Structure-guided engineering of: Meso -diaminopimelate dehydrogenase for enantioselective reductive amination of sterically bulky 2-keto acids

Article abstract of DOI:10.1039/c8cy01426d

meso-Diaminopimelate dehydrogenase (DAPDH) and mutant enzymes are an excellent choice of biocatalysts for the conversion of 2-keto acids to the corresponding d-amino acids. However, their application in the enantioselective reductive amination of bulky 2-keto acids, such as phenylglyoxylic acid, 2-oxo-4-phenylbutyric acid, and indole-3-pyruvic acid, is still challenging. In this study, the structure-guided site-saturation mutagenesis of a Symbiobacterium thermophilum DAPDH (StDAPDH) gave rise to a double-site mutant W121L/H227I, which showed dramatically improved enzyme activities towards various 2-keto acids including these sterically bulky substrates. Several d-amino acids were prepared in optically pure form. The molecular docking of substrates into the active sites of wild-type and mutant W121L/H227I enzymes revealed that the substrate binding cavity of the mutant enzyme was reshaped to accommodate these bulky substrates, thus leading to higher enzyme activity. These results lay a foundation for further shaping the substrate binding pocket and manipulating the interactions between the substrate and binding sites to access highly active d-amino acid dehydrogenases for the preparation of synthetically challenging d-amino acids.

Full text of DOI:10.1039/c8cy01426d

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Structure-guided
engineering
of
meso-diaminopimelate
dehydrogenase for enantioselective reductive amination of
sterically bulky 2-keto acids
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Xinkuan Cheng,a,b Xi Chen,a,b Jinhui Feng,a,b Qiaqing Wu,a,b,* Dunming Zhua,b,*
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a University of Chinese Academy of Sciences, No.19(A) Yuquan Road, Shijingshan
District, Beijing 100049, PR China
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b National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering
Research Center of Biocatalytic Technology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport
Economic Area, Tianjin 300308, PR China.
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Email: zhu_dm@tib.cas.cn and wu_qq@tib.cas.cn
Abstract
Mesoꢀdiaminopimelate dehydrogenase (DAPDH) and mutant enzymes are an
excellent choice of biocatalysts for the conversion of 2ꢀketo acids to the
corresponding
Dꢀamino acids. However, their application in the enantioselective
reductive amination of bulky 2ꢀketo acids, such as phenylglyoxylic acid, 2ꢀoxoꢀ4ꢀ
phenylbutyric acid, and indoleꢀ3ꢀpyruvic acid, is still challenging. In this study, the
structureꢀguided siteꢀsaturation mutagenesis of a Symbiobacterium thermophilum
DAPDH (StDAPDH) gave rise to a doubleꢀsite mutant W121L/H227I, which showed
1

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dramatically improved enzyme activities towards various 2ꢀketo acids including these
sterically bulky substrates. Several ꢀamino acids were prepared in optically pure
D
form. The molecular docking of substrates into the active sites of wildꢀtype and
mutant W121L/H227I enzymes revealed that the substrate binding cavity of the
mutant enzyme was reshaped to accommodate these bulky substrate, thus leading to
higher enzyme activity. These results lay a foundation for further shaping the
substrate binding pocket and manipulating the interactions between the substrate and
binding sites to access highly active
Dꢀamino acid dehydrogenases for the preparation
of synthetically challenging ꢀamino acids.
D
Keywords: reductive amination, enzyme engineering, mesoꢀdiaminopimelate
dehydrogenase, ꢀamino acids, bulky 2ꢀketo acids
D
1. Introduction
ꢀAmino acids are of growing interest due to their key roles in the industrial
D
production of drug intermediates, functional food, seasoning, cosmetic, and fine
chemicals 1, 2. Chemical and biological methods have been developed for the synthesis
of
D
ꢀamino acids 3ꢀ9. Among the biocatalytic methods, one of the straightforward
approaches is the reductive amination of 2ꢀketo acids catalyzed by
dehydrogenases 10ꢀ12. However,
Dꢀamino acid
D
ꢀamino acid dehydrogenases are rare in nature. The
most studied member of this class of enzymes is mesoꢀdiaminopimelate
dehydrogenase (DAPDH; EC 1.4.1.16). It catalyzes the reversible NADP+ꢀdependent
oxidative deamination of
Dꢀconfiguration center of mesoꢀdiaminopimelate (mesoꢀ
2

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DAP) to produce L
ꢀ2ꢀaminoꢀ6ꢀoxopimelate (Scheme 1) 13. According to a recent
phylogenetic analysis, the available 941 bacterial DAPDH sequences are classified
into two types: type I representing 68% of the sequences, shows an apparent
preference toward mesoꢀDAP as substrate and almost no ability toward amination of
2ꢀketo acids other than
Lꢀ2ꢀaminoꢀ6ꢀoxopimelate; whereas the remaining 32%
sequences (type II) exhibit obvious amination activity toward many 2ꢀketo acids 14.
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Scheme 1. The reversible reaction catalyzed by DAPDH
The mesoꢀdiaminopimelate dehydrogenase (CgDAPDH) from Corynebacterium
glutamicum is a typical type I DAPDH, and shows little or no activity for the
reductive amination of 2ꢀketo acids 15. Through three rounds of mutations and
screenings of CgDAPDH, some mutants (e.g. Q151L/D155G/T170I/R196M/H245N)
were obtained with dramatically enhanced activity toward some 2ꢀketo acids, and
applied in the synthesis of
Dꢀvaline, Dꢀleucine, Dꢀisoleucine, Dꢀcyclohexylalanine and
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ꢀphenylalanine 10, 12. Subsequently, similar mutations were carried out with other
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type I DAPDHs from Ureibacillus thermosphaericus strain A1 (UtDAPDH) and
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Clostridium
tetani
E88
(CtDAPDH)
.
Mutant
of
UtDAPDH
(D94A/Q154L/D158G/T173I/R199M/H248N) resulted in 8.3ꢀfold activity
enhancement (16.1 U/mg, 50 °C) toward phenylpyruvic acid 18. Mutant of CtDAPDH
(Q154L/T173I/R199M/P248S/H249N/N276S) showed 0.11 U/mg towards
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phenylpyruvic acid while wildꢀtype enzyme show no detectable activity toward the
same substrate 17.
Our previous studies have shown that a mesoꢀdiaminopimelate dehydrogenase from
Symbiobacterium thermophilum (StDAPDH) is a prototype of type II DAPDHs. This
enzyme catalyzes the reductive amination of some 2ꢀketo acids. For example, it
showed a specific activity of 2.87 U/mg towards pyruvic acid, which was converted to
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ꢀalanine in 68% yield and 99% ee 19. However, it showed 40 times less activity
toward phenylpyruvic acid than pyruvic acid 19. The siteꢀsaturation mutagenesis of
the Lꢀcenter binding sites (F146, T171, R181 and H227) of StDAPDH, gave rise to a
single amino acid variant, H227V, which had 35.1ꢀfold (2.39 U/mg) increased activity
towards phenylpyruvic acid 20. The structural analysis suggested that both an enlarged
substrate binding pocket and enhanced hydrophobicity of the pocket were mainly
responsible for the improved activity in reductive amination of phenylpyruvic acid.
Notwithstanding the successes of the various engineering efforts of DAPDH, some
stericallyꢀhindered 2ꢀketo acids such as 3,3ꢀdimethylꢀ2ꢀoxobutanoic acid,
phenylglyoxylic acid, and indoleꢀ3ꢀpyruvic acid have been rarely reported as the
substrate of DAPDHs. The corresponding
blocks for pharmaceutical molecules.
antiꢀinflammatory, and antiviral agents
Dꢀamino acids are important chiral building
D
ꢀtertꢀLeucine is used to synthesize antitumor,
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.
DꢀPhenylglycine is a significant precursor
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for semiꢀsynthetic antibiotics (penicillins and cephalosporins)
and actinomycete
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peptide antibiotics (streptogramin and vancomycin)
.
DꢀTryptophan is key
intermediate of tadalafil drug which is used to treat erectile dysfunction 25, 26. By
taking advantage of the structural information 27, semiꢀrational engineering of
StDAPDH was performed and the mutant libraries were screened using these
stericallyꢀbulky
Dꢀamino acids as the substrates. A set of StDAPDH variants were
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evolved to show improved activity toward these substrates with the enantioselectivity
intact. In addition, structural modeling of the mutant proteins and their docking with
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Dꢀamino acids were conducted to unravel the possible molecular basis for the
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enhanced activity.
2. Experimental
2.1 General
KOD Plus was purchased from TOYOBO Co., LTD. Dpn I was obtained from New
England Biolabs. Escherichia coli strain BL21(DE3) was bought from Novagen.
Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Roche.
LuriaꢀBertani (LB) broth and Terrific Broth (TB) medium were used for the culture.
All other chemicals were of analyticalꢀreagent grade or higher quality and were
purchased from commercial sources. Mesoꢀdiaminopimelate dehydrogenase (DAPDH)
from symbiobacterium thermophilum and glucose dehydrogenase (GDH) from
bacillus subtilis genes were preserved in our lab.
2.2 Substrate docking into StDAPDHs and selection of mutagenesis sites
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Based on the crystal structure of StDAPDH (PDB code 3WBB) , flexible dockings
of Dꢀamino acids into wildꢀtype enzyme and mutant W121L/H227I were performed
using a flexible docking protocol, and the structure of mutant W121L/H227I was
obtained by using the standard mutation protocol of Discovery Studio V4.1 (Bioviaꢀ
formerly Accelrys, USA). The protein active site was determined from PDB site
records. Other parameters were kept on default settings in flexible docking. The
docking poses were screened according to the score assessment option. Depending on
the distance and interaction between substrate and amino acid residues, the related
residues surrounding the binding pocket within 4 Å were identified (Table S1) and
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selected for mutagenesis.
2.3 Construction of StDAPDH mutant libraries
The mutant libraries of eight amino acid residues including D92, W121, F146, M152,
H154, T171, H227, and N253 were generated by applying siteꢀsaturation mutagenesis.
Briefly, single site mutations was introduced into the wildꢀtype gene using
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oligonucleotide primers containing an NNK degenerate codon at desired sites
.
Positive mutants obtained in the first round screening served as the template for
iterative saturation mutation with other sites, respectively. The primers used are listed
in the supplementary information (Table S2). All of the mutant libraries were
constructed by the PCRꢀbased QuickChange method 30. The 50 ꢁL reaction mixtures
typically contained 5 ꢁL of dNTPs (2 mM), 5 ꢁL of PCR buffer (10×), 3 ꢁL of
MgSO4 (25 mM), 1 ꢁL template DNA (50ꢀ100 ng), 1 ꢁL of forward primer (100 ꢁM),
1 ꢁL of reverse primer (100 ꢁM), 33 ꢁL of water, 1 ꢁL of KOD plus DNA
polymerase. The thermal cycling parameters were as follows: 95 °C, 5 min; 20 cycles
of (95 °C, 30 s; 63 °C, 30 s; 68 °C, 7 min); and final extension at 68 °C, 20 min. The
PCR products were purified with PCR purification kit. The purified PCR product was
transformed to E. coli BL21 (DE3) competent cells, and cultured at 37 °C in LB
medium with ampicillin (100 ꢁg/mL).
2.4 Library screening
Approximately 4000 individual colonies were picked into 96 deepꢀwell plates
containing 400 ꢁL of TB medium with 100 ꢁg/mL ampicillin and then incubated at 37
ºC for 12 hours. For each colony, 10 ꢁL culture was duplicated into the new 96 deepꢀ
well plates with fresh TB medium (800 ꢁL/well) and cultured for 1ꢀ1.5 hours under
the same conditions. For storage at ꢀ80 ºC, glycerol (final concentration of 20%) was
added to each seed plate. When the OD600 of new plate reached 0.6ꢀ0.8, the culture of
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StDAPDH was induced with 0.5 mM isopropyl βꢀDꢀthiogalactoside (IPTG) at 25 ºC
for additional 12 h. The cell pellets were harvested and washed with 200 ꢁL Na2CO3ꢀ
NaHCO3 buffer (100 mM, pH 9.0) by centrifugation at 4000 × g for 20 min. The
pellets were resuspended in 200 ꢁL of the same buffer with 2 mg/mL lysozyme and
0.1% Triton Xꢀ100 and the mixture was incubated at 37 °C for 2 h with shaking. Cells
debris were removed by centrifugation at 4000 × g for 30 min. The supernatant
obtained was analyzed for activity using a dye method where NADPH reacts with
nitro blue tetrazolium (NBT) to produce purple color formazan in the presence of
phenazine methosulfate (PMS) 10, 31. The reaction mixture was in 0.1 ml Na2CO3ꢀ
NaHCO3 buffer (100 mM, pH 9.5), containing 10 mM
and 10ꢀ40 µL supernatant. ꢀphenylglycine, ꢀtryptophan and
were used as the screening substrate for all of the mutant libraries. The 0.1 mL
screening system contained 10 mM ꢀamino acid, 10ꢀ40 µL supernatant and 0.5 mM
D
ꢀamino acid, 0.5 mM NADP+
D
D
Dꢀhomophenylalanine
D
NADP+ in Na2CO3ꢀNaHCO3 buffer (100 mM, pH 9.5). After the supernatant reacted
with substrate for 1 h at 37 °C, a same volume of dye mixture (0.01 mg/mL of PMS,
0.2 mg/mL of NBT) was added into the reaction mixture 10. Those wells that changed
color faster than the wildꢀtype control were noted as positive. To ensure 95%
coverage of each library, at least 95 clones were picked for each singleꢀsite mutant
library 32. The positive mutants were cultured and induced as described before. The
reductive amination activities were confirmed using 2ꢀketo acids (phenylglyoxylic
acid, indoleꢀ3ꢀpyruvic acid, and 2ꢀoxoꢀ4ꢀphenylbutyric acid) as substrate. All of the
positive mutants were sequenced.
2.5 Gene expression and protein purification
The E. coli BL21(DE3) strains harboring pETꢀ32a(+)ꢀDAPDHs plasmid of positive
mutants were cultured overnight at 37 °C with 250 mL shaking flasks containing 50
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mL of LB medium. 100 ꢁg/mL ampicillin was used in LB medium. The overnight
culture was inoculated into 800 mL of LB medium containing ampicillin (100 ꢁg/mL).
After the OD600 approximately reached 0.6, the cultures were induced with 0.5 mM
IPTG at 25 °C 200 rpm for 20 h. The cells were harvested by centrifugation at 6000 ×
g for 15 min at 4 °C and washed with TrisꢀHCl buffer (20 mM, pH 8.0). The cells
were resuspended with TrisꢀHCl buffer (20 mM, pH 8.0) containing 0.5 M NaCl, 5
mM βꢀmercaptoethanol, 5% glycerol and 10 mM imidazole, and lysed on the
homogenizer. The cell debris was removed by centrifugation at 9000 × g for 30 min at
4 °C. The soluble proteins were purified by an AKTA Purifier 10 system (GE
Healthcare) using a nickel affinity column (GE Healthcare). The column was preꢀ
equilibrated with 100 mL of equilibrium buffer (20 mM TrisꢀHCl, 0.5 M NaCl, 10
mM imidazole, pH 8.0). The sample (100 mL) was loaded with a flow rate of 1.0
mL/min. After washing with 200 mL of the equilibrium buffer, the bounded target
protein was washed with 10−500 mM imidazole solution containing 500 mM NaCl
and 20 mM TrisꢀHCl (pH 8.0). The target protein fractions were collected, desalted
and replaced with the Na2CO3ꢀNaHCO3 buffer (100 mM, pH 9.0) through
ultrafiltrations. Protein concentration was measured using the Bradford method 33.
2.6 Specific activities and kinetic constants of the wild-type and mutant enzymes
The specific activities of the wildꢀtype enzyme, single site mutants and double site
mutant toward phenylglyoxylic acid, phenylpyruvic acid, 2ꢀoxoꢀ4ꢀphenylbutyric acid,
and indoleꢀ3ꢀpyruvic acid were assayed spectrophotometrically by monitoring the
decrease (reductive amination reaction direction) in absorbance of NADPH at 340 nm.
The extinction coefficient of 6.22 mMꢀ1 cmꢀ1 was used to correlate absorbance to
concentration at 30 °C. In addition to the four aromatic 2ꢀketo acids, some aliphatic 2ꢀ
keto acids (pyruvic acid, 2ꢀketobutyric acid, 2ꢀoxovaleric acid, 3ꢀmethylꢀ2ꢀoxobutyric
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acid, 4ꢀmethylꢀ2ꢀoxovaleric acid, 3,3ꢀdimethylꢀ2ꢀoxobutanoic acid) were also tested
as the substrates. Apparent KM and Vmax for reductive amination were obtained by
nonlinear curve fit in term of the MichaelisꢀMenten equation. Initial velocities of the
enzymatic reactions were performed by varying the concentration of 2ꢀketo acids
(from 5 to 200 mM). A typical reductive amination reaction mixture comprised of 0.5
mM NADPH, 20 mM 2ꢀketo acid, 200 mM NH4Cl and appropriate amount of enzyme
in 0.2 mL Na2CO3ꢀNaHCO3 buffer (100 mM, pH 9.0) 19. The amount of enzyme
consuming 1 µmol/min of NADPH was defined as one unit (U). All experiments were
conducted in triplicate.
2.7 Reductive amination of 2-keto acids
Biotransformation reactions were carried out as follows 19: For 3,3ꢀdimethylꢀ2ꢀ
oxobutanoic acid, indoleꢀ3ꢀpyruvate and phenylglyoxylic acid, the reaction mixture
was in 1 mL of Na2CO3ꢀNaHCO3 buffer (200 mM, pH 8.5), containing 20 mM 2ꢀketo
acids, 60 mM NH4Cl, 80 mM glucose, 8 mg glucose dehydrogenase (GDH), 0.5 mM
NADP+ and 1 mg StDAPDH. The reaction was carried out at 37 °C for 24 h. After
that, 20 µL of the reaction mixture was mixed with 180 µL H2O, 400 µL of 1ꢀfluoroꢀ
2ꢀ4ꢀdinitrophenylꢀ5ꢀLꢀalanine amide (FDAA, 0.2%) in acetone solution before adding
80 µL of 1 M NaHCO3. Subsequently, the above mixture was heated at 40 °C for 1 h.
Then, 40 µL of 2 M HCl was added, the obtained mixture was ready for high
performance liquid chromatography (HPLC) analysis (see Supporting Information).
HPLC was performed on an Agilent 1200 system equipped with an Eclipse XDBꢀC18
column (4.6 × 150 mm, 5 µm) and a variable wavelength detector at the wavelength
of 340 nm. A mixture of triethylamine phosphate (50 mM, pH 3.0): acetonitrile = 6:4ꢀ
7:3 was used as mobile phase at flow rate of 0.8 mL/min.
For other 2ꢀketo acids, 1 mmol 2ꢀketo acid, 3 mmol NH4Cl, 4 mmol Dꢀglucose, 10
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ꢁmol NADP+, 10 mg StDAPDH, and 40 mg glucose dehydrogenase (GDH) were
added to 10 mL Na2CO3ꢀNaHCO3 buffer (200 mM, pH 8.5). The reaction mixture
was also incubated at 37 °C, 200 rpm for 24 h. Similarly, after FDAAꢀderivatization,
the reaction solution was subjected to HPLC analysis of ee value (see Supporting
Information). Then, 1.3 mmol Nꢀ(9ꢀfluorenylmethoxycarbonyloxy)succinimide
(FmocꢀOSu) in tetrahydrofuran solution was added to the above remaining mixture,
while Na2CO3 was added to maintain the solution pH at 8.0 to 9.0. After 37 °C, 200
rpm for 10 h, the mixture was washed with methyl tertꢀbutyl ether three times and the
pH of the aqueous phase was adjusted to 2 ~ 3 by addition of 6 N HCl. The aqueous
phase was then extracted by ethyl acetate for three times. The organic extracts were
combined and dried over anhydrous Na2SO4. The solvent was removed and the
product was purified through column chromatography and characterized by 1H NMR.
FmocꢀD
ꢀalanine: 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 4.0 Hz, 2H), 7.61 (m, 2H),
7.42 (t, J = 4.4 Hz, 2H), 7.33 (t, J = 4.4 Hz, 2H), 4.40ꢀ4.43 (m, 2H), 4.32 (t, J = 4.0
Hz, 2H), 4.23ꢀ4.26 (m, 1H), 1.51 (d, J = 2.0 Hz, 3H)
FmocꢀD
ꢀ2ꢀaminobutyrate: 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 4 Hz, 2H), 7.61
(m, 2H), 7.42 (t, J = 4.4 Hz, 2H), 7.33 (t, J = 4.4 Hz, 2H), 4.42 (d, J = 21.6 Hz, 2H),
4.36 (t, J = 4 Hz, 2H), 4.23ꢀ4.26 (m, 1H), 1.45 (q, J = 21.6 Hz), 0.99 (d, J = 4.8 Hz,
3H)
1
Fmocꢀ
D
ꢀnorvaline: H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 4 Hz, 2H), 7.61 (m,
2H), 7.40 (t, J = 4.4 Hz, 2H), 7.31 (t, J = 4.4 Hz, 2H), 4.35ꢀ4.47 (m, 3H), 4.23ꢀ4.26
(m, 1H), 1.62ꢀ1.95 (m, 2H), 1.38ꢀ1.41 (q, , J = 4.0, 8.0 Hz, 2H), 0.95 (d, J = 4.8 Hz,
3H)
1
Fmocꢀ
D
ꢀvaline: H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 4 Hz, 2H), 7.62 (m, 2H),
7.42 (t, J = 4.4 Hz, 2H), 7.33 (t, J = 4.4 Hz, 2H), 4.44 (d, J = 21.6 Hz, 2H), 4.36 (t, J
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= 4 Hz, 2H), 4.23ꢀ4.26 (m, 1H), 1.03 (d, J = 4.0 Hz, 3H), 0.97 (d, J = 4.0 Hz, 3H)
ꢀleucine: 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 4 Hz, 2H), 7.62 (m, 2H),
Fmocꢀ
D
7.42 (t, J = 4.4 Hz, 2H), 7.33 (t, J = 4.4 Hz, 2H), 4.40ꢀ4.46 (m, 3H), 4.23ꢀ4.26 (m,
1H), 1.68ꢀ1.75 (m, 2H), 1.56ꢀ1.63 (m, 1H), 0.99 (d, J = 4.0 Hz, 6H)
FmocꢀD
ꢀphenylalanine: 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 4 Hz, 2H), 7.62 (m,
2H), 7.28ꢀ7.44 (m, 6H), 7.14ꢀ7.18 (m, 2H), 4.40ꢀ4.46 (m, 2H), 4.18ꢀ4.35 (m, 1H),
4.19ꢀ4.26 (m, 1H), 3.20ꢀ3.26 (m, 1H), 3.10ꢀ3.17 (m, 1H)
1
D
ꢀhomophenylalanine: H NMR (400 MHz, DMSO) δ 7.14ꢀ7.34 (m, 5H), 2.67ꢀ2.74
(m. 1H), 2.58ꢀ2.66 (m, 1H), 1.99ꢀ2.07 (m, 1H), 1.89ꢀ1.97 (m, 1H)
257 3. Results and discussion
258
259
260
261
262
263
264
265
266
267
268
269
270
3.1 Choice of amino acid sites for saturation mutagenesis
Our previous studies suggest that StDAPDH catalyzes the oxidative
deamination/reductive amination by an ordered kinetic catalytic mechanism in that the
enzyme binds the cofactor first, and subsequently interacts with the substrate to
initiate catalysis. In order to select appropriate amino acid residues for mutation,
Dꢀ
homophenylalanine, ꢀphenylglycine and ꢀtryptophan were docked into the crystal
D
D
structure of StDAPDH containing the coenzyme NADP+. Eight amino acid residues
(D92, W121, F146, M152, H154, T171, H227, N253) around the substrate were
chosen for singleꢀsite saturation mutagenesis (Figure 1).
Dꢀhomophenylalanine, Dꢀ
phenylglycine and ꢀtryptophan in the active center interacted with amino acid
D
residues were summarized in supplementary information Table S1.
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272
Figure 1. Docking of Dꢀhomophenylalanine into the crystal structure of wildꢀtype
StDAPDH. Coenzyme NADP+ is colored pink, and Dꢀhomophenylalanine is colored
light blue. The selected amino acid residues (D92, W121, F146, M152, H154, T171,
H227, and N253) are displayed in green, cyan, blue, yellow, red, grass green, moss
green and purple.
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
3.2 Single site saturation mutagenesis and activity screening
Eight siteꢀsaturation mutagenesis libraries were generated, and the resulting mutant
libraries were screened with Dꢀphenylglycine, Dꢀtryptophan and Dꢀhomophenylalanine
as the substrates by monitoring the formation of a colored product as described in
experimental section. Several beneficial mutants with higher activity toward oxidative
deamination of the specific Dꢀamino acids than the wildꢀtype enzyme were identified
from the mutant libraries of sites W121, T171 and H227. No mutant library had clone
with significant activity toward Dꢀtryptophan. For Dꢀ2ꢀphenylglycine and Dꢀ
homophenylalanine, there were positive clones from libraries of positions W121,
T171 and H227. They were sequenced and noted as W121L, T171H and H227I.
These mutant proteins were purified (Figure S1), and the pure mutant enzymes were
tested for their activity toward the reductive amination of the corresponding 2ꢀketo
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313
314
acids. The specific activities of mutant H227V obtained in our previous study toward
these substrates were also determined. The results are shown in Table 1. No positive
mutant was found in the mutant libraries of D92, F146, M152, H154, and N253. In
the previous siteꢀsaturation mutation and screening of F146, we also did not find any
20
beneficial mutant for the reductive amination of phenylpyruvic acid . Our screening
results of D92 is contrary to Hayashi and Sakuraba’s study, in which mutant D94A
(D92 in StDAPDH numbering) of UtDAPDH exhibited dramatically improved
activity toward phenylpyruvic acid and 2ꢀoxoꢀoctanoic acid because the mutation
enlarged the substrateꢀbinding pocket and enhanced the hydrophobicity of the pocket
18. We created the D92A mutant of StDAPDH corresponding to D94A of UtDAPDH.
The results showed that D92A did not exhibit improved activity for all the selected
substrates (Data not shown). This may be due to that StDAPDH belongs to type II
DAPDH, while UtDPADH is a type I enzyme, although this site is conserved cross
both types of DAPDHs 14. In our previous siteꢀsaturation mutagenesis on T171, the
best results with phenylpyruvic acid as the substrate were obtained with T171P/S
20
(~0.2 U/mg) , whilst T171H was the best mutant with this substrate (~0.9 U/mg) in
the present study. We then speculate that the best mutant T171H for phenylpyruvic
acid was missed in our previous work. It should be noted that different screening
methods are used in these studies. Positions 171 and 227 have been reported
previously to be beneficial with phenylpyruvate 20 . In addition to this observation, the
present study shows that mutations on these positions are also beneficial for
phenylglyoxylic acid and 2ꢀoxoꢀ4ꢀphenylbutyric acid. Although no beneficial single
mutant was obtained for indoleꢀ3ꢀpyruvic acid in the present screening studies, the
previously identified mutant H227V 20 showed activity toward this substrate.
3.3 Iterative saturation mutation
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320
321
322
323
324
325
326
327
328
329
To further improve the catalytic activity, iterative saturation mutation was performed
using W121L, T171H and H227I, respectively, as the template for siteꢀsaturation
mutagenesis of the other two sites separately. Two 96ꢀwell plates (total 184 clones)
were screened for each library with Dꢀ2ꢀphenylglycine, Dꢀhomophenylalanine or Dꢀ
tryptophan as the substrate. For template T171H, no positive mutant was obtained
from the saturation libraries of W121 or H227. For W121L or H227I as the template,
a double mutant W121L/H227I with improved activity toward the oxidative
deamination of these substrates was obtained in both cases. When W121L/H227I was
used as the template, no positive mutant was obtained from the siteꢀsaturation
mutagenesis of T171. For further siteꢀsaturation mutagenesis and screening at D92,
F146, M152, H154 and N253, no positive mutant was obtained. The mutant
W121L/H227I protein was purified and its reductive amination activity toward the
bulky 2ꢀketo acid substrates were measured. The results are summarized in Table 1.
Table 1 Specific activity of wildꢀtype and mutant DAPDHs toward bulky 2ꢀketo acids
specific activity (U/mg)
enzyme
WT
W121L
ꢀ a
0.07±0.01
0.98±0.05
0.91±0.04
2.41±0.30
2.40±0.10
4.96±0.57
0.20±0.01
0.38±0.01
0.89±0.07
1.61±0.06
1.07±0.16
6.89±0.32
ꢀ
0.018±0.001
0.015±0.002
0.017±0.004
ꢀ
ꢀ
T171H
ꢀ
H227I
ꢀ
H227V
0.010±0.001
0.015±0.002
W121L/H227I
0.24±0.02
a
330
No measurable activity.
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332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
As shown in Table 2, wildꢀtype StDAPDH showed no detectable activity toward
phenylglyoxylic acid and indoleꢀ3ꢀpyruvic acid, but 0.20 U/mg specific activity
toward 2ꢀoxoꢀ4ꢀphenylbutyric acid. Both single site mutants H227I and H227V
20
exhibited high activity toward phenylpyruvic acid and 2ꢀoxoꢀ4ꢀphenylbutyric acid .
It is interesting that H227I was active toward phenylglyoxylic acid, but no activity
was observed for indoleꢀ3ꢀpyruvate acid. In contrast, H227V showed activity toward
indoleꢀ3ꢀpyruvic acid, but phenylglyoxylic acid was not its active substrate. During
the further mutation and screening, a mutant W121L/H227I was found to have 34
folds and 70 folds improved enzyme activity toward 2ꢀoxoꢀ4ꢀphenylbutyric acid and
phenylpyruvic acid, respectively, compared to the wildꢀtype enzyme. This mutant was
active toward both phenylglyoxylic acid and indoleꢀ3ꢀpyruvic acid with the specific
activity of 0.24 U/mg and 0.015 U/mg, respectively. Although mutants W121L and
H227I showed no activity for reductive amination of indoleꢀ3ꢀpyruvic acid,
W121L/H227I was active toward this substrate. An additive effect was observed for
W121L and H227I.
3.4 Substrate spectra of the mutant W121L/H227I
Since mutant W121L/H227I showed highest enzyme activity toward bulky 2ꢀketo
acids (Table1), its enzyme activities toward other 2ꢀketo acids were also measured
(Table 2). As shown in Table 2, mutant W121L/H227I showed an improved specific
activity toward all the tested 2ꢀketo acids compared to wildꢀtype enzyme. For most
keto acids, the specific activity increased more than 10 folds or from undetectable to
some activity. The mutant enzyme exhibited similar activity toward pyruvic acid, 2ꢀ
oxobutanoic acid and 2ꢀoxovaleric acid, although their side chain became longer
(entry 1ꢀ3). When the side chain of the substrate became branched, the mutant
enzyme activity decreased significantly, especially for 3ꢀmethylꢀ2ꢀoxobutanoic acid
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362
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365
366
367
368
369
370
371
372
373
374
and 3,3ꢀdimethylꢀ2ꢀoxobutanoic acid (entry 4 and 5). Adding a methylene group
between the isopropyl and 2ꢀcarbonyl group, the specific activity was increased from
1.02 to 9.91 U/mg (entry 4 and 6). This trend was also observed when methylene
group was inserted between the phenyl group and 2ꢀcarbonyl group, 28.7 folds higher
activity was observed for 2ꢀoxoꢀ4ꢀphenylbutyric acid than phenylglyoxylic acid.
For substrate 2ꢀoxoꢀ4ꢀphenylbutyric acid, mutant W121L/H227I showed nearly 55
times enhancement of kcat than wildꢀtype enzyme (0.23 sꢀ1), but wildꢀtype enzyme
showed lower
showed a nearly 9ꢀfold increase in kcat and 1.6ꢀfold increase in KM than wildꢀtype
(Table S3), while W121L showed a 5ꢀfold lower KM and slight enhancement in kcat
KM (15.56 mM). This is mainly due to the contribution of H227I. H227I
.
This was more obvious for phenylpyruvic acid, toward which H227I had a KM of
12.00 mM and a kcat of 4.28 sꢀ1 (Table 2 and Table S4). In conclusion, the enzyme
kinetic parameters showed that the mutant W121L/H227I increased catalytic velocity,
but exerted less effect on the affinity between the enzyme and the substrate. The
enhancement in enzyme activity of W121L/H227I was mainly caused by the increase
in catalytic velocity.
Table 2 Mutant W121L/H227I catalyzed the reductive amination of various 2ꢀketo
acids
wildꢀtype
W121L/H227I
entry
substrate
specific activity specific activity
kcat
(sꢀ1)
KM
yield
(%)
ee
(U/mg)
(U/mg)
(mM)
(%)
1
31.35±1.24
87 b (49) c >99
2.94
±
±
0.08
45.22
±
±
1.59 48.46
±
±
4.17
2
32.16±1.84
>99 (80)
>99
1.62
0.09
35.38
0.90 35.12
2.53
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4
5
30.55±1.24
1.02±0.07
0.006
>99 (58)
>99 (52)
44
>99
>99
>99
1.25
0.62
±
0.13
0.04
38.70
±
1.06 23.50
±
2.11
4.29
±
5.17
±
0.18 46.10
±
ꢀ a
ꢀ
ꢀ
6
7
9.91±0.67
0.24±0.02
>99 (68)
75
>99
>99
0.53
±
0.02
13.49
±
0.66 17.72
±
2.92
ꢀ
ꢀ
ꢀ
8
9
0.07±0.01
0.20±0.01
4.96±0.57
6.89±0.32
>99 (85)
75 (42)
>99
>99
6.20
±
0.60 12.20
±
±
3.60
6.91
12.60
±
1.07 33.83
10
ꢀ
0.015±0.002
ꢀ
ꢀ
46
>99
a
c
375
376
377
378
379
380
381
382
383
384
385
386
Not measured because of low enzyme activity; b HPLC yield; Isolated yields in
parentheses.
3.5 Preparation of D-amino acids by mutant W121L/H227I
The reductive amination reactions of the 2ꢀketo acids in Table 2 were conducted, and
the yields and ee values of the products were determined by HPLC analysis after
derivatization. In all the cases, the corresponding Dꢀamino acids were obtained
with >99% ee. The W121L/H227I mutant catalyzed the reductive amination of 2ꢀ
ketobutyric acid, 2ꢀoxovaleric acid, 3ꢀmethylꢀ2ꢀoxobutyric acid, 4ꢀmethylꢀ2ꢀ
oxovaleric acid, and phenylpyruvic acid to yield the corresponding Dꢀamino acids
with excellent HPLC yield (>99%). In addition, Dꢀ2ꢀaminobutyric acid and Dꢀ
phenylalanine were isolated in 80% and 85% yield after FmocꢀOSu derivatization.
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389
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391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
Moreover, bulky 3,3ꢀdimethylꢀ2ꢀoxobutanoic acid, phenylglyoxylic acid and indoleꢀ
3ꢀpyruvic acid were also converted to the corresponding Dꢀamino acids (Dꢀtertꢀleucine,
Dꢀphenylglycine, and Dꢀtryptophan), although lower conversion was observed under
the same reaction conditions. These results indicate that various sterically bulky Dꢀ
amino acids can be successfully synthesized from the corresponding 2ꢀketo acids by
employing the evolved mutant DAPDH.
3.6 Molecular insights into the substrate specificity
In order to explore the molecular basis for the increased enzyme activity of mutant
W121L/H227I, Dꢀhomophenylalanine, Dꢀ2ꢀamino pentanoic acid, Dꢀtryptophan were
docked into the substrateꢀbinding site of wildꢀtype and mutant W121L/H227I
enzymes. The most possible conformations of substrate in the active center of wildꢀ
type enzyme and mutant W121L/H227I were obtained, respectively (Fig. 2). For Dꢀ
homophenylalanine, the docking results showed that the distances between the
αꢀ
hydrogen atom of Dꢀhomophenylalanine and C4 of nicotinamide mononucleotide
(NMN) was 3.41 Å in wildꢀtype enzyme (Fig. 2A), whereas the corresponding
distance in mutant W121L/H227I was shortened to 3.11Å (Fig. 2B). This is in
agreement with the improved activity observed for mutant W121L/H227I. It is very
interesting that the phenyl group of Dꢀhomophenylalanine was away from the amino
acid residues W121 and H227 in wildꢀtype enzyme, while it was directed to the amino
acid residues L121 and I227 in mutant W121L/H227I, and the side chains of leucine
35
and isoleucine might provide CH/π interaction with the phenyl group of substrate
.
We speculate that the steric effect might result in the change of the substrate
orientation, affecting its position related to the cofactor and thus its activity.
When Dꢀ2ꢀamino pentanoic acid was docked into the active center, the steric effect of
W121 and H227 also forced the alkyl side chain of Dꢀ2ꢀaminopentanoic acid away
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414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
from the amino acid residues 121 and 227 (Fig. 2C). However, for mutant
W121L/H227I, the alkyl side chain of Dꢀ2ꢀaminopentanoic acid was directed to the
amino acid residues L121 and I227, and the side chains of leucine and isoleucine
might provide van der Waals interaction with the alkyl side chain of Dꢀ2ꢀ
aminopentanoic acid. As such, the distance between the αꢀhydrogen atom of Dꢀ2ꢀ
aminopentanoic acid and C4 of cofactor NMN was shorted from 4.31 Å (wildꢀtype
enzyme) to 2.99 Å (Fig. 2D), resulting in 24 times higher activity of mutant
W121L/H227I than the wildꢀtype enzyme.
When Dꢀtryptophan was docked in the wildꢀtype enzyme, the distance between the αꢀ
hydrogen atom of Dꢀtryptophan and C4 of cofactor NMN was 4.92 Å (Fig. 2E). For
mutant W121L/H227I, although the smaller leucine and isoleucine made some space
for indoleꢀgroup of the substrate, the steric effect of the Dꢀtryptophan itself caused the
distance between the αꢀhydrogen atom of Dꢀtryptophan and C4 of cofactor NMN was
still large (4.71 Å, Fig. 2F). Therefore, low activity was observed for mutant
W121L/H227I toward indoleꢀ3ꢀpyruvic acid. This suggests that the interaction
between the active sites and indoleꢀ3ꢀpyruvic acid should be further manipulated to
make the substrate closer to cofactor, leading to higher enzyme activity.
429
430
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432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
Figure 2. Dꢀamino acids (AA) were docked into the substrateꢀbinding site. A) Dꢀ
homophenylalanine in wildꢀtype enzyme; B) Dꢀhomophenylalanine in mutant
W121L/H227I; C) Dꢀ2ꢀaminopentanoic acid in wildꢀtype enzyme; D) Dꢀ2ꢀ
aminopentanoic acid in mutant W121L/H227I; E) Dꢀtryptophan in wildꢀtype enzyme;
F) Dꢀtryptophan in mutant W121L/H227I. NMN: nicotinamide mononucleotide.
4. Conclusions
Based on the crystal structure of StDAPDH and enzymeꢀsubstrate docking studies, the
substrate binding amino acid residues were selected for siteꢀsaturation mutagenesis.
Screening of the mutant libraries using Dꢀhomophenylalanine, Dꢀphenylglycine and Dꢀ
tryptophan as the substrate resulted in three positive mutants W121L, T171H and
H227I. The subsequent iterative saturation mutation generated a double mutant
W121L/H227I with significantly enhanced enzyme activity towards sterically bulky
2ꢀketo acids. Kinetic studies showed that the higher activity of mutants H227I and
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448
449
450
451
452
453
454
455
456
457
458
459
460
W121L/H227I was mainly contributed by mutation at H227, which greatly increased
the kcat. Compared to the wildꢀtype enzyme, W121L/H227I possessed increased
activity toward a set of aliphatic and aromatic 2ꢀketo acids, and catalyzed their
enantioselective reductive amination to afford the corresponding Dꢀamino acid
with >99% ee. The results suggested W121L/H227I as a versatile biocatalyst for the
synthesis of Dꢀamino acids with diverse structures via asymmetric reductive
amination of the corresponding 2ꢀketo acids. The enzymeꢀsubstrate docking studies
with the representative Dꢀamino acids suggested that mutations at W121 and H227
reshaped the substrate binding pocket and the orientation of the substrate was altered
to accommodate the substrate in appropriate position for the hydride transfer from/to
the coꢀfactor. Therefore, this study deepens our understanding of the substrateꢀbinding
and substrate specificity of DAPDHs at molecular level, provides guidance for our
future engineering of this enzyme to obtain active mutant enzyme for our challenging
synthetic purposes.
461 Conflicts of interest
462
There are no conflicts of interest to declare.
463 Acknowledgements
464
465
466
467
468
This work was financially supported by the National Natural Science Foundation of
China (No. 21778072) and the Tianjin Municipal Science and Technology
Commission (15PTCYSY00020 and 15PTGCCX00060). We thank Professor Peter C.
K. Lau for constructive advices on this article.
469 References
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Catalysis Science & Technology
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DOI: 10.1039/C8CY01426D
Structure-guided
engineering
of
meso-diaminopimelate
dehydrogenase for enantioselective reductive amination of
sterically bulky 2-keto acids
Xinkuan Cheng, Xi Chen, Jinhui Feng, Qiaqing Wu, Dunming Zhu
Structure-guided reshaping the substrate-binding pocket of a meso-diaminopimelate
dehydrogenase (StDAPDH) led to a mutant W121L/H227I, which catalyzed the
enantioselective reductive amination of some sterically bulky 2-keto acids.