Aminolytic reaction catalyzed by d-stereospecific amidohydrolases from Streptomyces spp

Article abstract of DOI:10.1016/j.biochi.2011.04.020

From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized. Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with respect to temperature and pH. Among hydrolytic activities toward d-aminoacyl-pNAs, the enzymes showed strict specificity toward d-Phe-pNA, but showed broad specificity toward d-aminoacyl esters. The specific activity for d-Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As a second function, each enzyme showed peptide bond formation activity by its function of aminolysis reaction. Based on results of d-Phe-d-Phe synthesis under various conditions, we propose a reaction mechanism for d-Phe-d-Phe production. Furthermore, the enzymes exhibited peptide elongation activity, producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which revealed that the primary structure of each enzyme showed 30-60% identity with those of peptidases belonging to the clan SE, S12 peptidase family categorized as serine peptidase with d-stereospecificity.

Full text of DOI:10.1016/j.biochi.2011.04.020

Biochimie 93 (2011) 1460e1469
Contents lists available at ScienceDirect
Biochimie
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Research paper
Aminolytic reaction catalyzed by -stereospecific amidohydrolases
D
from Streptomyces spp
,
a
b
a
Jiro Arima a , Hitomi Ito , Tadashi Hatanaka , Nobuhiro Mori
*
a Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan
b Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze
Received 17 September 2010
Accepted 25 April 2011
Available online 5 May 2011
D
-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The
enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized.
Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with
respect to temperature and pH. Among hydrolytic activities toward
showed strict specificity toward -Phe-pNA, but showed broad specificity toward
specific activity for -Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As
a second function, each enzyme showed peptide bond formation activity by its function of aminolysis
reaction. Based on results of -Phee -Phe synthesis under various conditions, we propose a reaction
mechanism for -Phee -Phe production. Furthermore, the enzymes exhibited peptide elongation activity,
D
-aminoacyl-pNAs, the enzymes
Keywords:
Amidohydrolase
D
D-aminoacyl esters. The
D
D
-Amino-acid
Aminolysis
Streptomyces
Peptide bond formation
D
D
D
D
producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which
revealed that the primary structure of each enzyme showed 30e60% identity with those of peptidases
belonging to the clan SE, S12 peptidase family categorized as serine peptidase with
D-stereospecificity.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
enzymatic synthesis of
b-amino acid containing peptides [13,14],
diverse prolyl peptides [15], and
D-alanine oligopeptides [8]. Their
Peptides in nature incorporating
microbial compounds and signal molecules. They are expected to
widen and extend the functional variety and applications of peptides
D
-amino-acids are useful as anti-
simple kinetics and the obviation of expensive additives make this
peptide synthetic system easy to use [16,17].
In this study, we identified
D-stereospecific amidohydrolases
[1e5]. Although
D-amino-acid-containing peptides have been
(D-aminopeptidase) in the culture supernatant of Streptomyces spp.
produced using organic synthesis [6,7], the complicated reaction steps
and the racemization of amino acids are persistent difficulties asso-
ciated with their organic synthesis. Therefore, enzyme-catalyzed
peptide synthesis is widely regarded as an alternative means of
chemical synthesis. Recent reports have described enzymatic
syntheses using peptidase [8,9], esterase [10], aminoacyltransferase
82F2 and 83D12, the activities of which were inhibited by phe-
nylmethylsulfonyl fluoride (PMSF). The enzyme is of particular
interest as a useful tool for synthesis of various peptides incorpo-
rating D-amino-acid because it has a function of aminolysis reac-
tion. This report describes the mechanism of peptide bond
formation, differences in properties of obtained enzymes, and
sequence analysis data.
[11], and D-alanineeD-alanine ligase [12]. Among them, aminolysis
reactions, reactions of an amine with a carboxylic acid derivative to
form an amide, of serine amidohydrolases containing peptidases,
esterases, and aminoacyl transferases have increasingly attracted
attention as tools for peptide synthesis. Recent reports describe
2. Materials and methods
2.1. Materials and bacterial strain
Abbreviations: 82F2-DAP,
D-stereospecific amidohydrolases from Streptomyces
Peptides, aminoacyl p-nitroanilides (pNAs), and other amino-
acyl derivatives were purchased from Bachem AG, Aldrich Chemical
Co. Inc., Sigma Chemical Co., Novabiochem Corp., and Wako Pure
Chemical Industries, Ltd. Streptomyces spp. 82F2 and 83D12 were
isolated from soil and identified using 16S ribosomal DNA analysis.
sp. 82F2; 83D12-DAP, -stereospecific amidohydrolases from Streptomyces sp.
D
83D12; PMSF, phenylmethylsulfonyl fluoride; pNA, p-nitroanilide; OBzl, benzyl
ester; OMe, methyl ester; tBu, tert-butyl ester.
* Corresponding author. Tel.: þ81 857 31 5363.
E-mail address: arima@muses.tottori-u.ac.jp (J. Arima).
0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2011.04.020

J. Arima et al. / Biochimie 93 (2011) 1460e1469
1461
2.2. Enzyme assay
of-flight mass spectrometry (MALDIeTOF MS). Gel filtration was
performed as described in the Supplemental material. MAL-
DIeTOF MS was performed using Autoflex TOF (Bruker Daltonics
For routine assay, the enzyme activity was determined using
continuous spectrophotometric assay with a
D
-Phe-pNA substrate.
Inc.). For sample preparation, the purified enzymes (5 mg mlꢁ1
)
In the assay, 0.1 ml of substrate solution (10 mM) was added to
were desalted using dialysis against Milli-Q water. For the
molecular mass determination, we chose 2,5-dihydroxybenzoic
acid as the MALDI matrix. Effects of pH on hydrolytic activity and
the pH stabilities of the enzymes were tested as described in
Supplemental materials. Thermostability was tested by incu-
0.9 ml of a mixture containing 200 mM Trisemalate (pH 6.5) and
10
m
g/ml of enzyme at 25 ꢀC. Increased absorption at 405 nm
caused by the release of p-nitroaniline per minute was monitored
continuously using a spectrophotometer (U2800; Hitachi, Ltd.). The
initial rate was determined from a linear segment of the optical
density profile (3405 nm ¼ 10,600 Mꢁ1 cmꢁ1 [18]).
bating 100
temperatures of 30e70 ꢀC. Residual activity was measured
using
-Phe-pNA at 25 ꢀC. Fluorescence emission spectra of the
m
l of a sample (1 mg mlꢁ1 protein) for 30 min at
D
2.3. Purification of the enzymes from culture supernatant
enzymes treated with respective temperature for 30 min were
recorded using an F4500 fluorescence spectrophotometer (Hita-
chi, Ltd.). The excitation wavelength was 284 nm, and emission
spectra were recorded between 300 and 400 nm in cuvettes with
1 cm path length. The kinetic parameters were determined at
25 ꢀC in a mixture containing 0.1 ml of an enzyme solution
Streptomyces spp. 82F2 and 83D12 were grown aerobically in
400 ml of culture medium containing 0.8% K2HPO4, 2.0% glucose,
0.05% MgSO4$7H2O, 0.5% polypeptone, and 0.5% yeast extract at
25 ꢀC for 6 days. The culture supernatant was brought to 45%
saturation using ammonium sulfate. Then the solution was loaded
onto a column (Butyl-Sepharose Fast Flow; GE Healthcare) equili-
brated with 100 mM TriseHCl (pH 8.0) containing 45% ammonium
sulfate. The column was washed with the same buffer containing
20% ammonium sulfate. Then the enzyme was eluted using
100 mM TriseHCl. Fractions exhibiting high activity were pooled
and then dialyzed against 20 mM TriseHCl (pH 8.0). Next, the
dialysate was loaded onto a spin column (Vivapure-Q; Sartorius AG)
equilibrated with 20 mM TriseHCl (pH 8.0). After washing with the
same buffer, the bound protein was eluted with the same buffer
containing 0.2 M NaCl. Fractions exhibiting high activity were
pooled and then dialyzed against 20 mM sodium acetate (pH 5.5).
The dialysate was loaded onto a spin column (Vivapure-S; Sartorius
AG) equilibrated with 20 mM sodium acetate (pH 5.5). After
washing with the same buffer containing 0.1 M NaCl, the bound
protein was eluted with the same buffer containing 0.4 M NaCl. The
homogeneity of purified proteins was confirmed using 12%
SDSePAGE under denaturing conditions [19].
(0.1 mg mlꢁ1), 0.1 ml of a 0.1e10 mM
D-Phe-pNA solution, and
0.8 ml of 200 mM Trisemalate (pH 6.5). Inhibition of the activity
by penicillin G was determined by treating each enzyme with
0.1e10 mM penicillin G at pH 6.5 and 25 ꢀC for 5 min. Residual
activity was measured using D
-Phe-pNA at pH 6.5 and 25 ꢀC.
2.6. Peptide bond formation by aminolysis reaction
Peptide synthesis by aminolysis reaction was performed as
follows: 2
added to 46
initiated by adding 2
m
l of aminoacyl esters dissolved in DMSO (0.5 M) was
l of 0.25 M TriseHCl (pH 9.0). The reaction was
l of 0.2 mg mlꢁ1 purified enzyme. The
m
m
reaction was then continued at 25 ꢀC for an appropriate time
(5 mine72 h). The reaction was terminated by adding 0.05 ml of 3%
formic acid. The molecular mass of peptides synthesized by the
enzymes was determined using ESIeTOF MS, for which the reaction
mixture was diluted with 200 or 4000-fold volume of 0.1% formic
acid. After the solution was filtered, 5
analyzed using an ESIeTOF MS system (LCT Premier XE; Waters
Corp.). Synthesized -Phee -Phe, -Phee -Phee -Phe (( -Phe)3),
hydrolysate -Phe, and substrate -Phe-OMe were quantified using
ml of each sample was
2.4. Hydrolytic activity toward aminoacyl esters and peptides
D
D
D
D
D
D
D
D
The hydrolytic activity toward aminoacyl derivatives and
peptides was determined through quantitative assay of amino
acids, N-acetylated amino acids, and deesterified peptides derived
UPLC e ESIeTOF MS equipped with a C18 reverse-phase system.
The reaction mixture was diluted with 4000-fold volume of 0.1%
formic acid and filtered; then 5
chromatography. Each sample was eluted and analyzed as
described in Section 2.4. For quantifying the synthesized ( -Phe)3,
a standard curve for ( -Phe)3 was used instead of a standard for
-Phe)3.
ml of each sample was subjected to
from the hydrolyzed substrates. To 46
acid (pH 6.5), 2
l of an enzyme solution (0.05 or 0.2 mg mlꢁ1) and
l of a substrate solution (0.5 M) were added. The reaction
mixture was incubated at room temperature for 5 min. Then the
reaction was stopped by the addition of 50 l of 3% formic acid. The
ml of 200 mM Trisemaleic
m
D
2
m
L
(D
m
liberated amino acid was quantified using ultrahigh-performance
liquid chromatography (UPLC)-electrospray ionization time-of-
flight mass spectrometry (ESIeTOF MS) equipped with a C18
reverse-phase system (Acquity UPLC; Waters Corp.). The reaction
mixture was diluted with 200-fold or 4000-fold volume of 0.1%
2.7. Analysis of length of peptide synthesized from D-Tyr-OMe
A precipitate produced in the 3 h reaction using
collected by centrifugation, washed twice with distilled water, then
dissolved in 100 l of 50% acetic acid containing 0.1% formic acid.
D-Tyr-OMe was
m
formic acid and filtered. Then 5
m
l of each sample was subjected to
The obtained samples were analyzed using Autoflex TOF (Bruker
Daltonics Inc.). For the molecular mass determination, we chose
2,5-dihydroxybenzoic acid as the MALDI matrix.
chromatography. Each sample was eluted with solvent A e solvent
B of 95:5 for 2 min, solvent A e solvent B of 80:20 for 1 min, solvent
A e solvent B gradient of 80:20 to 50:50 for 2 min, and solvent A e
solvent B of 20:80 for 2 min, where solvent A was Milli-Q water
containing 0.1% formic acid and solvent B was acetonitrile con-
taining 0.1% formic acid. The data were processed using a computer
program (MassLynx; Waters Corp.).
2.8. Specificity for acyl acceptor
Specificity for acyl acceptor in the aminolysis reaction was
evaluated as follows: 2
(0.5 M) was added onto 46
taining 20 mM acetylated (Ac-)
initiated by adding 2
m
l of aminoacyl esters dissolved in DMSO
l of 0.25 M TriseHCl (pH 9.0) con-
-Phe-OMe. The reaction was
m
2.5. Biochemical studies
D
m
l of 0.2 mg mlꢁ1 purified enzyme. The
The molecular mass of the enzymes was determined using gel
filtration and matrix-assisted laser desorption ionization e time-
reaction was then continued at 25 ꢀC for 1 h. The reaction was
terminated by adding 0.05 ml of 3% formic acid. The molecular mass

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J. Arima et al. / Biochimie 93 (2011) 1460e1469
Table 1
Purification of 82F2-DAP and 83D12-DAP from the culture supernatant.
82F2-DAP
83D12-DAP
Total protein Total activity Specific activity Purification Yield
Total protein Total activity Specific activity Purification Yield
(mg)
(U)
(U/mg)
(fold)
(%)
(mg)
(U)
(U/mg)
(fold)
(%)
Culture supernatant 263
284
233
130
1.08
6.66
10.4
18.3
1
100
82
45.8
21.5
252
41.3
16.1
62.0
31.2
15.7
0.25
0.76
0.98
1.72
1
100
50.3
25.3
14.0
Butyl-sepharose
Vivapure-Q spin
Vivapure-S spin
35.0
12.5
3.35
6.2
9.6
16.9
3.1
4.0
7.0
61.2
5.06
8.69
Enzyme activity was determined using 1 mM D-Phe-pNA and 200 mM Trisemalate (pH 6.5). The protein concentration was determined using the Bradford method.
of peptides synthesized by the enzymes was determined using
ESIeTOF MS, as described in Section 2.6.
2.9. Determination of N-terminal and internal amino
acid sequences
The purified enzyme was blotted onto a PVDF membrane after
12% SDSePAGE under denaturing conditions [19]. The membrane
was then stained with Coomassie Brilliant Blue. The protein band
was excised from the membrane. Subsequently, the protein band
was sent to APRO Life Science Institute, Inc., from whom we
requested determination of the N-terminal and internal sequence
using Edman degradation.
2.10. Sequencing of the gene encoding
amidohydrolases
D-stereospecific
Chromosomal DNA was prepared using the method described
by Saito and Miura [20]. To obtain gene encoding -stereospecific
D
amidohydrolases from Streptomyces spp., two primer sets were
designed (82IntF-82IntR and 83NF-83IntR) using the N-terminal and
internal amino acid sequences for respective amplification of the
Fig. 2. Thermal stability of 82F2-DAP and 83D12-DAP. (A) Residual activity of enzymes
after treatment at respective temperatures for 30 min. Each value is the average of
three independent experiments ꢂ the standard deviation. (B) Fluorescence spectra of
enzymes after treatment at respective temperatures for 30 min: 30, 40, 50, 55, 60, and
65 ꢀC. Upper panel: spectra of 82F2-DAP after treatment at 30 ꢀC and 40 ꢀC are shown
as black lines. Those at 30e65 ꢀC are shown as gray lines. Lower panel: spectra of
83D12-DAP after treatment at 30e55 ꢀC are shown as black lines; those of 60 ꢀC and
65 ꢀC are shown as gray lines.
Fig. 1. MALDIeTOF MS analysis of purified 82F2-DAP and 83D12-DAP.

J. Arima et al. / Biochimie 93 (2011) 1460e1469
1463
Table 2
Substrate specificities for hydrolytic activities of 82F2-DAP and 83D12-DAP.
amidohydrolase that exhibited
D
-aminopeptidase activity: Strep-
tomyces spp. 82F2 and 83D12. Their activities were inhibited by
PMSF. Therefore, we assumed that the enzymes were serine-type
amidohydrolases. We purified the enzymes from the culture
supernatants to obtain information related to their characteristics.
Substrate
82F2-DAP
mol minꢁ1 mgꢁ1
83D12-DAP
mol minꢁ1 mgꢁ1
)
(
m
)
(
m
Nitrocefina
n.d.c
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Gly pNAa
As Table
1 shows, three purification steps (butyl-sepharose,
L
-Ala-pNAa
Vivapure-Q, and Vivapure-S) were performed after ammonium
D
-Ala-pNAa
-Leu-pNAa
-Leu-pNAa
-Phe-pNAa
-Phe-pNAa
Ac-DL-Phe-pNAa
L
D
L
0.2 ꢂ 0.04
D
18.3 ꢂ 2.1
n.d.
1.7 ꢂ 0.1
n.d.
n.d.
D
D
D
-Alae
-Alae
-Alae
L
-Alab
n.d.
D
D
-Alab
1.5 ꢂ 0.2
1.6 ꢂ 0.2
-Ala-OMeb
n.d.
n.d.
(detection of free
D
-Ala)
-Ala)
D
-Alae
D
-Ala-OMeb
2.9 ꢂ 0.5
3.2 ꢂ 0.3
(detection of
-Ala-Glyb
D
-Alae
D
D
D
D
D
D
D
D
0.3 ꢂ 0.1
0.4 ꢂ 0.1
n.d.
0.4 ꢂ 0.03
0.3 ꢂ 0.1
-Ala-Gly Glyb
-Ala-
L
-Leub
n.d.
-Ala-
L
-Pheb
n.d.
n.d.
n.d.
n.d.
-Phee
D
-Pheb
-Ala-OMeb
-Val-OMeb
0.6 ꢂ 0.1
16.6 ꢂ 2.9
1.8 ꢂ 0.4
64.1 ꢂ 7.1
20.5 ꢂ 1.5
15.0 ꢂ 1.5
518 ꢂ 15
86.3 ꢂ 7.7
83.0 ꢂ 8.7
135 ꢂ 24
105 ꢂ 5
7.5 ꢂ 1.8
6.3 ꢂ 1.0
3.3 ꢂ 0.3
4.5 ꢂ 0.2
0.7 ꢂ 0.1
17.2 ꢂ 3.3
3.8 ꢂ 1.2
87.2 ꢂ 12.6
37.4 ꢂ 2.7
9.2 ꢂ 1.0
365 ꢂ 19
47.4 ꢂ 4.0
123 ꢂ 14
109 ꢂ 3
L
-Leu-OMeb
D
-Leu-OMeb
-Leu-OBzlb
D
L
-Phe-OMeb
D
-Phe-OMeb
-Phe-OBzlb
D
Ac-D
-Phe-OMeb
D
D
D
D
D
D
-Trp-OMeb
-Tyr-OMeb
-Pro-OMeb
-Ser-OMe
-Ser-OBzlb
-His-OMe
44.7 ꢂ 5.7
7.7 ꢂ 0.6
4.3 ꢂ 0.5
2.6 ꢂ 0.4
5.0 ꢂ 0.3
All data are expressed as the mean ꢂ standard deviation of three independent
experiments.
a
Reactions using aminoacylepNA derivatives were performed at a final substrate
concentration of 1 mM at pH 6.5.
b
Reactions using aminoacyl esters and peptides were performed at a final
substrate concentration of 20 mM at pH 6.5 and assayed using UPLC-TOF/MS
analysis.
c
n.d. not detectable (velocity is less than 0.1 m
mol minꢁ1 mgꢁ1).
genes encoding 82F2-DAP and 83D12-DAP (see Table S1 in
Supplemental materials). After PCR amplification using chromo-
somal DNA, we determined partial sequences using the amplified
DNA fragments. Next,
a digoxigenin-labeled PCR probe was
synthesized using a synthesis kit (PCR DIG Probe; Roche Molecular
Biochemicals). Approximately 2.0-kb fragments of chromosomal
DNA of Streptomyces sp. 82F2 digested by SmaI or SacI and 1.0-kb
fragments of chromosomal DNA of Streptomyces sp. 83D12 digested
by SmaI or SacII were hybridized using the probe. The positive
fragments were recovered and self-ligated. Then the ligation
products were amplified using PCR with the primer set, 82InverseF-
82InverseR for use of 2.0-kb fragments from Streptomyces sp. 82F2
chromosomal DNA and 83InverseF-83InverseR for using 1.0-kb
fragments from Streptomyces sp. 83D12 chromosomal DNA. The
PCR products were then used for sequencing. The DNA sequences
were assigned accession nos. AB531389 and AB581523 in the DDBJ
database.
Fig. 3. D-PheeD-Phe synthesis by 82F2-DAP and 83D12-DAP. (A) MS of product
synthesized with D-Phe-OMe as
a substrate. For both investigations, the final
concentration of D-Phe-OMe was 20 mM. The reaction was performed at 25 ꢀC and pH
6.5 and 8.0 for 1 h b. The panels portray profiles of the reaction mixture with (w/82F2-
DAP or w/83D12-DAP) and without (w/o enzyme) the enzyme for comparison of
product synthesis by each enzyme with that of a negative control. (B) Effect of pH on D-
3. Results
3.1. Purification of D-stereospecific amidohydrolases
PheeD-Phe synthetic activity. The reaction was performed at 25 ꢀC in 0.2
Trisemalate (pH 6.0e8.0, closed circles) and TriseHCl (pH 8.0e9.0, open circles). Each
value is the average of three independent experiments ꢂ the standard deviation.
M
By screening for enzymes with
among 2000 soil isolates, we obtained two strains that secreted an
D-Phe-pNA hydrolytic activity

1464
J. Arima et al. / Biochimie 93 (2011) 1460e1469
Fig. 4. Time dependence on the products by hydrolytic and aminolytic reaction. (A) Profiles in the reaction using 82F2-DAP. (B) Profiles in the reaction using 83D12-DAP. The
reaction was continued up to 1 h. As substrate, D-Phe-OMe at 20 mM was used. The reaction was performed at pH 9.0 and 25 ꢀC. Each value represents the average of three
independent experiments ꢂ the standard deviation.
sulfate precipitation. Both enzymes were purified to homogeneity,
as determined using SDSePAGE (data not shown). The enzyme
showed molecular masses of approximately 37 kDa using MAL-
DIeTOF MS analysis (Fig. 1) and 35e37 kDa by gel filtration (see
Fig. S1 in Supplemental materials), suggesting that the enzymes are
using
substrate was used, deesterification activity was observed instead
of the -Ala releasing activity.
In contrast to the results described above, each enzyme showed
broad specificity toward -aminoacyl ester derivatives including
D-alanyl peptides as substrates. When a D-AlaeD-Ala-OMe
D
D
monomers. We renamed the enzymes with
D
-aminopeptidase
benzyl esters (eOBzls) and methyl esters (eOMes). Although the
activity as “82F2-DAP” and “83D12-DAP”.
enzymes show no activity toward acetylated (Ac-) DL-Phe-pNA, Ac-
D
-Phe-OMe was hydrolyzed by each enzyme. Similarly, a difference
in hydrolytic activity was observed between the structures of
esterified moiety. The hydrolytic activity of each enzyme toward
aminoacyl-OMe was 2e8-fold higher than that toward -amino-
acyl-OBzl (Table 2). These results reflect that the flanking moiety
affects the hydrolytic activities of the enzymes toward aminoacyl
derivatives.
3.2. Properties of purified enzymes
D
-
D
We next characterized the purified 82F2-DAP and 83D12-DAP.
As shown in Fig. S2 in Supplemental materials, each enzyme is
stable at pH 5.0e10.5; they exhibited moderate stability with
respect to temperature (up to 45 ꢀC) from the assay of residual
activity after heat treatment (Fig. 2A). The maximum of fluores-
cence emission spectra of 82F2-DAP shifted approximately 10 nm
to the long wavelength side after treatment at 50 ꢀC for 30 min,
indicating that the overall structure was changed by the treatment
(Fig. 2B). In contrast, the spectra of 83D12-DAP shifted slightly to
the short wavelength side after treatment at 60 ꢀC for 30 min. The
results indicate that 83D12-DAP has slightly higher structural
stability than that of 82F2-DAP.
3.3. Peptide bond formation by aminolysis reaction
We evaluated whether the obtained enzymes catalyze the
peptide synthetic activity using D-Phe-OMe as a substrate under the
condition of pH 6.0 and 8.0 for 1 h. As portrayed in Fig. 3A, the MS
The activities for D-Phe-pNA hydrolysis were optimum at pH
5.5e6.5. However, for pH higher than 8.0, a gradual increase in
activity was observed following the increase of pH in each enzyme.
Investigation of substrate specificity using aminoacyl-pNAs showed
that the enzymes specifically hydrolyzed
hydrolysis rate of 82F2-DAP toward -Phe-pNA was approximately
ten-fold higher than that of 83D12-DAP. In addition to the reaction
rate difference, we found that the Km value for -Phe-pNA hydro-
lytic activity of 82F2-DAP was approximately seven-fold higher
than that of 83D12-DAP (the Km values for -Phe-pNA hydrolytic
D-Phe-pNA (Table 2). The
D
D
D
activity of 82F2-DAP and 83D12-DAP are, respectively, 1.3 ꢂ 0.1 and
0.18 ꢂ 0.02 mM). In addition, 82F2-DAP exhibited little activity
toward L-Phe-pNA, indicating that the enantioselectivity of the
Fig. 5. Effect of D-PheeD-Phe concentration on the (D-Phe)3 synthesis. D-Phe-OMe at
20 mM and D-PheeD-Phe at 0e10 mM were used as substrates. Open circles, 82F2-
DAP; closed circles, 83D12-DAP. For investigations, the reaction was performed at 25 ꢀC
for 1 h. Each value represents the average of three independent experiments ꢂ the
standard deviation.
enzymes is not strict toward
a
-carbon of
a-amino acid. The
enzymes have no activity toward any other
L-aminoacyl-pNAs,
nitrocefin, or N-blocked DL-aminoacyl-pNAs. In the investigation
using peptides, little activity was observed in the examination

J. Arima et al. / Biochimie 93 (2011) 1460e1469
1465
Fig. 6. Proposed mechanism for the catalytic reaction of 82F2-DAP and 83D12-DAP using D-Phe-OMe as substrate. The reaction presented in the lower half is the D-PheeD-Phe
production by the hydrolysis of (D-Phe)3, which is synthesized from D-PheeD-Phe (acyl acceptor) and D-Phe-OMe (acyl donor).
spectrum exhibits apparent product peaks with m/z of approxi-
mately 313.2 and 460.4, which are similar to the theoretical
was much lower than that of pH 8.0 in the investigation using each
enzyme. The pH-dependence curve for the aminolysis reaction
molecular masses of
D
-Phee
D
-Phe and
D
-Phee
D
-Phee
D-Phe ((
D-
differed from that for the
D-Phe-pNA hydrolytic activity: The
Phe)3). The ion intensity of
D
-Phee -Phe using the sample of pH 6.0
D
maximum velocity was observed at pH 7.0 (Fig. 3B). The hydrolytic
Table 3
Tested chemicals and homopeptides synthesized using 82F2-DAP and 83D12-DAP.
Substrate
Product
Amino acid
moiety
Type of derivatization
of carboxyl group
82F2-DAP
(homopeptides)
(w/Ac-
n.d.
D
-Phe-OMe)
83D12-DAP
(homopeptides)
(w/Ac-
n.d.
D-Phe-OMe)
Gly
eOMe
eOMe
eOBzl
eOMe
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
D
-Ala
Ac-
Ac-
D
-F-
-F-
D
-A-OMe
-A-OBzl
Ac-
Ac-
D
-F-
-F-
D
-A-OMe
D
D
D
D-A-OBzl
D
-Val
D
-V-
D
D
-V
-V
D
-V-
Ac-
-V-
Ac-
D
-V, Ac-
-F- -V-OMe
-V, Ac- -F-
-F- -V-OBzl
-L, Ac- -F-
-L, Ac- -F-
D
-F-
D
D
-V
-V
D
-V-
D
D
-V
-V
D
-V-
Ac-
-V-
Ac-
D
-V, Ac-
-F- -V-OMe
-V, Ac- -F-
-F- -V-OBzl
-L, Ac- -F-
-L, Ac- -F-
D
-F-
D
D
-V
-V
D
D
D
D
eOBzl
D
-V-
D
D
D
D
-V-
D
D
D
D
D
D
D
D
D
D
-Leu
-Ile
eOMe
eOBzl
eOBzl
D
D
-L-
D
-L, (
-L, (
D
D
-L)3
-L)3
D
-L-
D
D
D
-L
D
D
-L-
D
-L, (
-L, (
D
D
-L)3
-L)3
D
-
L-D
D
D-L
-L-D
D
-
L-
D
D
D
-L
-L-D
D
-L-D
D
D
-L
n.d.
Ac-
Ac-
D
-F-
-F-
D
-I
-I-OBzl
n.d.
Ac-
Ac-
D
-F-
-F-
D
-I
-I-OBzl
D
D
D
D
-Phe
eOH
e
n.d.
e
n.d.
eOMe
eOBzl
eOMe
D
D
D
-F-
-F-
-W-
D
D
-F, (
-F
D
-F)3
D
D
D
-F-
-F-
-W-
D
D
-F, Ac-
-F, Ac-
D
D
-F-
D
-F
-F
D
D
D
-F-
-F-
-W-
D
D
-F, (
-F
D
-F)3
D
D
D
-F-
-F-
D
D
-F, Ac-
-F, Ac-
D
D
-F-
-F-
D
D
-F
-F
-F-D
D-Trp
D
-W
D
-W, Ac-
D
D
-F-
D
-W
-W
D
-W
-W-
-F-
-W- -W,
Ac- -F- -W, Ac-
-Y- -Y, ( -Y)3,
Ac- -F- -Y, Ac-
D
-W,
-W, Ac-
-W-D
D
-W-
D
-W-OMe,
-F- -W-OMe
-W-OBzl,
-F- -W-OBzl
-Y- -Y-OMe,
-F- -Y-OMe
Ac-
D
D
D
D
D
eOBzl
eOMe
D
D
-W-
D
-W
D
D
-W-
D
-W, Ac-
-F-
D
D
D
-W-
D
-W
D
D
D
D
D
D
D
-Tyr
-Y-
-Y-
D
D
-Y, (
D
-Y)3, (
D
D
-Y)4
-Y)4
-Y-
-Y-
D
D
-Y, (
-Y, (
D
D
-Y)3, Ac-
-Y)3, Ac-
D
-F-
D
D
-Y
-Y
-Y-
-Y)4,
-Y)3-OMe, (
-Y- -Y, ( -Y)3,
-Y)4, -Y- -Y-OBzl,
-Y)3-OBzl, ( -Y)4-OBzl
D
-Y, (
D
-Y)3,
-Y-OMe,
-Y)4-OMe
D
D
D
D
D
(D
D
-Y-
D
D
D
D
D
(D
D
eOBzl
D
-Y, (
D
-Y)3, (
D
D
-F-
D
D
D
D
-Y-
D
D
-Y, (
D
-Y)3,
D
-Y-
D
-Y-OBzl,
(D
D
D
Ac-
-F-
D
-Y, Ac-D-F-
D-Y-OBzl
(D
D
D
D
-Pro
-Ser
eOMe
eOBzl
eOMe
eOBzl
eOMe
eOMe
eOMe
eOMe
eOH
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Ac-
Ac-
Ac-
D
-F-
-F-
-F-
D
D
D
-S, Ac-
-S
-H, Ac-
D
-F-
-F-
-F-
D
-S-OMe
Ac-
Ac-
Ac-
Ac-
Ac-
D
-F-
-F-
-F-
-F-
-F-
D
D
D
D
D
-S-OMe
D
D
D
D
D
D
-S, Ac-
-H, Ac-
-R, Ac-
-K, Ac-
D
-F-
-F-
-F-
-F-
D-S-OBzl
D
D
D
D
D
-His
-Arg
-Lys
-Asp
-Phee
D
D
-H-OMe
D
D
-H-OMe
n.d.
D
D-R-OMe
D-K-OMe
Ac-
n.d.
n.d.
D
-F-
D
-K, Ac-
D
D
-K-OMe
D
n.d.
n.d.
D
-Phe
n.d., not detected (the area of ion intensity was less than 30).

1466
J. Arima et al. / Biochimie 93 (2011) 1460e1469
activity decreased concomitantly with increasing pH. Therefore, the
maximum of the ratio of velocity (aminolysis/hydrolysis) was
observed at pH 9.0.
Fig. 6. As the mechanism for (
as an acyl acceptor and -Phe-OMe served as an acyl donor. After
the production of ( -Phe)3, the enzymes might hydrolyze ( -Phe)3
to produce -Phee -Phe (Fig. 6).
D-Phe)3 production, D-PheeD-Phe acts
D
D
D
We verified whether the free amino group is necessary for the
D
D
molecule to act as a donor. After 20 mM of Ac-
added to the reaction mixture for -Phee -Phe production (the
reaction mixture containing 20 mM of -Phe-OMe), we confirmed
the production of Ac- -Phee -Phe (see Fig. S3 in Supplemental
D-Phe-OMe was
D
D
3.6. Variety of homopeptides synthesized by
82F2-DAP and 83D12-DAP
D
D
D
materials). The result indicates that the free amino group is not
necessary for the molecule to act as a donor.
During investigation of homopeptide synthetic activity, homo-
peptide was detected when using -Val-derivatives, -Leu-deriva-
tives, -Phe-derivatives, -Trp-derivatives, and -Tyr-derivatives as
substrates among 21 aminoacyl derivatives (Table 3). The result
indicates that the aminoacyl derivatives presented above can act as
either an acyl donor or acceptor. The only difference in the product
D
D
D
D
D
3.4. Mechanism for
D
-PheeD-Phe production
We synthesized
D
-Phee
-Phe that was liberated from the hydrolysis of
Phe derivative acted as an acyl acceptor. However, -Phee -Phe
production levels of respective enzymes were unaffected by the
addition of free -Phe for -Phee -Phe production (see Fig. S4A in
Supplemental materials). Similarly, no production of -Phe- -Trp
was observed when free -Trp was added to the reaction mixture
(see Fig. S4B in Supplemental materials). Furthermore, no
production occurred when Ac- -Phe-OMe and free -Phe were
used, respectively, as acyl donors and acceptors (data not shown).
The results indicate that free -amino-acid cannot act as an acyl
acceptor. In contrast, -Phee -Phe-OMe was detected in the
investigation of the product dependence on the reaction time using
D
-Phe from -Phe derivatives. We first
D
inferred that free
D
D-
between 82F2-DAP and 83D12-DAP was observed when D-Tyr ester
D
D
derivatives were used as substrates (bold type in Table 3). Homo-
peptidyl esters remained in the reaction mixture of 83D12-DAP.
Therefore, it is regarded as resulting from the weak hydrolytic
ability of 83D12-DAP toward homopeptidyl ester derivatives.
In the reaction mixtures of each enzyme, precipitates were
D
D
D
D
D
D
produced after 3 h reaction when D-Tyr ester derivatives were used
D
D
as the substrates (data not shown). These precipitates were
analyzed using MALDIeTOF MS. Results demonstrated that the
precipitates were 3e6 mer of Tyr (Fig. 7), indicating that each
enzyme can use the Tyr homooligomer as an acyl acceptor in
peptide bond formation activity. Although Table 3 shows that the
homopeptidyl esters remained in the reaction mixture of 83D12-
DAP, the ester derivatives were not detected in this investigation.
D
D
D
20 mM
product
D
-Phe-OMe as a substrate at pH 9.0 (Fig. 4A and B). The
-Phee -Phe-OMe emerged first in 5 min; then it vanished
D
D
in a few minutes. Based on this result, DAP might have a function of
deesterification activity toward -formed dipeptidyl esters.
D,D
3.7. Specificity for acyl acceptor
As portrayed in Fig. 4A and B, the concentration of hydrolysate,
-Phe, increased rapidly for 20 min. Subsequently, it was increased
D
We evaluated the synthesis of heteropeptides by reacting Ac-D-
slightly. The final concentration of
12 mM. Similarly, -Phee -Phe emerged up to 20 min; then the
concentration was increased slightly. The final concentration of
Phee -Phe was approximately 2 mM. The results show that the
enzymes catalyze hydrolysis more efficiently than aminolysis.
However, the concentration of -Phee -Phe was not reduced for
72 h reaction (data not shown), indicating that the product,
-Phee -Phe, was not hydrolyzed by the enzymes themselves.
D-Phe was approximately
Phe-OMe with various aminoacyl derivatives as substrates at pH 9.0
for 1 h. Although only a few substrates were used for homopeptide
synthesis, the results of this investigation indicate that the enzyme
D
D
D
-
D
can use various D-aminoacyl derivatives as acyl acceptors (Table 3).
D
D
D
D
3.5. Mechanism for (D-Phe)3 production
In the investigation of Fig. 3A, the tripeptide (
detected in addition to the main product: -Phee -Phe. The tri-
peptide ( -Phe)3 was synthesized efficiently up to 20 min. Then the
product decreased gradually, concomitantly with a slight increase
in -Phe and -Phee -Phe (Fig. 4A and B). For clarification of the
mechanism for trimer formation, we investigated the effect of
Phee -Phe addition on the reaction using 20 mM -Phe-OMe as
a substrate at pH 9.0 for 1 h. As presented in Fig. 5, the production of
the tripeptide ( -Phe)3 increased following the increase of
Phee -Phe concentration. The results indicate that -Phee -Phe
acts as an acyl acceptor when -Phe-OMe served as an acyl donor.
Results show that the production level of ( -Phe)3 of 83D12-DAP
was approximately double that of 82F2-DAP. We further investi-
gated the effect of -Phee -Phe addition on the reaction using
20 mM Ac- -Phe-OMe as an acyl donor. However, we were unable
D-Phe)3 was
D
D
D
D
D
D
D
-
D
D
D
D-
D
D
D
D
D
D
D
D
to detect the new product through this investigation (data not
shown).
As the mechanism for peptide synthesis by both DAPs, it is
suggested that the enzyme uses
donor and acceptor, thereby producing
Phee -Phe-OMe is regarded as hydrolyzed to
proposed mechanism for -Phee -Phe production is presented in
D
-Phe-OMe initially as an acyl
-Phee -Phe-OMe. Next,
-Phee -Phe. The
D
D
D-
D
D
D
Fig. 7. MALDIeTOF MS analysis of the reaction mixture catalyzed by 82F2-DAP and
83D12-DAP using D-Tyr-OMe as substrate. The reaction was performed for 3 h.
D
D

J. Arima et al. / Biochimie 93 (2011) 1460e1469
1467
The difference in the product between 82F2-DAP and 83D12-DAP
was observed in using several substrates (bold type in Table 3). It is
regarded as resulting from the weak hydrolytic ability of 83D12-
DAP toward homopeptidyl ester derivatives: a similar inference to
that made for homopeptide synthesis.
which belong to the clan SE, S12 peptidase family categorized as
serine peptidase with -stereospecificity (Fig. 8). The SXTK motif,
involving the active site residues Ser and Lys, and motifs found in the
R61 -peptidase, YSN and HXG, exist in their internal regions. The
calculated molecular masses from the deduced amino acid sequence
without a signal sequence were 37,394 and 37,892, respectively, for
82F2-DAP and 83D12-DAP.
D
D,D
3.8. Sequence analysis
b-lactam compounds inhibit members of S12 peptidase family.
Therefore, we tested the inactivation of both enzymes by treatment
with penicillin G. According to this investigation, the residual
activity of both enzymes after the treatment of 0.1 mM, 1 mM and
10 mM penicillin G for 5 min were 54.6 ꢂ 3.2%, 18.6 ꢂ 2.7%, and
4.8 ꢂ 2.5%, respectively, for 82F2-DAP, and 84.6 ꢂ 1.0%, 46.1 ꢂ 1.1%,
and 8.8 ꢂ 1.8%, respectively, for 83D12-DAP. Those results show
that both enzymes are sensitive to penicillin, as are all members of
the S12 peptidase family.
Using Edman degradation, the N-terminal amino acid sequences
of the enzymes were determined to be APA for 82F2-DAP, and
AAPAKKDHAATQAALQAN for 83D12-DAP. The internal amino acid
sequences were also determined: ITVRQLLNHTSG and LSRDVNAPVH
for 82F2-DAP, and ITVRQLLNHTSG for 83D12-DAP. Using this
sequence information, we analyzed the sequence of the gene
encoding the obtained peptidase using an inverse PCR technique.
As presented in Fig. 8, the N-terminal and internal sequences
determined using Edman degradation were found in the deduced
amino acid sequences of each enzyme. The enzymes possess signal
sequences that might be divisible into an Arg cluster region and
a hydrophobic region. The deduced amino acid sequence of each
4. Discussion
Several stereospecific peptidases reportedly recognize an amide
enzyme showed 30e60% identity with those of putative alkaline
D-
bond involving
D-amino-acid residues [21e29]. They are regarded as
peptidases, and -carboxypeptidases from Streptomyces or Bacillus,
D,D
associated mainly with biosynthesis and remodeling of peptidoglycan.
Fig. 8. Alignments of entire ORF sequences of 82F2-DAP, 83D12-DAP, and family S12 peptidases. Multiple sequence alignment was performed using the CLUSTAL algorithm:
asterisks (*) denotes conserved residues; colons (:) denotes conservative substitutions in all sequences, and periods (.) signifies semi-conservative substitutions in all sequences. The
determined N-terminal and internal sequences of each enzyme are underlined. The signal sequence, which was divisible into an Arg cluster region (Arg residues are shown in bold)
and a hydrophobic region, is dotted-underlined. The SXTK motif and motifs found in the R61 D,D-peptidase, YSN and HXG, are boxed. Proteins: 82F2, 82F2-DAP; 83D12, 83D12-DAP;
DADA B.cer., D-AlaeD-Ala carboxypeptidase from Bacillus cereus; d-pep B.cer., D-peptidase from B. cereus; d-end S.cla., putative D-stereospecific endopeptidase from Streptomyces
clavuligerus; d-pep S.cla., putative alkaline D-peptidase from S. clavuligerus; DADA S.cla., putative D-AlaeD-Ala carboxypeptidase from S. clavuligerus; DADA S.R61, D-AlaeD-Ala
carboxypeptidase from Streptomyces sp. R61.

1468
J. Arima et al. / Biochimie 93 (2011) 1460e1469
Among them, peptidases such as
D
,
D
-carboxypeptidase from Strepto-
Their primary structure exhibits approximately 80% mutual identity
(Fig. 8). Therefore, their differences in hydrolytic ability might be
attributable to the 20% difference in their primary structures. To
develop potent biocatalysts through modification of their properties
using protein engineering techniques, information of key residues
associated with hydrolytic activity is important. The primary struc-
tures of the enzymes are very similar. Therefore, chimeric analysis
might be useful for identifying key regions or residues for hydrolytic
activity without loss of function. The construction of chimeras of
82F2-DAP and 83D12-DAP is in progress.
myces sp. R61 [30],
D-aminopeptidase from Ochrobactrum anthropi [8],
and -peptidase from Bacillus cereus [9] have the function of ami-
D
nolysis reaction. The enzymes have catalytic serine residues and
belong to the clan SE, S12 peptidase family. Because few data are
available, obtaining additional information related to such enzymes,
including their detailed functions of their regions and residues, is
expected to be useful to develop potent biocatalysts through modifi-
cation of their properties using protein engineering techniques. This
paper describes one example of the function of this class of enzymes.
Some serine peptidases exhibit peptide bond formation from
aminoacyl derivatives, through aminolysis of esters, thioesters, and
amides, in accordance with their hydrolytic activity [31,32]. In
enzymatic peptide synthesis by reverse reaction, not by the ami-
nolysis reaction, peptides that act as good substrates in hydrolysis
are appropriate targets of synthesis [33e35]. In contrast, this report
Acknowledgment
Acknowledgment of grant support: This research was supported
by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
describes that the product, D-PheeD-Phe was not hydrolyzed by the
enzymes themselves. A similar phenomenon was observed in
Appendix. Supplementary material
a family S9 aminopeptidase from Streptomyces [14,15]; the enzyme
can synthesize
b-Ala containing peptides and diverse prolyl
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biochi.2011.04.020.
peptides. Family S9 aminopeptidase has catalytic Ser residue in its
active centers [36,37]; it shows an aminolysis reaction. Family S9
aminopeptidase from Streptomyces cannot hydrolyze carnosine (b-
Ala-His), but it can synthesize carnosine efficiently by its aminolytic
reaction [14]. In fact, the hydrolytic activities of 82F2-DAP and
83D12-DAP toward the peptide substrate were very low (Table 2).
The results indicate that, instead of water molecules, the enzyme
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(Fig. 6). In the proposed mechanism, only -Phe-OMe was used
initially as an acyl donor and acceptor. The result presented in
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The enzymes have no hydrolytic activity toward -Phee -Phe, but
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D
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D
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D-Phe)3, as
D
From a comparison of molecular masses obtained from the
sequence analysis and MS analysis, differences between the theo-
retical mass and measured values were observed, for 536 and 231
for 82F2-DAP and 83D12-DAP, respectively. Because the N-termini
have been determined, their differences can be attributed to resi-
dues at the C-terminal ends and modification of protein. Assuming
the C-terminal TAPKK of 82F2-DAP was truncated, the calculated
molecular mass of 82F2-DAP was 36,868, a difference from the
measured mass was 10 (Fig. 1). The difference of mass is within the
margin of error of the instrument. Therefore, we speculated that
the C-terminal TAPKK of 82F2-DAP might have been truncated
during or after the process of translation and secretion. In contrast,
if the C-terminal KK of the 83D12 was truncated, then the calcu-
lated molecular mass of 83D12-DAP was 37,635, a difference from
the measured mass was 26 (Fig. 1), and larger than 82F2-DAP. We
consider that the difference in molecular mass results from
unspecific modification of protein such as oxidation of Cys residue.
However, the cause of the difference between the calculated and
measured molecular masses remains unknown.
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