Deracemization and stereoinversion to aromatic d-amino acid derivatives with ancestral l-amino acid oxidase

Article abstract of DOI:10.1021/acscatal.9b03418

Enantiomerically pure amino acid derivatives could be foundational compounds for peptide drugs. Deracemization of racemates to l-amino acid derivatives can be achieved through the reaction of evolved d-amino acid oxidase and chemical reductants, whereas deracemization to d-amino acid derivatives has not progressed due to the difficulty associated with the heterologous expression of l-amino acid oxidase (LAAO). In this study, we succeeded in developing an ancestral LAAO (AncLAAO) bearing broad substrate selectivity (13 l-amino acids) and high productivity through an Escherichia coli expression system (50.7 mg/L). AncLAAO can be applied to perform deracemization to d-amino acids in a similar way to deracemization to l-amino acids. In fact, full conversion (>99% ee, d-form) could be achieved for 16 racemates, including nine d,l-Phe derivatives, six d,l-Trp derivatives, and a d,l-phenylglycine. Taken together, we believe that AncLAAO could be a key enzyme to obtain optically pure d-amino acid derivatives in the future.

Full text of DOI:10.1021/acscatal.9b03418

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Article
Deracemization and Stereoinversion to Aromatic D-Amino
Acid Derivatives with Ancestral L-Amino Acid Oxidase
Shogo Nakano, Yuki Minamino, Fumihito Hasebe, and Sohei Ito
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b03418 • Publication Date (Web): 27 Sep 2019
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1 Deracemization and Stereoinversion to Aromatic D-Amino Acid
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2 Derivatives with Ancestral L-Amino Acid Oxidase
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Shogo Nakanoa, *, Yuki Minaminoa, Fumihito Hasebe and Sohei Ito*
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Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of
Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
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Correspondence: *snakano@u-shizuoka-ken.ac.jp., *itosohei@u-shizuoka-ken.ac.jp
aThese authors contributed equally to this work.
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ABSTRACT
Enantiomerically pure amino acid derivatives could be foundational compounds for
peptide drugs. Deracemization to L-amino acid derivatives from racemates can be achieved
through the reaction of evolved D-amino acid oxidase (DAAO) and chemical reductants,
whereas deracemization to D-amino acid derivatives has not progressed due to the difficulty
associated with heterologous expression of L-amino acid oxidase (LAAO). In this study, we
succeeded in developing an ancestral LAAO (AncLAAO) bearing broad substrate selectivity
(13 L-amino acids) and high productivity through an E. coli expression system (~50.7 mg/L).
AncLAAO can be applied to perform deracemization to D-amino acids in a similar way to
deracemization to L-amino acids. In fact, full conversion (>99% ee, D-form) could be
achieved for 16 racemates, including nine D,L-Phe derivatives, seven D,L-Trp derivatives,
and a D,L-phenylglycine. Taken together, we believe that AncLAAO could be a key enzyme
to obtain optically pure D-amino acid derivatives in the future.
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Keywords: L-amino acid oxidase, deracemization, D-amino acid derivatives, ancestral
sequence reconstruction, artificial protein
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INTRODUCTION
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Enantiomerically pure compounds could potentially be precursors in the synthesis of
commercial products such as pesticides, pharmaceuticals, and antibiotics 1. Dynamic kinetic
resolution (DKR) and deracemization has previously been applied to enantiomerically pure
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compounds to obtain a theoretical 100% yield of chiral compounds from racemates . To
date, several enzymes have been used in this process as they are superior to conventional
chemical catalysts. These enzymes are eco-friendly and can synthesize enantiomerically
pure compounds under standard conditions 2-6. The application of these enzymes requires
that they be optimized in order to be suitable for use. Optimization of productivity, solubility,
thermostability, reactivity, and broadening of substrate selectivity is required. Many protein
engineering methods have been developed to perform this optimization and to enhance
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7-9
enzymatic functions. These methods can be categorized as sequence-based methods
and structure-based methods 10.
Deracemization to chiral amino acids from D,L-mixtures is a popular area of research at
this time due to its potential application to commercial products 11. Currently, many
biocatalytic methods used to obtain chiral amino acids have been reported including: i)
conversion of α-keto acid to D-amino acids utilizing engineered D-amino acid
dehydrogenase and glucose dehydrogenase 12, ii) synthesis of L-amino acids with amination
of cinnamic acid catalyzed by phenylalanine ammonia lyase 13, 14, iii) the hydantoinase
process which utilizes racemase/hydantoinase/carbamoylase 15, 16, iv) N-acetyl amino acid
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racemase/acylase biotransformation 17, v) transamination of α-keto acid 18, and vi)
deracemization of racemic amino acids utilizing a combination of deaminase and oxidase
and chemical reductants 19, 20. L-amino acids can be obtained by chemoenzymatic reactions
catalyzed by D-amino acid oxidase (DAAO) and chemical reductants (NH3:BH3, NaBH4, etc.)
19, 21. DAAO oxidizes the main chain amide group of D-amino acids 22 and produces imino
acid, and this product is continuously reduced to the D,L-mixture by the reductant. Only L-
amino acids are accumulated by repeating this process. In addition, DAAO generates H2O2
as a by-product. This H2O2 can be detected by the Trinder reaction. This creates a simple
mode of detection to assess whether substrate candidates are oxidized by evolved DAAO
produced by directed evolution and rational design 2, 3. As an example, R-selective amine
oxidase was designed from DAAO using a directed evolution approach; the evolved DAAO
(Y228L/R283G mutant) bearing amine oxidase activity was screened by detecting the
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generation of H2O2 through the addition of (R)-phenylethylamine 23, 24
.
Theoretically, deracemization to D-amino acids from D,L-racemates could be achieved by
replacing DAAO with an L-amino acid oxidase (LAAO) exhibiting broad substrate selectivity.
LAAO would catalyze the oxidation of L-amino acids through a reaction mechanism similar
to that of DAAO 25. However, the deracemization to D-amino acids has been slow to progress
due to the difficulty associated with heterologous overexpression of LAAOs in prokaryotic
hosts, such as E. coli. These difficulties have been recognized by many research groups 2,
25. As an alternative to LAAOs, L-amino acid deaminases (LAADs) could be applied in the
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deracemization to D-amino acids 11, 20, 26. However, unlike LAAOs, LAADs require an
exogenous electron transfer system to oxidize the FADH2 generated by the reaction. Thus,
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the reaction is often carried out using an entire cell expressing LAAD or through the addition
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of an artificial electron acceptor 2, 27. Furthermore, LAADs generate very little H2O2
,
indicating that the approach which is utilized in the design of evolved DAAOs cannot be
applied to the design of LAADs. LAAOs can complement these points, and therefore we
believe that the development and design of novel LAAOs will contribute to the ability to
perform deracemization of D,L-amino acids to D-amino acids.
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RESULTS & DISCUSSION
To find novel LAAOs, we focused
on our previous study that followed
the evolutionary track of orphan L-
arginine oxidase (AROD) through
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the design and functional analysis
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of ancestral AROD (AncAROD)
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This study suggested that native
AROD from Oceanobacter kriegii
(OkAROD) exhibits high specificity
toward L-Arg, whereas AncAROD
acquired broad substrate selectivity
towards 5 L-amino acids. Here,
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FIGURE 1. Schematic model showing how to
design AncLAAO from AncAROD. The design
was performed using the following procedure:
paralog search (a), finding PpLAAO sequence (b),
collection of six LAAO candidate sequences (Table
S1) including PpLAAO (c), and design of AncLAAO
(Table S2) with ASR (d).
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sequence identity between AncAROD and OkAROD was about 60% , suggesting that a
40% displacement of the native AROD sequence could broaden the selectivity. Based on
these results, we hypothesized that a paralogous gene of the native AROD may have
different selectivity in a similar way to AncAROD. To prove this point, we attempted to find
new paralogs of AROD via the procedure described in Figure 1. A total of 10 AROD
candidates were assigned in the previous study and homology searches were performed for
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all of these AROD candidates in their respective genomes (a in Figure 1). A paralog
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sequence bearing moderate sequence similarity to AROD from Pseudoalteromonas
piscicida (PpAROD, NCBI Reference Number: WP_045988413.1) was found only in a
genus of Pseudoalteromonas (b in Figure 1); the E-value calculated by Blastp was < 1.0E-
25. The existence of LAAOs in this genus has been implicated in previous studies 25, 29. Based
on these analyses, we predicted that the paralog had the potential to oxidize certain L-amino
acids, and it was therefore named PpLAAO. However, we did not confirm LAAO activity of
PpLAAO at this time for multiple reasons including lower expression and solubility, and
inappropriate usage of the expression host. Give these restrictions, we chose to perform
experiments utilizing homologous sequences of PpLAAO. Ancestral sequence
reconstruction (ASR) is one of the options that could be used to obtain the homologous
sequences. To undertake this task, we attempted to redesign the sequence using ASR as
many groups have reported that protein functions such as solubility and productivity can be
improved through ASR without losing native activity 30-32. After collecting sequences of six
LAAO candidates bearing >60% genetic resemblance to PpLAAO (c in Figure 1), ancestral
LAAO (AncLAAO) was designed by following the procedure described in the Materials &
Methods (d in Figure 1). Here, no enzymatic functions of the other five LAAO candidates
(Table S1) are estimated by experimental approaches, and only sequence data are currently
available. Multiple sequence alignments amongst the six AROD, AncLAAO, and AncAROD
sequences suggested that AncLAAO shares 27% sequence identity with AncAROD (Figure
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S1). Phylogenetic analysis indicated that AncLAAO was positioned on the ancestral node of
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the LAAO family (Figure S2). This approach can clearly achieve an improvement in
overexpression of LAAO. Up to 50 mg (672 units) of AncLAAO per 1 L simple batch
cultivation can be obtained after purification with Ni affinity chromatography utilizing the
BL21(DE3) expression system (Table S3).
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Enzymatic properties of AncLAAO were characterized by biochemical analysis, and the
results indicated that AncLAAO is an FAD-dependent LAAO bearing broad substrate
selectivity. All of the enzymatic reactions were performed at a fixed oxygen concentration.
Thus, the kinetic values obtained in this study are all apparent values. Analysis of the UV-
Vis spectrum showed that two characteristic peaks derived from oxidized FAD (390 and 450
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Scheme 1. Deracemization reaction cascades to obtain enantiomerically pure D-1.
nm) could be confirmed for the purified AncLAAO sample, and the peak disappeared with
the addition of the substrate L-Met (Figure S3A) to the sample. This disappearance occured
because oxidized FAD converted to the reduced form with the progression of the reaction.
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The thermostability of AncLAAO is not high as the relative activity decreases to <50% on
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incubation at 40 °C (Figure S3B). The loss of AncLAAO activity at 30 °C and 35 °C was
negligible (Figure S3B). The optimal pH value was 7.0 (Figure S3C). Substrate selectivity
was estimated by utilizing 20 L- and D-amino acids as substrates. The results indicated that
AncLAAO can react with 13 L-amino acids that are evenly distributed between hydrophilic
and hydrophobic residues, whereas there is no activity towards any D-amino acids (Table
1). Enzyme kinetic parameters of AncLAAO were calculated for the six L-amino acids (L-Gln,
L-Met, L-Leu, L-Glu, L-Phe, and L-Trp) which exhibited the highest activity (Figure S4). The
measured parameters indicated that the kcat values ranged from 6.0 to 33.0/s (Table 2).
These values were comparable to the ones derived from other LAAOs. The kcat and kcat/Km
values of L-Met were the highest in the tested L-amino acids (Table 2).
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As discussed above, a large amount of AncLAAO can be obtained to perform
deracemization to D-amino acids from D,L-racemates. Thus, deracemization was performed
by using the combination of AncLAAO and the chemical reductant, NH3:BH3 (scheme 1).
Firstly, stereoinversion of two L-amino acids, L-Phe (L-1a) and L-Trp (L-1k), to D-amino acids
was performed. Chiral HPLC analysis indicated that both L-1a (red line, Figure 2A) and L-1k
(Figure 2B) can be completely converted into a D-enantiomer (> 99% ee) with the addition
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of 0.6 mg of AncLAAO (blue line
in Figure 2A, B). The time course
of D-1a and D-1b production from
the D,L-mixture was monitored as
shown in Figure 2C and 2D,
respectively. The results suggest
that both D,L-mixtures are
completely converted into D-
amino acids and the times
required to complete their
conversions are dependent on
the compounds and the addition
of 0.6 mg of AncLAAO. In fact,
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FIGURE 2. Chiral HPLC chromatograms of
compounds before (red line) and following
stereoinversion (blue) by AncLAAO (A, B). The
stereoinversions for L-1a and L-1k are represented in
A and B, respectively. In both cases, HPLC
chromatograms were collected for racemates (black
line). Through the addition of AncLAAO to L-1a and
1k (red line), the peak for the L-enantiomer
disappeared, whereas the peak for the D-enantiomer
appeared after the stereoinversion reaction (blue
line). Time course of D-1a (C) and D-1b (D)
production from D,L-mixture by deracemization
reaction.
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D,L-1a completely converted within 3 h (Figure 2C), whereas the conversion of D,L-1b
required approximately 24 h (Figure 2D). AncLAAO was stable during the deracemization
process in spite of its low thermostability.
The deracemization reaction was applied to a total of 18 D,L-amino acid derivatives
(Figure 3). Chiral HPLC chromatograms for each compound are represented in Figure S5.
For D,L-Phe derivatives (D,L-1a to 1j), 9 of 10 D,L-mixtures were completely converted to D-
enantiomers (>99% ee) within 24 h. Specific activity of D,L-1g (0.33 U/mg, Figure 3) was
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more than one order lower than
those of other D,L-Phe
derivatives; and this may be
one of the reasons for the
incomplete deracemization in
this instance. For D,L-Trp
derivatives (D,L-1k to 1q), six of
the seven tested D,L-mixtures
were completely converted to
D-enantiomers (> 99% ee).
Specific activity ranged from
0.63 to 12.6 U/mg, and the
addition of the chemical group
at the 7‘ position of the indole
ring appeared to decrease the
activity. In fact, the activity of
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FIGURE 3. Deracemization to D-1a-r amino acid
derivatives from D,L-mixtures. The “R” in the figure
represents amino acid side chain. Reaction conditions
for the deracemization are as follows: 100 mM KPB (pH
8.0), 5 mM substrate, 150 mM NH3:BH3, 200 U catalase
(only for 1g-1r), 10 U (1a-1q), or 20 U (1r) AncLAAO,
30 °C, 150 rpm. Conversion rate and enantio excess
were detemined as per the equation in the supporting
information. Chromatography conditions and retention
time for the compounds are listed in Table S4.
Summarizing the results, 6.9-8.6 μM of the products, D-
amino acid derivatives, were produced in 24 h by the
addition of 0.6 mg (D-1a-q) and 1.1 mg (D-1r) purified
AncLAAO.
D,L-1o (0.63 U/mg) was approximately one order lower than that of D,L-1k (6.4 U/mg, Figure
3). D,L-phenylglycine (D,L-1r) was also completely converted to the D-enantiomer despite
lower specific activity (0.21 U/mg). This is remarkable for AncLAAO because the activities
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of the reported LAADs toward D,L-1r were too weak to apply to the deracemization process
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In order to prove that AncLAAO can be applied to deracemize aromatic D,L-amino acids
to D-amino acid derivatives at preparative scale, the following five D,L-amino acid derivatives
were deracemized following the methods of the previous study 20: D,L-2-fluoro phenylalanine
(183 mg, 10 mM, 100 mL), D,L-3-fluoro phenylalanine (183 mg, 10 mM, 100 mL), D,L-4-nitro
phenylalanine (210 mg, 10 mM, 100 mL), D,L-3,4,5-trifluoro phenylalanine (219 mg, 10 mM,
100 mL), and D,L-phenylglycine (151 mg, 10 mM, 100 mL). In the presence of 150 mM
NH3:BH3, a total of 7 mg of AncLAAO was utilized to deracemize four D,L-phenylalanine
derivatives, whereas a total of 70 mg of AncLAAO was utilized to deracemize D,L-
phenylglycine. Enantiopure D-forms (> 99% ee) were obtained for the four phenylalanine
derivatives after a 24 h-reaction time (Figure S6A-D), whereas the D-form of phenylglycine
was obtained at 84% ee after 48 h (Figure S6E). This indicated that improvement of
AncLAAO reactivity toward L-phenylglycine is required to obtain enantiopure D-
phenylglycine at preparative scale. The conversion to the D-forms was evaluated by HPLC
and LC-HRMS analysis (Figure S6).
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Taken together, we succeeded in the design of AncLAAO, which is a notable development
given its broad substrate selectivity and higher productivity in the E. coli expression system
(~50.7 mg/L). Phylogenetic tree analysis for homologs of AncLAAO in the genus of
Pseudoalteromonas indicated that AncLAAO is located near the ancestor of three gene
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clusters (red-filled circle in blue-squared region, Figure S7). AncLAAO can be applied to
deracemize several aromatic D,L-amino acids to D-amino acids at preparative scale as well
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as previously reported enzymes . LAAOs from several fungi have been used to attempt
expression in the prokaryotic system, however, the expression levels of native LAAOs are
quite low and cleavage of the MBP tag and SDS treatment were required for their activation
33, 34. In contrast, no protein fusion tag or activation process was required for AncLAAO,
which makes it highly preferable. Recently, several groups have synthesized enantiopure D-
amino acids using the multienzyme reaction system, and LAADs and specific LAAOs were
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incorporated into this system
36. LAADs are especially favorable to synthesize
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enantiopure D-tryptophan derivatives at preparative scale 35. Along with these enzymes, we
believe that in the future, AncLAAO could be an important contributing component in the
process of deracemization to D-amino acids.
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Materials & Methods
Reconstruction of ancestral L-amino acid oxidase
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After the assignment of paralog sequences of L-arginine oxidase from
Pseudoalteromonas piscicida (PpAROD), we selected the six homolog sequences of the
paralog (listed in Table S1). With reference to the selection procedure indicated in a previous
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study , we selected the sequences bearing >60% identity to each other. The sequences
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were aligned with MAFFT software ; the aligned sequences were represented in Figure
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S1. The aligned sequences were analyzed by MEGA6 , and the phylogenetic tree was
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generated by the maximum-likelihood method. The aligned sequences and phylogenetic
data were submitted to the FastML web server 39; JTT empirical model was adopted for the
analysis. Finally, we were able to obtain one ancestral L-amino acid oxidase (AncLAAO);
the sequence is indicated in Table S2. Posterior probabilities (PPs) would be a criterion for
whether the designed ancestral sequence provides a plausible estimation of ancestral
proteins 31, 40. The average PPs for AncLAAO was 0.92, suggesting that the value was not
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inferior to other ancestral proteins 31, 40
.
Overexpression and purification of AncLAAO
Genes encoding AncLAAO were synthesized and cloned into the pET28a vector via the
NcoI and XhoI sites by ordering in GeneWiz. The plasmids were transformed into the
BL21(DE3) strain. The strain was cultivated in 1 L of LB broth at 37 ºC. The temperature
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was decreased to 23 ºC when the OD600 reached to 0.5–0.8, and then IPTG (isopropyl-β-D-
thiogalactopyranoside) was added to a final concentration of 0.5 mM. After the cells were
grown overnight, they were harvested by centrifugation. The cells were suspended in buffer
A (20 mM Tris-HCl [8.0] and 10 mM NaCl). After sonication of the cells, the supernatant was
collected by centrifugation at 11000 g for 35 min. The supernatant was applied to the HisTrap
HP column (GE Healthcare, Uppsala, Sweden), and the column was washed with 30 mL of
buffer A containing 10 mM imidazole. The samples were eluted utilizing 15 mL of buffer A
containing 70 mM and 300 mM imidazole, and the latter samples were utilized for further
purification. The eluted samples were concentrated and applied to the Superdex 200
Increase column (GE Healthcare, Uppsala, Sweden), equilibrated by buffer A. AncLAAO
purity was determined by SDS-PAGE. The production of AncLAAO in the E. coli expression
system was quantified by measuring specific activity of the cell-free extract and sample
towards L-Met, which applied to the His-tagged purification system (Table S3). The purified
AncLAAO was utilized in subsequent biochemical assays.
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Estimation of thermal stability and pH dependency of AncLAAO
The thermal stability of AncLAAO was determined as follows. The enzyme solution was
incubated at 30–80 ºC for 10 min without substrate. The heat-treatment samples were
moved to an ice-bath. The remaining activity was measured by applying a procedure
identical to that of the measurement of substrate specificity (Figure S3B). The pH
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dependency of AncLAAO was determined under the same conditions, except that sodium
acetate (pH 3.5 to 5.5), bis-Tris HCl (pH 6.0 to 7.0), HEPES-NaOH (pH 7.5 to 8.0), and
CAPSO (pH 9.7 to 10.5) were used as buffers (Figure S3C).
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Estimation of substrate selectivity and kinetic parameters for AncLAAO
AncLAAO activity was measured by quantifying the amount of H2O2 produced by
enzymatic reaction using a color-developed method. The assay buffer was composed of the
following reagents: 1.5 mM aminoantipyrine, 2 mM phenol, 50 U/mL horseradish peroxidase,
and 100 mM bis-Tris HCl (pH 7.0). Specific activity of AncLAAO was measured by adding
10 mM of substrate (20 L-amino acids and 20 D-amino acids) and a purified AncLAAO
sample to the assay buffer. The initial velocity of AncLAAO was calculated by monitoring the
time-dependent absorption change at 505 nm which was derived from N-ethyl-N-(2-hydroxy-
3-sulfopropyl)aniline bearing Ɛ505 = 12,700 M-1 cm-1 with a UV-Vis spectrometer (UV-2450,
Shimazu). The relative activity of AncLAAO towards 20 L-amino acids was calculated
relative to the activity of L-Met (Table 1).
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The kinetic parameters for six L-amino acids (L-Met, L-Glu, L-Phe, L-Leu, L-Trp, and L-
Gln) were measured by utilizing the following substrate concentrations: 0.1–10 mM L-Met
(Figure S4A), 0.3–10 mM L-Leu (Figure S4B), 0.1–10 mM L-Phe (Figure S4C), 0.5–15 mM
L-Trp (Figure S4D), 0.1–10 mM L-Gln (Figure S4E), and 0.5–13 mM L-Glu (Figure S4F). The
procedure to determine the initial velocity was identical to that for the measurement of
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substrate specificity. The parameters were estimated by fitting the initial velocity to the
Michaelis-Menten equation and by applying the non-linear least-squares method (Table 2).
Deracemization and stereoinversion of D,L-amino acid derivatives to D-amino acids
Deracemization of D,L-amino acids (D,L-1a to 1r) was performed by the following
procedure. We first prepared the reaction buffers composed of 100 mM KPB (pH 8.0), 150
mM NH3:BH3, 200 U/mL Catalase (only for the case of D,L-1g to 1r), and 5 mM substrates
(D,L-1a to 1r). The deracemization reaction was started by adding 0.6 mg (D,L-1a to 1q, 10
units) or 1.1 mg (D,L-1r, 20 units) of purified AncLAAO. The reactions progressed for 20 h at
30 ºC to determine the conversion rate and ee (%) value. After the reaction was complete,
80 μL of the reaction mixture were quenched by adding 720 μL of 1.15% (w/v) HClO4
solution; the supernatant was applied to subsequent HPLC analyses. Stereoinversion of L-
1a (Figure 2a) and L-1k (Figure 2b) was performed by applying the identical procedure.
The time course of deracemization of D,L-1a (Figure 2c) and D,L-1b (Figure 2d) was
determined by sampling 100 μL of the reaction mixture after incubation for 0, 10, 20, 40, 60,
90, 180, and 360 min, and 24 h. In the measurement, the amount of AncLAAO was changed
to 5 U/mL and other components of the reaction buffer were identical to that of the
deracemization condition. The mixture was incubated at 90 ºC for 10 min to stop the reaction,
and a total 40 μL of the mixture was dissolved by adding 360 μL of 1.15% (w/v) HClO4
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solution; the supernatant was applied to HPLC analyses.
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HPLC analyses
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Reverse phase HPLC analyses were performed on the Shimadzu apparatus
(Prominence) equipped with a UV-Vis detector (SPD-20AV, Shimadzu) and CROWNPAK
CR-I(+) column, length/internal diameter = 150/3.0 mm (DAICEL, Osaka, Japan). Elution of
the compounds was monitored by detecting UV changes at 210 nm. Mobile phase condition
was 1.15% (w/v) HClO4, and column temperature was set to 30 ºC. Other conditions, such
as retention time and flow rate for each of the compounds (1a-1r), are summarized in Table
S4. HPLC chromatograms for all of the compounds are shown in Figure S5. Conversion
rates and enantiomer excesses (ee(%)) were calculated by the following equation:
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[(
)
(
)]
퐷푎푟푒푎 − 퐿푎푟푒푎
푒푒 (%) =
⁄
× 100 (After reaction)
[(
)
(
)]
퐷푎푟푒푎 + 퐿푎푟푒푎
(
)
퐷푎푟푒푎 푎푓푡푒푟 푟푒푎푐푡푖표푛
( )
Conversion rate % =
315
⁄
[(
)
(
)]푏푒푓표푟푒 푟푒푎푐푡푖표푛
퐷푎푟푒푎 + 퐿푎푟푒푎
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Here, Darea and Larea represent the peak areas of HPLC corresponding to the D- and L-
enantiomers, respectively.
Qualitative analysis of products by LC-HRMS
LC-HRMS analysis (Figure S6) was carried out using Q Exactive (Thermo Fisher Scientific,
MA, USA), equipped with an electrospray ionization module. To detect the products by LC-
HRMS, the following columns which are jointed into the LC-HRMS system were utilized: a
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UPLC column (XBridge® BEH Amide XP column [length, 2.1 x 50 mm; inner diameter (i.d.),
2.5 μm; Nihon Waters K.K., Tokyo, Japan]) equipped with a guard column (XBridge® BEH
Amide XP VanGuard cartridge [length, 2.1 x 5 mm; i.d., 2.5 μm; Nihon Waters K.K., Tokyo,
Japan]). The column was maintained at 40°C. 5 mM ammonium formate in 90% acetonitrile
(solvent A) and 5 mM ammonium formate in 50% acetonitrile (solvent B) were used as
mobile phases for the gradient elution of products at a flow rate of 0.4 mL/min. Products
were eluted as follows: 0% B for 1 min, 0–100% B over 4 min, 100% B for 2 min, and 0% B
for 5 min.
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Supporting Information Available
Supporting information includes the following figures: Multiple sequence alignment of
homologous sequences of PpLAAO; Phylogenetic analysis of the AncLAAO, AncAROD,
and six homologous sequences; UV-Vis spectrum changes, thermal stability, and optimal
pH of AncLAAO; Enzyme kinetics of AncLAAO toward six L-amino acids; Chiral HPLC
chromatograms of compounds from D,L-1a to 1r; LC-HRMS analysis of D-amino acid
derivatives deracemized at preparative scale; and Phylogenetic analysis for paralogs of
AncLAAO in genus of Pseudoalteromonas. The Supporting Information contains the
following tables: Sequence lists used to design AncLAAO; Sequence of AncLAAO;
Summary of purification of AncLAAO from 1 L cultivation; and LC condition and retention
time for amino acid derivatives. This information is available free of charge on the ACS
Publications website.
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Acknowledgements
This work was supported by JSPS KAKENHI grant numbers 16K18688, 17K06931, and
18K14391. S.N. designed ancestral LAAO (AncLAAO). S.N., Y.M., and F.H. performed the
experiments. S.N., M.N., and S.I. performed the data analysis. S.N. and S.I. designed the
research study. S.N. wrote the manuscript.
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TABLES
Table 1. Relative activity of AncLAAO towards 20 L-amino acidsa
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Substrate
Relative activity (%)
L-Glutamine
L-Methionine
L-Leucine
101.0±1.8
100±2.1
85.2±1.7
65.9±1.4
52.9±0.3
40.8±0.5
25.8±1.1
8.8±0.2
5.5±0.2
1.5±0.2
1.3±0.4
1.3±0.4
0.8±0.6
< 0.0
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L-Glutamic Acid
L-Phenylalanine
L-Tryptophan
L-Tyrosine
L-Isoleucine
L-Arginine
L-Proline
L-Valine
L-Alanine
L-Lysine
L-Cysteine
L-Aspartic Acid
Glycine
< 0.0
< 0.0
L-Histidine
L-Asparagine
L-Serine
< 0.0
< 0.0
< 0.0
L-Threonine
< 0.0
477
478
479
a There is no activity toward any of the 20 D-amino acids.
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Table 2. Enzyme kinetic parameters of AncLAAO towards L-Met, L-Leu, L-Phe, L-Trp,
L-Gln, and L-Glua
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Compounds
kcat
Km
kcat/Km
/s
mM
/(s·mM)
Hydrophobic amino acids
L-Met
22.0 ± 1.1
7.5 ± 0.5
0.4 ± 0.1
0.6 ± 0.1
55.0
12.5
L-Leu
Aromatic amino acids
L-Phe
12.6 ± 0.4
6.4 ± 0.6
3.7 ± 0.3
3.1 ± 0.6
3.4
2.1
L-Trp
Hydrophilic amino acids
L-Gln
L-Glu
13.3 ± 0.2
33.0 ± 3.5
1.5 ± 0.1
8.9
2.9
11.5 ± 2.2
483
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aThe measurement of enzyme kinetic parameters was performed independently three
times (n=3).
486
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TOC Figure
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