Improving and Inverting Cβ-Stereoselectivity of Threonine Aldolase via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening

Article abstract of DOI:10.1021/acscatal.9b00859

Threonine aldolase (TA)-catalyzed aldol condensation is a powerful tool for C-C bond formation under mild conditions, but the low C_β-stereoselectivity has hampered its wide application. A stepwise visual screening method was developed to measure the activity and stereoselectivity of threonine aldolase-catalyzed aldol condensation by employing a stereoselective phenylserine dehydratase, enabling direct selection of mutants with higher C_β-stereoselectivity. Mutants of l-PsTA from Pseudomonas sp. with improved or inverted stereoselectivity toward aromatic aldehydes were obtained by simultaneously mutating amino acid residues which interact with the amino and hydroxyl groups of the substrate and screening the resulting mutant libraries with this method. The mutation and enzyme-substrate docking studies provided some insights into the regulation of the C_β-stereoselectivity by the enzyme-substrate interactions. This study offers a tool and useful guidance for further engineering of TAs to address the C_β-stereoselectivity problem.

Full text of DOI:10.1021/acscatal.9b00859

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Article
Improving and Inverting C#-stereoselectivity of Threonine Aldolase via
Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening
Qijia Chen, Xi Chen, Jinhui Feng, Qiaqing Wu, Dunming Zhu, and Yanhe Ma
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b00859 • Publication Date (Web): 09 Apr 2019
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Improving and Inverting C -stereoselectivity of Threonine
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Aldolase via Substrate-Binding-Guided Mutagenesis and a
Stepwise Visual Screening
Qijia Chen, 1,2 Xi Chen, 1,2 Jinhui Feng, Qiaqing Wu, Dunming Zhu1,2,*, Yanhe Ma1,2
1,2
1,2
1
University of Chinese Academy of Sciences, No.19(A) Yuquan Road, Shijingshan District,
Beijing 100049, China
2
National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Research
Center of Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308,
China
*
Email: zhu_dm@tib.cas.cn
ABSTRACT: Threonine aldolase-catalyzed aldol condensation provides one of the powerful
tools for C-C bond formation under mild conditions, but the low C -stereoselectivity has

hampered its wide application. A stepwise visible screening method was developed to measure
the activity and stereoselectivity of threonine aldolase-catalyzed aldol condensation by
employing a stereoselective phenylserine dehydratase, enabling direct selection of mutants with
higher C -stereoselectivity. Mutants of L-PsTA from Pseudomonas sp. with improved or inverted

stereoselectivity towards aromatic aldehydes were obtained by simultaneously mutating the
amino acid residues which interact with the amino and hydroxyl groups of the substrate and
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screening the resulting mutant libraries with this method. The mutation and enzyme-substrate
docking studies provided some insights into the regulation of the C -stereoselectivity by the
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enzyme-substrate interactions. This study offers a tool and useful guidance for further
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engineering of TAs to address the C -stereoselectivity problem.
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KEYWORDS: Threonine aldolase, High-throughput screening, Stereoselectivity, Enzyme
Engineering, C-C bond formation
INTRODUCTION
Threonine aldolases (TAs), a class of pyridoxal 5'-phosphate (PLP)-dependent enzymes,
catalyze the aldol condensation of aldehydes with glycine, providing one of the useful tools to
construct two stereogenic centers in a single reaction under mild conditions.1-2 The resulting
products -hydroxy-α-amino acids are valuable precursors for the active ingredients of
pharmaceuticals and agrochemicals such as thiamphenicol, L-3,4-dihydroxyphenylserine.3-4
Several TAs from different origins have been isolated and characterized.4-11 L-TAs catalyze the
aldol addition of glycine to aldehydes to give both L-threo and L-erythro products, while a
mixture of D-threo/D-erythro products are obtained for D-TAs (Scheme 1).12,13 They are
generally stereoselective for the α-carbon (>99% ee), but suffer from moderate to poor
stereoselectivity at the β-carbon.12 The insufficient C -stereoselectivity has limited the
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application of TAs in organic synthesis.14
OH
α COOH
OH
α COOH
O
O
Aldol condensation
Retro-aldol
H N
2
or
R β
R β
OH
R
NH2
NH2
L-threo/erythro by L-TAs D-threo/erythro by D-TAs
Scheme 1. Aldol and retro-aldol reaction catalyzed by L- or D-TAs
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Although enzyme engineering is a powerful strategy for generating mutants with excellent
properties,15,16 it has been rarely applied to improve the C -stereoselectivity of TAs with limited
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success.6,14 This is largely due to the lack of suitable screening method for determining the
enzyme activity and the C -stereoselectivity at same time, and the variation of C -
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stereoselectivity during the reaction process of kinetic or thermodynamic control. Given the
importance of TAs in potential industrial applications, several efforts have been made to increase
the enzyme stability by protein engineering or enzyme immobilization. In these studies, the
screening methods were based on detecting the released aldehyde in the retro-aldol reaction of -
hydroxy-α-amino acids 6,17-19 or the consumption of toxic aldehyde in the aldol reaction, and the
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mutants with higher activity or stability could be selected. Following the First Law of Directed
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Evolution-‘you get what you screen for’, the direct measurement of the stereoselectivity in the
TA-catalyzed formation of -hydroxy-α-amino acids would be very important. There is no high-
throughput screening (HTS) method for the selection of TA mutants based on the
stereoselectivity, presenting the experimental bottleneck in the engineering of this enzyme. At
the initial stage of TA-catalyzed aldol condensation, the reaction is under kinetic control and a
higher C -stereoselectivity can be obtained. However, as the reaction proceeds, it often leads to a
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thermodynamically controlled low ratio of product diastereomers. This variation presents another
great challenge to identify desired C -stereoselective TAs.
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In this context, we have developed a stepwise visual screening (SVS) method that incorporates
a stereoselective phenylserine dehydratase (PSDH) for determining both the conversion and the
de value of the product. By employing this SVS method, mutant enzymes with improved or
inverted C -stereoselectivity and enhanced activity towards aromatic aldehyde were obtained
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from an L-TA of Pseudomonas sp.
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RESULTS AND DISCUSSION
In our search for potential catalysts for asymmetric synthesis of -hydroxy-α-amino acids,
twenty L-TAs from different origins were cloned, out of which eighteen L-TAs were expressed as
soluble proteins (Table S1 and Figure S1). Whole cell biotransformations were performed using
benzaldehyde and glycine, but all the L-TAs showed poor stereoselectivity at β-carbon (14-46%
de).
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A suitable HTS method greatly contributes to acquisition of the mutants with high activity and
stereoselectivity against particular substrates.22,23 We attempted to develop a HTS method, that
could measure the activity and stereoselectivity of TAs, by employing a stereoselective enzyme
involved in the transformations of -hydroxy-α-amino acid. According to the analysis of
functional groups, -hydroxy-α-amino acid can undergo dehydrogenation, dehydration and
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decarboxylation (Scheme S1). Phenylserine dehydrogenase and decarboxylase
are deemed
not suitable for this purpose because they are not able to differentiate the chirality at the -carbon
of -hydroxy-α-amino acids. The PLP-dependent phenylserine dehydratase catalyzes the
dehydration reaction of phenylserine to form phenylpyruvate. This enzyme is stereoselective at
β-carbon, and only D- or L-threo isomer can be dehydrated.27,28 Therefore, phenylserine
dehydratase might be coupled with aldol condensation reaction to develop a HTS method for
screening TA mutants based on the C -stereoselectivity.
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The bacterial strains available in our laboratory were screened for the dehydratase activity and
the strain Paraburkholderia xenovorans (DSM 17367) was found to have dehydration activity
towards DL-threo-phenylserine. Based on the P. xenovorans genome information, the
dehydratase gene (GenBank: AIP32383) was amplified and expressed (Figure S2). The
stereoselectivity measurement of the recombinant dehydratase PxPSDH showed that only L-
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threo-phenylserine was completely dehydrated (Figure S3), indicating that it is a promising
enzyme for our purpose. Substituted phenylserine derivatives were produced in high conversion
(50%) and ee (>99%) for substrates with ortho- and para-substituents (o-F, Cl, Br, p-F, Cl, Br),
although meta-substituted phenylserines (m-F, Cl, Br) were obtained with modest ee (Table S2).
As such, L-PxPSDH possessed a relatively wide substrate scope and was selected as a tool
enzyme for developing the screening method.
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L-TA from Pseudomonas sp. (L-PsTA) was chosen to demonstrate the reliability of the HTS
method because it had a wide range of substrate specificity towards aromatic aldehydes, and
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would be a good template for the further engineering. The aldol condensation reaction of
benzaldehyde (10 mM) and glycine (100 mM) was carried out with purified L-PsTA. After the
reaction was terminated and the reaction mixture was extracted with dichloromethane, the
dehydration reaction was performed by adding L-PxPSDH into the aqueous solution. The
unreacted benzaldehyde and the generated phenylpyruvate were detected by the 2,4-
dinitrophenylhydrazine (DNPH) method after 40 times dilution to quantitatively measure the
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carbonyl group of the molecules (Figure S4). HPLC analysis was also carried out to measure
the amounts of the L-β-phenylserine. The results measured by colorimetric assay and HPLC
analysis indicated that they followed a similar trend. Accordingly, a stepwise screening method
was established for determining conversion and product de value of TA-catalyzed aldol reaction
by coupling with the dehydration (Scheme 2), where the consumption of aldehyde and the
production of phenylpyruvate could be measured in terms of the yield of L-threo/erythro-
phenylserine and L-threo-phenylserine, respectively. The screening assay of D-TAs can be
established in the same way by using the reported D-PSDH which specifically catalyzes
dehydration of D-threo-phenylserine.27
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OH
O
OH O
L-TA
OH
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NH2
NH2
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P
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H
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NH2
Scheme 2. Principles of screening assay coupled with stereoselective PSDH
Restricted by the interference between aldehyde carbonyl and ketone carbonyl and the
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detection range of DNPH method, purpald and Fe were investigated as alternative reagents for
the determination of benzaldehyde and phenylpyruvate, respectively.30,31 As phenylpyruvate can
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form a coplanar enol structure, it can react with Fe to form a complex with cyan color (Figure
1A). Multi-wavelengths scanning showed that 540 and 640 nm were suitable detection
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wavelength for the purpald and Fe colorimetric assays, respectively (Figure 1B). Furthermore,
the target detection compounds could be measured without the dilution and extraction operations
as the detection ranges were up to 10 mM (Figure 1C-D). The aldol addition reactions were
carried out by whole cell biotransformation of eighteen newly cloned L-TAs and the results of
conversion and de measured by this method followed a similar trend as those obtained by HPLC
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analysis (Figure 1E). Because the purpald and Fe complexes display distinct colors (Figure 1D),
this stepwise visible screening (SVS) method offers a rapid HTS, in which the entire processing-
time of one 96 deep-well plate is no more than 5.5 h compared to 30 h by HPLC (Figure S5). It
should be noted that by choosing suitable stereoselective dehydratases of diverse substrate
profiles, this screening method could be useful in the screening of TAs toward a wide range of
target substrates.
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Figure 1. (A) Reactions of colorimetric assays for benzaldehyde and phenylpyruvic acid. (B)
Multi-wavelength scanning. (C) Calibration curves. (D) Photos of colorimetric assays at different
concentrations. (E) Conversion and de values of whole cell biotransformation with newly cloned
TAs measured by HPLC and SVS assay.
This screening method was then applied in the mutation of L-PsTA in an effort to obtain the
mutant enzymes with improved C -stereoselectivity. A structural model of L-PsTA was
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into the homology model using Discovery Studio 4.1 (Accelrys). Initially, the site-saturation
mutagenesis of the amino acid residues lining the substrate-binding pocket (Figure 2A) was
performed and the resulting mutant libraries were screened by this SVS method. The aldol
condensation reactions were carried out for 12 h to ensure that the reactions were under
thermodynamic control.32,33 Unfortunately, no mutant with improved activity and
stereoselectivity was obtained. Our results are consistent with the previous kinetic and
crystallographic studies of E. coli L-TA, which suggest that the substrate preference between the
threo- and erythro- isomers of L-TAs may be determined by the overall microenvironment that
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surrounds the substrate bound at the enzyme active site rather than by one specific residue. We
thus assumed that single amino residue might not exert meaningful effect on the activity and
stereoselectivity. As such, to examine the cooperative effects of the residues, the adjacent
residues (S10/N12, A34/Y35, H89/D93, I132/H133, T146/E147, A176/R177, T206/K207,
R321/M323) were grouped together35-37 and the combinatorial active-site saturation libraries
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were constructed. The following screening also resulted in no mutant with drastically improved
catalytic activity and stereoselectivity.
Figure 2. Target amino acid residues chosen for mutagenesis marked in the homology mode of
L-PsTA. (A) The initial selected ten amino acid residues (S10, N12, Y35, H89, D93, H133, E147,
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R177, K207 and R321) are displayed in lemon. The extended eighteen sites which showed in
cartoon are displayed in red. (B) Amino acid residues selection based on interaction with
different functional groups of substrate (black dashed lines).
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As shown in Figure 2B, the carboxylate and hydroxyl group of the substrate interacts with
R177 and R321 or D93, respectively. E147 was close to the amino group of substrate without
direct interaction with each other due to the steric hindrance. After the above unsuccessful
attempts, that usually work well for other enzymes,37,39 we envisioned that in order to improve
the stereoselectivity of TAs, it might be necessary to tune the interactions of the amino acid
residues with the different functional groups of the substrate by simultaneously mutating the
amino acid residues which interact with different functional groups of the substrate. To prove
this hypothesis, three saturation mutagenesis libraries (D93/E147, E147/R321 and R177/R321)
were created and screened by the SVS method using benzaldehyde (1) and o-fluorobenzaldehyde
(2) as the aldehyde substrate. Positive mutants from D93/E147 mutant library exhibited
improvement or reversal of C -stereoselectivity. The wild-type enzyme catalyzed the aldol

addition of 2 and glycine to afford the product with 31% de, whereas D93F/E147D,
D93H/E147D and D93N/E147D catalyzed the reaction with de of 68%, 69% and -40%,
respectively (Table 1). D93S/E147D with lower de was also identified as the variant had higher
activity. It has been reported that E90 and D126 of L-TA from A. jandaei, which were located
close to H85 and H128, respectively, were observed to play important roles in aiding these two
histidine residues during the recognition of substrate.40 In our case, D93 is close to H89
(corresponding to H85), while E147 is not near H133 (corresponding to H128) as shown in the
Figure S6. The mutation of D147 may not directly aid H133 but affect the overall
microenvironment during the recognition of substrate.
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4
4
4
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5
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Table 1. Synthesis of -hydroxy-α-amino acids employing L-PsTA and its mutantsa
O
OH
OH
CO H
2
CO H
CO H
2
2
R
R
R
NH2
NH2
L-threo
3 R=m-F
7 R=o-Br
NH2
L-erythro
0
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9
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1
5
R=H
R=o-Cl
2 R=o-F
6 R=m-Cl
4 R=p-F
8 R=m-Br
9 R=o-NO2
10 R=m-NO2 11 R=p-NO2
Substrate
WT
D93F/E147D
42, 12 (erythro)
80, 68 (threo)
54, 20 (threo)
84, 18 (threo)
34, 39 (threo)
73, 25 (threo)
46, 64 (threo)
64, 9 (erythro)
60, 36 (threo)
62, 40 (threo)
45, 47 (threo)
D93H/E147D
62, 30 (threo)
84, 69 (threo)
43, 40 (threo)
84, 26 (threo)
91, 71 (threo)
91, 26 (threo)
81, 68 (threo)
66, 9 (threo)
62, 45 (threo)
63, 38 (threo)
29, 55 (threo)
D93N/E147D
36, 74 (erythro)
40, 40 (erythro)
65, 59 (erythro)
61, 43 (erythro)
43, 59 (erythro)
33, 49 (erythro)
38, 49 (erythro)
30, 44 (erythro)
13, 24 (erythro)
41, 38 (threo)
b
c
1
2
55 , 19 (threo)
55, 31 (threo)
47, 36 (threo)
54, 36 (threo)
75, 48 (threo)
64, 26 (threo)
62, 33 (threo)
54, 59 (threo)
50, 33 (threo)
43, 41 (threo)
42, 28 (threo)
3
4
5
6
7
8
9
10
1
1
46, 37 (threo)
aThe reactions were carried out as follows: aldehyde (100 mM), glycine (1 M), PLP (10 μM),
1
0% DMSO (v/v) and purified mutants (1 mg/mL) were mixed in Tris-HCl buffer (100 mM, pH
b
c
7.5). The reactions were performed at 25°C for 12h. Yield in percent. de value in percent.
A series of aromatic aldehyde substrates was investigated with purified wild-type enzyme or
mutants (Figure S7). High stereoselectivity at -carbon for threo enantiomer products was
retained with D93H/E147D, especially up to nearly 70% de towards benzaldehydes with an
ortho-halogen substitution on the phenyl ring (Table 1 and Figures S8-12). Surprisingly,
D93N/E147D dramatically inverted the C -stereoselectivity in preference for the erythro

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enantiomer product, especially for benzaldehyde or benzaldehyde with 3′-fluoro- and 2′-chloro-
substituent (Table 1 and Figures S8-12). Benzaldehyde with nitro substitution on the phenyl ring
had little effect on C -stereoselectivity. It is interesting that the difference of a single amino acid

1
1
1
1
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1
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at 93 results in such reversal of C -stereoselectivity for a TA. Although this has been observed

for other enzymes,41-43 this is the first example for TAs. In addition, this study also represents the
rare example of improvement of C -stereoselectivity catalyzed by an engineered L-TA at high

conversion reported so far.
Although all the obtained mutants share one single point mutation E147D, the mutant E147D
itself has lower activity. That is why this variant was not screened by SVS assay of the site-
saturation mutant library. As Asp93 had remarkable effect on the activity and stereoselectivity,
Asp93 was substituted with other amino acids on the basis of E147D, but these mutants were not
better than the obtained mutants. These results also demonstrate the viability of the SVS method.
In order to gain the molecular insights for the alteration of stereoselectivity, the structural
models of mutant D93H/E147D and D93N/E147D were constructed based on the known crystal
structure (PDB: 1V72), and L-β-phenylserine and L-β-o-fluorophenylserine were docked into the
substrate-binding site of wild-type and mutant enzymes. The previous studies have shown that
the water molecules interacted with the phosphate of PLP and a water molecule interact with the
hydroxyl group of threonine and the phosphate of PLP in the crystal structure of E. coli L-TA
complex with L-threonine.34 Since L-β-phenylserine is bigger and more hydrophobic than L-
threonine, water molecules were omitted in our docking studies. The lowest-energy docking
conformations of enzyme-substrate complexes were selected by using the score functions of
flexible docking program. In these conformations, H89 forms π-π stacking interaction with PLP-
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cofactor and the distances between the amino group of substrate and aldehyde group of cofactor
PLP are in the range (2.4-4.2 Å) for the formation of the imine intermediate.
In previous studies, two conserved histidine residues, H83 and H126, or H85 and H128 were
shown to interact with the hydroxyl group of L-threonine or L-allo-threonine.34,40 The interaction
of the two histidine residues with the hydroxyl group of the substrate was proposed to activate
the hydroxyl group and facilitate the reaction. Although the sizes of aromatic compounds are
quite different from the threonine, we thought that the proposed reaction mechanism might apply
in our case, and H89 and H133 played similar roles in the catalytic reaction. The docking studies
confirmed that the substrate bindings of L-threo-phenylserine, L-threonine and L-allo-threonine
in the catalytic center of wild-type L-PsTA were similar (Figure S13). Therefore, we compared
the distance between imidazole of H133 or H89 and hydroxyl of substrate in lowest-energy
docking conformations of enzyme-substrate complexes with the experimentally observed activity.
The similar distances between the hydroxyl of threo- or erythro- isomers and H133 indicate that
the stereoselective control of the wild-type enzyme at -carbon may be poor (Figure 3A-B and
Figure S14). This is in agreement with the experimental data. In variant D93H/E147D, the amino
acid residues H93 and D147 interact with hydroxyl and amino group of the substrate,
respectively, resulting in the π-π stacking interaction between H133 and substrate benzene ring
0
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(Figure 3C-D). For L-threo-phenylserine, the distance between imidazole of H133 and hydroxyl
of substrate is shortened to 4.6 Å compared to 5.5 Å in wild-type enzyme (Figure 3C). The
corresponding distance is further shortened to 3.6 Å for L-threo-o-fluorophenylserine as the
hydrogen bond is formed between fluorine atom at 2-position of phenyl ring and hydrogen atom
on imidazole of His89 (Figure 3D). As such, D93H/E147D exhibits higher C -stereoselectivity

than the wild-type enzyme, especially for the substrate 2.
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It was particularly worthy noting that D93N/E147D showed a greatly increased preference for
erythro isomers. The hydroxyl and amino group of erythro isomer of substrate 1 are directed to
the amide side chain of N93, and the phenyl group forms a CH-π interaction with the benzene
ring of Y312 (Figure 3E). As such, the distance between hydroxyl group of L-erythro-
phenylserine and imidazole of H133 is shortened from 7.3 Å (wild-type enzyme, Figure S14) to
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
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3
.9 Å (Figure 3E). This is consistent with the high de (74%) for erythro isomer. For L-erythro-o-
fluorophenylserine, the ortho-fluoro group forms a hydrogen bond with imidazole of H89,
weakening the CH-π interaction between the phenyl group of the substrate and the benzene ring
of Y312. The distance between the hydroxyl group of substrate and the imidazole of H133 is
thus increased to 5.1 Å (Figure 3F), leading to a lower de than the substrate 1. The docking
results also show the shortened corresponding distances between the imidazole of H89 and the
hydroxyl of substrate for the mutants D93H/E147D and D93N/E147D.
After being mutated to Asp, D147 shows interaction with the -amino group of L-β-
phenylserine or L-β-fluorophenylserine in either mutants, the distance between hydroxyl group of
D147 and -amino group of substrate are in the range (2.3-2.8 Å) compared to 6.6 Å in the wild-
type enzyme. Therefore, mutations at D93 and E147 affect the interactions of the substrate with
the amino acid residues in the catalytic site, thus resulting in the change of the distances between
the hydroxyl group of substrate and the imidazoles of H133 and H89, which may play an
important role in regulating the stereoselectivity at -carbon by tuning the deprotonation of the
hydroxyl group in the retro-aldol reaction or the protonation of the aldehyde group in the aldol
addition.
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Figure 3. Flexible docking into the active sites of wild-type and mutant enzymes. (A) and (B)
Catalytically active conformations of wild-type enzyme complexed with L-threo-phenylserine
and L-threo-o-fluorophenylserine, respectively. (C) and (D) Catalytically active conformations of
mutant D93H/E147D complexed with L-threo-phenylserine (cyan) and L-threo-o-
fluorophenylserine (green), respectively. (E) and (F) Catalytically active conformations of
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mutant D93N/E147D complexed with L-erythro-phenylserine (yellow) and L-erythro-o-
fluorophenylserine (salmon), respectively. PLP is shown as green in lines.
CONCLUSION
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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9
0
In summary, a SVS method was developed for measuring the conversion and stereoselectivity
of TA-catalyzed aldol condensation reaction by detecting the consumption of aldehyde and the
generation of keto acid from one stereoisomer of the aldol condensation product. The optimized
SVS method provide a simple and rapid strategy suitable for HTS in the discovery of TAs with
desired synthetic activity and stereoselectivity. By using this method, mutants of L-PsTA with
improved or inverted stereoselectivity towards aromatic aldehydes were obtained. The
commonly used site-saturation mutagenesis of the amino acid residues lining the substrate-
binding site and neighbouring residue grouping failed to generate benefit mutant, suggesting that
the stereoselective control mechanism of TAs is unique from other enzymes and the interactions
of the substrate with enzyme should be manipulated as a whole to achieve the desired
stereoselectivity. By simultaneously mutating the amino acid residues which interact with the
amino and hydroxyl groups of the substrate, we successfully obtained mutants with improved or
inverted C -stereoselectivity towards aromatic aldehydes. The molecular modelling studies

suggested that the mutations tuned the distances between hydroxyl group of the substrate and
imidazole groups of H133 and H89 by altering the interactions of the substrate with the amino
acid residues in the catalytic site, thus resulting in the changes of the C -stereoselectivity. This

study not only provides a new SVS method for identifying beneficial stereoselective mutant TAs,
but also acquires mutant enzymes with improved or inverted C -stereoselectivity, offering some

hints for the direction of further enzyme engineering to address the challenge of stereochemical
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control at the -carbon in the TA-catalyzed aldol condensation reaction. To fully understand the
molecular basis for the substrate-binding and stereoselectivity control mechanisms, and
ultimately address the C -stereoselectivity problem, more studies such as the crystal structure

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determination of wild-type L-PsTA and mutant enzymes are needed, and this is being pursued in
our laboratory.
ASSOCIATED CONTENT
Supporting Information
The experimental details, source of L-threonine aldolases, primers used for mutagenesis,
purification of enzymes and mutants, docking analysis, and the HPLC spectra of the aldol
addition reaction. This material is available free of charge via the Internet at http://pubs.acs.org.
Corresponding Author
*
Corresponding authors: Dunming Zhu
Telephone: 86-22-84861962
Fax: 86-22-84861996
E-mail: zhu_dm@tib.cas.cn
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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We thank Prof. Peter C. K. Lau for helpful discussions and constructive suggestions. This
work was financially supported by the National Natural Science Foundation of China
(No.21602246), the STS Program of Chinese Academy of Sciences (KFJ-SW-STS-164) and
Tianjin Municipal Science and Technology Commission (15PTGCCX00060 and
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5PTCYSY00020).
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