are valuable precursors for promising antimicrobials against Plasmodium falciparum and multidrug-resistant Staphylococcus aureus, can be generated in good yields (up to 70 %) and excellent enantiopurity (>99 % ee). This newly developed cascade process may be tailored and used for the biocatalytic production of various vitamin B₅ derivatives by modifying the pantoyl or β-alanine moiety.

Full text of DOI:10.1002/chem.201804151

A Journal of
Accepted Article
Title: Modular enzymatic cascade synthesis of vitamin B5 and its
derivatives
Authors: Mohammad Zainal Abidin, Thangavelu Saravanan, Jielin
Zhang, Pieter Tepper, Erick Strauss, and Gerrit Jan
Poelarends
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201804151
Link to VoR: http://dx.doi.org/10.1002/chem.201804151
Supported by

Chemistry - A European Journal
10.1002/chem.201804151
COMMUNICATION
Modular enzymatic cascade synthesis of vitamin B
5
and its
derivatives
[
a]
[a]
[a]
[a]
[b]
Mohammad Z. Abidin, Thangavelu Saravanan, Jielin Zhang, Pieter G. Tepper, Erick Strauss,
Gerrit J. Poelarends*[a]
Abstract: Access to vitamin B
diastereoisomers of α-methyl-substituted vitamin B
(R)-2,4-dihydroxy-3,3-dimethylbutanamido)-2-methylpropanoic acid]
5
[(R)-pantothenic acid] and both
5
[(R)- and (S)-3-
(
has been achieved using a modular three-step biocatalytic cascade
involving 3-methylaspartate ammonia lyase (MAL), aspartate-α-
decarboxylase (ADC), β-methylaspartate-α-decarboxylase (CrpG) or
glutamate decarboxylase (GAD), and pantothenate synthetase (PS)
enzymes. Starting from simple non-chiral dicarboxylic acids (either
fumaric acid or mesaconic acid), vitamin
B
5
and both
, which are
5
Figure 1. Structures of vitamin B and its α-methyl-substituted derivative.
diastereoisomers of α-methyl-substituted vitamin B
5
valuable precursors for promising antimicrobials against Plasmodium
falciparum and multidrug-resistant Staphylococcus aureus, can be
generated in good yields (up to 70%) and excellent enantiopurity
PanAm analogues.[10] However, the more challenging chemical
syntheses of these compounds poses a significant barrier to the
discovery of their clinical potential. Therefore, the development
of an asymmetric biocatalytic synthesis strategy that provides
efficient and step-economic access to pantothenic acid (5a) and
both diastereoisomers of its α-methyl substituted derivative (5b,
Figure 1), avoiding (de-)protecting steps and intermediate
purifications, is of high interest. The desired PanAms can be
easily prepared from the corresponding pantothenic acids by
transforming the carboxylic acid group to an amide.[
(>99% ee). This newly developed cascade process might be tailored
and used for the biocatalytic production of various vitamin B
5
derivatives by modifying the pantoyl or β-alanine moiety.
Coenzyme A (CoA) is an essential enzyme cofactor in all
organisms, and its biosynthetic pathway enzymes have been
identified as attractive targets for new antimicrobial drugs.[1,2] An
interesting class of new antimicrobials that target CoA
biosynthesis is the pantothenamides (PanAms), which are
5
secondary or tertiary amides of pantothenic acid (vitamin B , 5a,
Figure 1), the biosynthetic precursor of CoA. Various PanAms
have been shown to possess potent antimicrobial activity
11]
We envisioned that pantothenic acid (5a) and its α-methyl
substituted derivative (5b) could be prepared from fumaric acid
(
1a) and mesaconic acid (1b), respectively, via a modular three-
step enzymatic cascade involving 3-methylaspartate ammonia
lyase (MAL), an appropriate decarboxylase such as aspartate-α-
against several organisms, including the pathogenic bacterium
Staphylococcus aureus[3] as well as the malaria parasite
Plasmodium falciparum.[4] However, pantetheinase enzymes
decarboxylase
(ADC),
β-methylaspartate-α-decarboxylase
(CrpG) or glutamate decarboxylase (GAD), and pantothenate
synthetase (PS) (Scheme 1). The expected chemoselectivity of
each biocatalyst could allow for a one-pot reaction sequence
due to the orthogonal reactivity of each enzyme. In this process,
the new stereogenic center in product 5b can be established by
either regio- and diastereoselective amination (as catalyzed by
MAL), or diastereospecific decarboxylation by one of the
decarboxylase enzymes.
that normally hydrolyse pantetheine in human serum also act on
the PanAms, thereby reducing their efficacy.[5,6] Interestingly,
pantetheinase-mediated hydrolysis of PanAms could be
prevented by modifying the β-alanine moiety of the
compounds.[ Indeed, a PanAm with an added α-methyl group
was shown to have superior antiplasmodial activity compared to
7,8]
[
9]
its parent molecule. However, such modifications introduces
stereochemical complexity to the molecules that recent results
have indicated is highly relevant to the antimalarial activity of
[a]
M. Z. Abidin, Dr. T. Saravanan, J. Zhang, 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
[
b]
Prof. Dr. E. Strauss
Department of Biochemistry
Stellenbosch University
Private Bag X1, Matieland 7602 South Africa
5
Scheme 1. Proposed enzymatic cascade synthesis of vitamin B and its
Supporting information for this article is given via a link at the end of
the document.
derivatives.
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Chemistry - A European Journal
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COMMUNICATION
MAL of Clostridium tetanomorphum is part of a catabolic
pathway for L-glutamate, where it catalyzes the conversion of L-
threo-3-methylaspartate to ammonia and mesaconate.[12] Using
a large molar excess of ammonia, the enzyme also efficiently
catalyzes the amination of mesaconate (1b) to give both
(>99% after 24 h) and good isolated yield of product 3a (85%)
were achieved (Table 1, Figure S16).
Unfortunately, ADC showed no decarboxylase activity
towards either L-threo-2b or L-erythro-2b. Therefore, we cloned,
expressed and purified the decarboxylase CrpG, and incubated
it with a 1:1 mixture of L-threo-2b and L-erythro-2b. Under these
conditions the L-erythro isomer was fully decarboxylated,
whereas the L-threo isomer was not converted, not even after
prolonged incubation for 7 d (Figure S11). This indicates that
CrpG is highly diastereoselective, with detectable activity only
towards the L-erythro isomer.
(
2S,3S)-3-methylaspartate
(L-threo-2b)
and
(2S,3R)-3-
methylaspartate (L-erythro-2b) as products (Scheme 1). It was
found that L-threo-2b is formed at a rate much faster than L-
erythro-2b, but at equilibrium (using a 65-fold molar excess of
ammonia over 1b at pH 9) the molar ratio of these
[
13]
diastereoisomers is approximately 1. In addition, MAL accepts
fumarate (1a) as substrate, which is converted to L-aspartate
Table 1. Two step enzymatic cascade synthesis of β-alanine (3a) and both
enantiomers of 3-amino-2-methylpropanoic acid (3b)[
(
(
2a). The mechanism-inspired engineering of a MAL mutant
H194A) with strongly enhanced diastereoselectivity in the
a]
amination of 1b, giving exclusively L-threo-2b, has been
reported previously.[13] Moreover, the substrate scope of MAL
has been expanded by structure-guided site-saturation
mutagenesis, allowing for the biocatalytic production of a broad
range of valuable 3-substituted L-aspartic acid derivatives.[14]
ADC of Escherichia coli is part of the biosynthesis pathway
for pantothenate, where it catalyzes the decarboxylation of 2a to
give β-alanine (3a).[15] This enzyme has a limited substrate
scope and showed no decarboxylation activity towards 2b.[16]
The enzyme β-methylaspartate-α-decarboxylase (CrpG) of
Nostoc sp. ATCC53789 is involved in a biosynthetic pathway for
cryptophycin, where it catalyzes the decarboxylation of L-
erythro-2b to yield (R)-3-amino-2-methylpropanoic acid (3b).[17]
CrpG is the only enzyme known that can catalyze the
decarboxylation of 2b, with significant activity towards the L-
erythro isomer only. GAD of the hyperthermophilic archaeon
Thermococcus kodakarensis has been reported to function as
Conversion
Isolated
yield (%)
ee (%)[d]
Product
Enzymes
[b]
[c]
(%)
MAL and
ADC
3a
> 99
> 99
75
85
-
MAL and
CrpG
(R)-3b
78
63
> 99
>99
MAL-H194A
and GAD
(S)-3b
[
a] For synthesis of 3a, the reaction mixture contained MAL (0.02 mol%),
ADC (0.6 mol%), 1a (25 mM), NH Cl (500 mM), and MgCl (25 mM) in 25
mL Tris-HCl buffer (pH 8, 100 mM). For synthesis of (R)-3b, the reaction
mixture contained MAL (0.02 mol%), CrpG (0.47 mol%), 1b (30 mM), NH Cl
(25 mM) in 25 mL potassium phosphate buffer (pH 8,
00 mM). For the synthesis of (S)-3b, the reaction mixture contained MAL-
Cl (500 mM), PLP
4
2
4
(
1
500 mM), and MgCl
2
H194A (0.04 mol%), TkGAD (0.6 mol%), 1b (10 mM), NH
4
(1 mM), and MgCl
2
(25 mM) in 25 mL potassium phosphate buffer (pH 8,
00 mM); [b] Conversion was analysed by
Products were purified by cation exchange chromatography; [d]
Enantiomeric excess (ee) was analysed by chiral HPLC.
1
1
H NMR spectroscopy; [c]
an ADC and is most likely responsible for the production of β-
alanine necessary for pantothenate biosynthesis.[18] In this study,
we demonstrate that this enzyme exhibits decarboxylase activity
towards 2b, but with highest activity towards the L-threo isomer.
Note that ADC and CrpG have to undergo self-processing
leading to formation of the catalytic pyruvoyl group, whereas
GAD is a PLP-dependent decarboxylase that does not require
autocatalytic self-processing.
Having established the preference of CrpG for L-erythro-
2b, a two-step enzymatic cascade reaction was performed at
analytical scale by incubation of 1b and NH Cl with MAL and
4
CrpG in one pot. Interestingly, full conversion of starting
Pantothenate synthetase (PS) of E. coli is involved in the
last step of pantothenate biosynthesis and catalyzes the ATP-
dependent condensation of (R)-pantoate (4) and β-alanine (3a)
substrate 1b was observed (Figure S4), yielding solely the (R)-
1
enantiomer of product 3b, as confirmed by
H
NMR
spectroscopy and chiral HPLC analysis. This is explained by a
to form (R)-pantothenate (vitamin B
5
, 5a). PS enzymes typically
transformation[20,21]
dynamic kinetic asymmetric
of the
accept a variety of β-alanine analogues in the condensation
reaction, albeit with reduced catalytic efficiency compared to that
with the natural substrate.[15,19]
Initially, we set out to combine MAL and ADC in one-pot to
4
prepare product 3a. Accordingly, substrate 1a and NH Cl were
diastereoisomeric mixture of 2b (Scheme 2). In the first cascade
step, MAL produces both L-erythro- and L-threo-2b as
intermediate products. Subsequently, in the second step CrpG
only decarboxylates L-erythro-2b to give exclusively (R)-3b. The
remaining L-threo-2b is also converted into L-erythro-2b by MAL,
leading to the full conversion of the starting material (1b) into the
desired product (R)-3b.
incubated with MAL and ADC, and the reaction was monitored
by TLC (Figure S3). After 24 h, 1a was completely converted
1
into product 3a, as confirmed by H NMR spectroscopy. These
Several control experiments were also performed. First,
initial results showed that the two enzymes MAL and ADC are
compatible for cascade synthesis in one pot. To further
demonstrate the preparative usefulness of this two-step cascade
system, a 100 mg-scale synthesis was performed. Accordingly,
substrate 1a (25 mM) and NH Cl (500 mM) were incubated with
4
MAL (0.02 mol%) and ADC (0.6 mol%) in one pot (25 mL of
buffer, pH 8). Under these conditions, excellent conversion
4
when 1b and NH Cl were incubated with MAL only, a ~1:1
mixture of L-threo-2b and L-erythro-2b was obtained (Figure
S12A). After removal of MAL from the reaction mixture by heat
inactivation and filtration, CrpG was added. After prolonged
incubation (7 d), a mixture of unreacted L-threo-2b and product
3b was obtained (Figure S12B). Second, incubation of 1b and
4
NH Cl with the diastereospecific mutant of MAL (MAL-H194A)
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Chemistry - A European Journal
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COMMUNICATION
and CrpG in one pot resulted in the formation of L-threo-2b but
did not yield product 3b, consistent with the inability of CrpG to
decarboxylate L-threo-2b (Figure S9). These results confirm that
MAL is responsible for both the synthesis and epimerisation of
L-threo- and L-erythro-2b, and that CrpG displays activity
towards L-erythro-2b only, allowing for the selective synthesis of
selected the GAD from the hyperthermophilic archaeon
Thermococcus kodakarensis (TkGAD), which has 71%
sequence similarity with PfGAD (Figure S2). TkGAD reportedly
catalyzes the decarboxylation of L-glutamate and L-aspartate.[
The gene encoding TkGAD was cloned and expressed, and the
corresponding enzyme purified, yielding soluble and active
protein (Figure S1). Initially, TkGAD activity was tested towards
L-aspartate (2a) and L-erythro- and L-threo-2b (Figure S10).
The enzyme completely converted 2a to 3a, whereas the
reaction with L-erythro-2b showed <10% conversion. To our
delight, L-threo-2b was also accepted as substrate by TkGAD,
yielding the desired (S)-3b with more than 70% conversion.
Because TkGAD displays activity towards both
23]
(
R)-3b starting from the simple non-chiral dicarboxylic acid 1b.
diastereoisomers of 2b,
a
dynamic kinetic asymmetric
transformation approach in which MAL and TkGAD are
combined in one pot would not yield enantiopure (S)-3b product.
Hence, for the one-pot, two-step enzymatic cascade synthesis of
(
S)-3b, TkGAD was used in combination with the
diastereospecific MAL-H194A mutant, which produces
exclusively L-threo-2b upon amination of 1b (Scheme 3 and
Figure S5). Accordingly, substrate 1b (10 mM) and NH
mM) were incubated with MAL-H194A (0.02 mol%) and TkGAD
0.3 mol%) in one pot (25 mL of buffer, pH 8). Because some
4
Cl (500
(
protein precipitation occurred (due to instability of TkGAD), the
same amount of each enzyme was added again after 24 h of
incubation. Using these conditions, good conversion (75% after
Scheme 2. One pot two-step enzymatic cascade reaction involving MAL and
CrpG that fully converts mesaconate (1b) to only (R)-3b. This is due to CrpG
only acting on L-erythro-2b, and the MAL-mediated dynamic kinetic
asymmetric transformation of the L-threo-2b into the desired diastereomer.
48 h), good isolated yield (63%), and excellent enantiopurity of
product (S)-3b (>99% ee) were achieved (Table 1, Figures S18
and S25).
To demonstrate the synthetic usefulness of this two-step
enzymatic cascade, a 100 mg-scale synthesis was performed.
Accordingly, substrate 1b (30.8 mM) and NH Cl (500 mM) were
4
incubated with MAL (0.02 mol%) and CrpG (0.47 mol%) in one
pot (25 mL of buffer, pH 8). High conversion (>99% after 7 d),
good isolated yield (78%), and excellent enantiopurity of product
(
R)-3b (>99% ee) were achieved (Table 1, Figures S17 and
S25).
CrpG displays activity towards L-erythro-2b, enabling the
enzymatic synthesis of (R)-3b. In order to synthesize the
opposite enantiomer of 3b, a decarboxylase with activity towards
L-threo-2b was required. Our attempts to obtain CrpG variants
by directed evolution through screening of single-site saturation
mutagenesis libraries did not yield any mutants with detectable
activity towards L-threo-2b. We therefore cloned and produced
two PLP-dependent GAD enzymes and tested their ability to
decarboxylate L-threo-2b. Initially, we worked on the GAD from
Pyrococcus furiosus (PfGAD), which was reported to accept L-
aspartate, L-glutamate and L-tyrosine as substrates.[22] Although
many different expression conditions were tested, we were not
able to produce PfGAD in a soluble form in an Escherichia coli
host. In an attempt to produce soluble protein, different
constructs were made as fusions with three solubility enhancers:
maltose-binding protein (MBP), small ubiquitin-like modifier
protein (SUMO) and Fh8, a small protein secreted by the
parasite Fasciola hepatica. However, inefficient solubilization of
PfGAD limited the effectiveness of this approach. Therefore, we
Scheme 3. The one pot two-step enzymatic cascade reaction that converts
mesaconate (1b) to only (S)-3b relies on a diastereospecific mutant MAL
(
MAL-H194A) and the newly discovered stereoselectivity of TkGAD towards L-
threo-2b.
Having developed one-pot, two-step enzymatic cascade
reactions for the production of 3a, (R)-3b and(S)-3b, we next
verified whether the PS enzyme is able to accept these
compounds as substrates in condensation with (R)-pantoate (4)
using small-scale (1 mL) reactions. We were pleased to find that
PS accepted 3a, (R)-3b and (S)-3b as substrates in the
condensation reaction, yielding the corresponding pantothenic
1
acid products 5a and 5b, as confirmed by H NMR spectroscopy
(
Figure S13).
Having established that PS can be used to set up a three-
step enzymatic cascade reaction, the enzymes MAL, ADC, and
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Chemistry - A European Journal
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COMMUNICATION
Tris-HCl buffer (100 mM, pH 9). [b] conversion was analysed by 1H NMR
spectroscopy; [c] product was purified by preparative HPLC; [d] product
was purified by silica gel column chromatography; [e] de and ee were
PS were combined in one pot. Initially, the reaction was tested
by adding the three enzymes simultaneously and using an
equimolar ratio of 1a, 4 and ATP. Although full conversion of the
starting substrate 1a was observed after 24 h, the desired
product (R)-5a was obtained in a crude yield of only 50% along
with accumulation of 3a. This indicated that further optimization
of the reaction conditions was necessary to improve the yield of
product (R)-5a. After testing different ratios of 4 and ATP, a
molar ratio of 2:3 was found to be best, which resulted in
excellent conversion of the starting substrate into the desired
product 5a (crude yield >99%; Figures S6 and S14). Hence,
under these conditions, no significant accumulation of
intermediate product 3a was observed.
1
analysed by H NMR spectroscopy and HPLC analysis.
In conclusion, we have successfully developed a one-pot
cascade process for the synthesis of enantiomerically pure
vitamin B starting from fumarate and utilizing MAL, ADC, and
5
PS enzymes. Starting from mesaconic acid, the stereoselective
synthesis of both diastereoisomers of α-methyl-substituted
5
vitamin B , an important antibiotic precursor, was achieved by
using either the CrpG or GAD enzyme instead of ADC, with one
stereogenic center being set by the selected combination of
MAL/CrpG or MAL-H194A/GAD and the other derived from one
of the starting substrates. Given the availability of engineered
MAL mutants and natural PS enzymes with a broad substrate
scope, work is in progress to expand the substrate scope of
CrpG and GAD by protein engineering.
While the decarboxylation step actually is stereochemically
deconstructive, with the loss of one chiral centre, the cascade
approach strongly benefits from the use of stereo-divergent
decarboxylases. These enzymes not only allow the synthesis of
To assess the performance of this one-pot multi-enzymatic
cascade, a 32 mg-scale synthesis was performed. The three-
step cascade reaction was performed by addition of all
components simultaneously including the three enzymes. After
2
(
4 h, the starting material 1a was completely consumed
conversion >99%, Figure S15A) and the desired product (R)-5a
was obtained in good isolated yield (70%) and with excellent ee
>99%) (Table 2, Figures S19 and S20). Importantly, the
(
modularity of this enzymatic cascade approach also allows for
the facile synthesis of both diastereoisomers of α-methyl-
5
both diastereoisomers of α-methyl-substituted vitamin B , but
substituted vitamin B
5
, that is (2R,2'R)-5b and (2S,2'R)-5b
they also provide a strong driving force to pull the equilibrium of
the MAL-catalyzed reaction towards product formation. The use
of an irreversible decarboxylation step, with stereochemical
kinetic distinction, is an important strategy in biocatalytic
cascade synthesis to overcome thermodynamic limitations and
maximize product yield.[24,25]
A possible constraint on the use of the developed cascade
for large-scale transformations would be the dependence of the
PS enzyme on the expensive cofactor ATP. This could be
addressed by the incorporation of an auxiliary enzyme-catalyzed
step for efficient ATP recycling. Several ATP recycling enzyme
systems are available and a few have already been successfully
implemented in preparative biocatalysis.[26-28] However, as the
starting materials (fumarate and mesaconate) can be efficiently
(
Figures S7 and S8), with one stereogenic center being set by
the selected combination of enzymes and the other by the
substrate (R)-pantoate (4). Under suitable reaction conditions
(
for details, see section 8.2 in SI), and using the appropriate
combination of enzymes in one-pot, the desired products
2R,2'R)-5b and (2S,2'R)-5b were obtained with excellent de
(
and ee values (>99%) and in 46-49% isolated yield (Table 2,
Figures S15, S21-S24, and S26).
Table 2. Three-step enzymatic cascade synthesis of pantothenic acid (5a)
and both diastereoisomers of its α-methyl-substituted derivative 5b[a]
Conv
Isolated yield
(%)
de and ee
Product
Enzymes
(
%)[
b]
(%)
[e]
produced in high yields by large-scale fermentation using
metabolically engineered E. coli strains,[
29,30]
we envision to co-
MAL, ADC
and PS
express the enzymes employed for the cascade in such a
(
R)-5a
> 99
70[c]
> 99
fermentation host in order to directly obtain vitamin B and its α-
5
methyl-substituted derivatives from cheap carbon and nitrogen
sources. Indeed, such a cell-based approach would eliminate
the need for ATP recycling.
MAL, CrpG
and PS
(
2R,2'R)-5b
> 99
75
49[c]
46[d]
> 99
>99
MAL-H194A,
GAD and PS
(
2S,2'R)-5b
Acknowledgements
[
9
a] The enzymes were found to be compatible for cascade synthesis at pH
(MAL, MAL-H194A and PS, optimum pH: 9.0-10.0; ADC, CrpG and
The authors thank Sabry Younes, Hans Raj, Harshwardhan
Poddar and Marianne de Villiers for insightful discussions.
Robbert Cool is acknowledged for his expert assistance in
construction and purification of the fusion proteins. We
acknowledge financial support from the Netherlands
Organization of Scientific Research (VICI grant 724.016.002)
and from the European Research Council (PoC grant 713483).
Mohammad Z. Abidin acknowledges funding from the Indonesia
Endowment Fund for Education (LPDP) and Jielin Zhang
acknowledges funding from the China Scholarship Council.
GAD, optimum pH: 7.5-8.0). The amounts of applied enzymes were
adjusted such that high conversions were achieved. For synthesis of 5a,
the reaction mixture contained MAL (0.01 mol%), ADC (0.3 mol%), PS
(
0.07 mol%), 1a (10 mM), 4 (20 mM), ATP (30 mM), NH
MgCl (10 mM) in 20 mL Tris-HCl buffer (100 mM, pH 9). For synthesis of
2R,2'R)-5b, the reaction mixture contained MAL (0.01 mol%), CrpG (0.7
mol%), PS (0.07 mol%), 1b (10 mM), 4 (20 mM), ATP (30 mM), NH Cl
500 mM) and MgCl (10 mM) in 20 mL Tris-HCl buffer (100 mM, pH 9).
For synthesis of (2S,2'R)-5b, the reaction mixture contained MAL-H194A
0.04 mol%), TkGAD (0.6 mol%), PS (0.07 mol%), 1b (10 mM), 4 (20 mM),
ATP (30 mM), PLP (1 mM), NH Cl (500 mM) and MgCl (10 mM) in 20 mL
4
Cl (500 mM) and
2
(
4
(
2
(
4
2
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Chemistry - A European Journal
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COMMUNICATION
Keywords: biocatalysis, enzyme cascade, pantothenic acid
Appl. Biol. Chem. 2012, 55, 213–218.
[
24]
J. Steinreiber, M. Schürmann, M. Wolberg, F. van Assema, C.
Reisinger, K. Fesko, D. Mink, H. Griengl, Angew. Chem. Int. Ed.
2007, 46, 1624-1626.
[
[
[
1]
2]
3]
C. Spry, K. Kirk, K. J. Saliba, FEMS Microbiol. Rev. 2008, 32, 56-
06.
W. J. A. Moolman, M. de Villiers, E. Strauss, Biochem. Soc. Trans.
014, 42, 1080-1086.
1
[25]
E. Ricca, B. Brucher, J. H. Schrittwieser, Adv. Synth. Catal. 2011,
353, 2239 – 2262.
2
M. de Villiers, L. Barnard, L. Koekemoer, J. L. Snoep, E. Strauss,
FEBS J. 2014, 281, 4731–4753.
[26]
[27]
J. N. Andexer, M. Richter, ChemBioChem. 2015, 16, 380 – 386.
H. Zhao, W. A. van der Donk, Curr. Opin. Biotechnol. 2003, 14,
583–589.
[
[
4]
5]
K. J. Saliba, C. Spry, Biochem. Soc. Trans. 2014, 42, 1087-1093.
C. Spry, C. Macuamule, Z. Lin, K. G. Virga, R. E. Lee, E. Strauss, K.
J. Saliba, PLoS One 2013, 8, e54974.
[28]
[29]
[30]
A. Alissandratos, K. Caron, T. D. Loan, J. E. Hennessy, C. J. Easton,
ACS Chem. Biol. 2016, 11, 3289-3293.
[
[
[
[
6]
7]
8]
9]
S. Duprè, M. A. Rosei, L. Bellussi, E. Del Grosso, D. Cavallini, Eur.
J. Biochem. 1973, 40, 103–107.
C. W. Song, S. Y. Lee, Appl. Microbiol. Biotechnol. 2015, 99, 8455–
8464.
M. de Villiers, C. Macuamule, C. Spry, Y.-M. Hyun, E. Strauss, K. J.
Saliba, ACS Med. Chem. Lett. 2013, 4, 784–789.
J. Wang, K. Zhang, Metab. Eng. 2015, 30, 190–196.
L. Barnard, K. J. Mostert, W. A. L. van Otterlo, E. Strauss, ACS
Infect. Dis. 2018, 4, 736–743.
C. J. Macuamule, E. T. Tjhin, C. E. Jana, L. Barnard, L. Koekemoer,
M. de Villiers, K. J. Saliba, E. Strauss, Antimicrob. Agents
Chemother. 2015, 59, 3666–3668.
[
10]
J. Schalkwijk, E. L. Allman, P. A. M. Jansen, L. E. de Vries, S.
Jackowski, P. N. M. Botman, C. A. Beuckens-Schortinghuis, K. M.
Koolen, J. M. Bolscher, M. W. Vos, K. Miller, S. Reeves, H. Pett, G.
Trevitt, S. Wittlin, C. Scheurer, S. Sax, C. Fischli, G. Josling, T. W. A.
Kooij, R. Bonnert, B. Campo, R. H. Blaauw, F. P. J. T. Rutjes, R.
Sauerwein, M. Llinas, P. H. H. Hermkens, K. J. Dechering, bioRxiv
2018, doi: http://dx.doi.org/10.1101/256669.
[
[
[
11]
12]
13]
J. L. Meier, A. C. Mercer, H. Rivera, M. D. Burkart, J. Am. Chem.
Soc. 2006, 128, 12174–12184.
H. A. Barker, R. D. Smyth, R. M. Wilson, H. Weissbach, J. Biol.
Chem. 1959, 234, 320–328.
H. Raj, W. Szymanski, J. De Villiers, V. Puthan Veetil, W. J. Quax, K.
Shimamoto, D. B. Janssen, B. L. Feringa, G. J. Poelarends, Chem.
Eur. J. 2013, 19, 11148–11152.
[
14]
H. Raj, W. Szymański, J. De Villiers, H. J. Rozeboom, V. Puthan
Veetil, C. R. Reis, M. De Villiers, F. J. Dekker, S. De Wildeman, W.
J. Quax, A. M. W. H. Thunnissen, B. L. Feringa, D. B. Janssen, G. J.
Poelarends, Nat. Chem. 2012, 4, 478–484.
[
[
[
[
[
[
[
[
[
15]
16]
17]
18]
19]
20]
21]
22]
23]
M. E. Webb, A. G. Smith, C. Abell, Nat. Prod. Rep. 2004, 21, 695–
7
21.
M. E. Webb, E. Stephens, A. G. Smith, C. Abell, Chem. Commun.
003, 2416–2417.
Z. Q. Beck, D. A. Burr, D. H. Sherman, Chembiochem 2007, 8,
373–1375.
2
1
H. Tomita, Y. Yokooji, T. Ishibashi, T. Imanaka, H. Atomia, J.
Bacteriol. 2014, 196, 1222–1230.
K. Miyatake, Y. Nakano, S. Kitaoka, J. Nutr. Sci. Vitaminol. 1978, 24,
2
43–253.
J. Steinreiber, K. Faber, H. Griengl, Chem. Eur. J. 2008, 14, 8060-
072.
8
B. M. Trost, R. C. Bunt, R. C. Lemoine, T. L. Calkins, J. Am. Chem.
Soc. 2000, 122, 5968-5976.
H. W. Kim, Y. Kashima, K. Ishikawa, N. Yamano, Biosci. Biotechnol.
Biochem. 2009, 73, 224–227.
S. J. Hong, I. Ullah, G. S. Park, C. Lee, J. H. Shin, J. Korean Soc.
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Chemistry - A European Journal
10.1002/chem.201804151
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
M. Z. Abidin,T. Saravanan, J. Zhang, P.
G. Tepper, E. Strauss, G. J. Poelarends*
Page No. – Page No.
Modular enzymatic cascade
synthesis of vitamin B
derivatives
5
and its
Enzymes show "Unity In Diversity": Starting from simple non-chiral dicarboxylic
acids enantiopure vitamin
B
5
derivatives, which are valuable antimicrobial
precursors, were prepared using a modular reaction cascade involving three
different enzymes in one-pot.
This article is protected by copyright. All rights reserved.