Engineered transaminopeptidase, aminolysin-S for catalysis of peptide bond formation to give linear and cyclic dipeptides by one-pot reaction

Article abstract of DOI:10.1039/b914320c

Aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 was engineered into transaminopeptidase and used to catalyze an aminolysis reaction to give linear and cyclic dipeptides from cost-effective substrates such as the ester derivatives of amino

Full text of DOI:10.1039/b914320c

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Engineered transaminopeptidase, aminolysin-S for catalysis of peptide
bond formation to give linear and cyclic dipeptides by one-pot reactionw
Hirokazu Usuki,a Yoshiko Uesugi,ab Jiro Arima,c Yukihiro Yamamoto,a
Masaki Iwabuchia and Tadashi Hatanaka*a
Received (in Cambridge, UK) 16th July 2009, Accepted 17th November 2009
First published as an Advance Article on the web 4th December 2009
DOI: 10.1039/b914320c
Aminopeptidase from Streptomyces thermocyaneoviolaceus
NBRC14271 was engineered into transaminopeptidase and used
to catalyze an aminolysis reaction to give linear and cyclic
dipeptides from cost-effective substrates such as the ester
derivatives of amino acids.
reactions.10,15–21 In both cases, their substrate recognition
properties toward the protecting group containing amino
acids or oligopeptides make the direct synthesis of small
dipeptides impossible. Therefore, an alternative strategy for
the enzymatic direct synthesis of dipeptides must be developed.
We tried to achieve a novel enzymatic method to overcome the
above problems. First, we used a data mining approach to
search for the parent aminopeptidase. Next, this parent
aminopeptidase was engineered into ‘‘transaminpeptidase’’
by the site-directed mutagenesis of catalytic residue. Its
dipeptide synthesis capability was then evaluated in this study.
Nishimura et al. recently reported the identification of
family S9 aminopeptidase, which shows broad substrate
specificity, from actinomycete Streptomyces morookaensis.22
As a rule, family S9 peptidases possess catalytic Ser residue
and show oligopeptidase activity, not aminopeptidase activity
(http://merops.sanger.ac.uk/). Therefore, this report is
important because it suggests the distribution of new S9
enzymes possessing aminopeptidase activity, rather than
oligopeptidase activity. Indeed, the wide distribution of such
S9 aminopeptidases (S9AP) in actinomycetes was confirmed in
our previous paper.23 The mutation of catalytic Ser to Cys or
another amino acid to engineer the ‘‘serine oligopeptidase’’
into ‘‘transpeptidase’’ for peptide bond formation has been
well characterized.18,24 These reports describe the general role
of the construction of transpeptidase from serine peptidase.
The reports also present a discussion of the limitations
hindering the enzymatic synthesis of short peptides including
dipeptides because of their substrate specificities toward
oligopeptides. The facts described above prompted us to
screen S9APs with broad substrate specificity from actino-
mycetes with the goal of engineering an aminopeptidase into
transaminopeptidase. A data mining approach for such an
aminopeptidase brought the discovery of a new amino-
peptidase from S. thermocyaneoviolaceus NBRC14271. The
enzymes showed aminopeptidase activity with moderately
broad substrate specificity. The aminopeptidase hydrolyzed
L-phenylalanine-p-nitroanilide (LF-pNA), LA-pNA, and
LP-pNA exhibited similar magnitudes (Fig. S1 of ESIw). The
site-directed mutagenesis of catalytic Ser502 into Cys caused
the disappearance of its aminopeptidase activity and the
appearance of peptide synthesis activity, as expected. The
S502C mutant recognized several ester derivatives of amino
acids as acyl donors and acyl acceptors to give diverse peptides
(Table S1 and Fig. S2s of ESIw).
Dipeptides and their analogues, including cyclic dipeptides,
are a specific and attractive group of biologically active
compounds. The most familiar dipeptide, aspartame, which
is used as a food additive, is known to be 180 times sweeter
than sucrose. In addition, cyclo(L-Arg-D-Pro) acts as a specific
inhibitor of family 18 chitinase and is considered the ideal lead
compound for the development of antifungal reagents and
insecticides.1,2 Moreover, dehydrophenylahistin, which is
prepared using enzymatic dehydrogenation of naturally
occurring phenylahistin, reportedly shows an extremely potent
inhibitory effect on cell division.3 Therefore, dipeptides
and their analogues can be regarded as ideal and simple
‘‘templates’’ for diverse biologically active compounds.
Enzymatic peptide synthesis is an attractive method because
of its simple procedure, mild reaction conditions, and ability
to directly obtain products without deprotection procedures.
Nevertheless, the reported enzymes are not ideal for dipeptide
synthesis for the following reasons. Non-ribosomal peptide
synthase (NRPS) or cyclodipeptide synthetase (CDPS) can
catalyze the synthesis of linear or cyclic dipeptides.4–9
However, these enzymes have required expensive substrates
such as ATP for NRPS or aminoacyl-tRNA for CDPS. A
method that uses peptidase/protease catalysis is now recognized
as an alternative and cost-effective method for peptide bond
formation.10,11 Such methods could mainly be divided into
two distinct strategies: the reverse reaction of hydrolysis,
which is mainly adopted for the endpeptidase reaction,11–14
and the aminolysis reaction, which is used for transpeptidase
a Research Institute for Biological Sciences (RIBS), Okayama,
7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
E-mail: hatanaka@bio-ribs.com; Fax: +81 866 56 9454;
Tel: +81 866 56 9452
b Department of Biomaterials, Field of Tissue Engineering,
Institute for Frontier Medical Sciences, Kyoto University,
53 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
c Department of Agricultural, Biological, and Environmental Sciences,
Faculty of Agriculture Tottori University, 4-101 Koyama-Minami,
Tottori 680-8553, Japan
w Electronic supplementary information (ESI) available: Detailed
procedures for cloning, overexpression of aminopeptidase and
aminolysin-S, and product characterization and semi-quantification
by MS/MS methods, in addition to the enzymatic characteristics of
aminolysin-S. See DOI: 10.1039/b914320c
Fig. 1 shows that the S502C mutant catalyzed the enzyme-
dependent dipeptide synthesis. In this experiment, 0.5 mM of
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Fig. 1 Peptide synthesis by aminolysin-S. (A) Enzyme-dependent
peptide synthesis by aminolysin-S. The reaction was conducted for
1.0 h at 50 1C with several amounts of enzyme. (B) Time course of
aminolysin-S catalyzed peptide synthesis. The reaction was conducted
with 10 mg of the enzyme at 50 1C.
L-phenylalanine ethyl ester (LF-O-Et) used as the acyl donor
and 60 mM of L-phenylalanine (LPhe) used as the acyl
acceptor were subjected to the reaction. The conversion
efficiency of LF-O-Et to L-phenylalanyl-L-phenylalanine (LF-LF)
was estimated to be approximately 50% (Fig. 1A and B). The
by-products cyclo(LF-LF) and LF-LF-O-Et were also observed
in these experiments. In panel [B], the ratio of the major
product LF-LF to by-product cyclo(LF-LF) after a 3 h reaction
was calculated to be 12.5. Herein, LF-LF existed stably in the
reaction medium, as shown in Fig. 1A and B.
Fig. 3 Dipeptide synthesis. A LF-O-Et as acyl donor and represented
amino acids as acceptors were subjected to the reaction. Left side panel
is the total ion chromatogram (TIC) of the reaction mixtures. The
produced dipeptides (LF-X) are indicated by arrows. The gray
characters are observed m/z of [M + H]+ ions. Asterisks denote the
by-products cyclo(LF-LF). The right side panel is its multiple reaction
monitoring (MRM) chromatogram. The gray characters are MRM
transitions for the monitoring of LF containing dipeptides.
In Fig. 1B, LF-LF-OEt reached the maximal amount at
30 min followed by a rapid decrease, and the cyclo(LF-LF)
increased linearly for 1 h followed by stable existence.
Therefore, the cyclo(LF-LF) would have originated from the
intramolecular aminolysis of LF-LF-O-Et. It is still unclear
whether the reaction is enzymatic or non-enzymatic. Herein,
the absence of LF-O-Et as an acyl donor prevented the
production of any products (data not shown), indicating that
the reaction mechanism was aminolysis, not a reverse reaction
of hydrolysis (also known as a dehydration/condensation
reaction). The production of the dipeptide was not observed
using a heat-inactivated enzyme (95 1C, 30 min), indicating
that the reaction was a so-called enzymatic one. The enzyme
was designated as aminolysin-S, which was derived from
its catalytic mechanism (aminolysis) and the genus of its
microorganism (Streptomyces). The parent aminopeptidase
containing the catalytic Ser502 was unable to catalyze the
peptide bond formation under the same assay conditions
(data not shown), underscoring the critical role of Cys502 in
the aminolysis reaction. Fig. 2 shows the proposed mechanism
of aminolysin-S catalysis.
of glycine as an acceptor (Table S2 and Fig. S3 of ESIw), its
a-allyl and a-propargyl derivatives were recognized, indicating
that hydrophobic or bulky moieties might be responsible for
its interaction with the enzyme. Such an explanation should be
adopted for the cases of LPhe and LTrp. Nevertheless, the
exceptional recognition toward LLeu and LMet remains to be
clarified.
The benzyl ester derivatives of amino acids were good
substrates for the one-pot synthesis of diverse cyclic
dipeptides. Fig. 4 presents experimental evidence for the
recognition of L- and D-amino acids in the aminolysin-S
catalyzed reaction. When a set of 0.5 mM LF-O-Et and either
0.5 mM L-tyrosine benzyl ester (LY-O-Bzl) or DY-O-Bzl was
used, the major products gave different retention times in the
Fig. 3 shows that aminolysin-S recognized diverse free
amino acids as acyl acceptors. Despite the non-recognition
Fig. 4 One-pot synthesis of cyclo(LF-LY) and cyclo(LF-DY) by
aminolysin-S. (A) LF-O-Et with LY-O-Bzl (A(1)) or LF-O-Et with
DY-O-Bzl (A(2)) were subjected to aminolysin-S catalysis. TIC of the
reaction mixtures are shown. Arrows indicate the corresponding cyclic
dipeptides produced. Asterisks denote the by-products, cyclo(LF-LF).
[B] MS and MS/MS spectra of produced cyclic dipeptides (a) and (b).
MS spectra are shown as inset small images.
Fig. 2 Proposed mechanism of aminolysin-S catalysis.
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ODS HPLC analysis, as indicated by the arrows in Fig. 4A(1)
and A(2). Fig. 4B shows that their MS and MS/MS spectra
were identical. Their planar structures can be reasonably
inferred to be cyclo(F-Y), as judged from the fragmentation
profiles in the MS/MS spectra (Fig. S6 of ESIw). From the
observation described above and the corresponding substrates
used, compounds (a) and (b) were identified, respectively, as
cyclo(LF-LY) and cyclo(LF-DY). In addition, the enzyme
recognized the diverse benzyl ester derivatives of D- and
L-amino acids needed to produce the corresponding cyclic
dipeptides. (Table S4 and Fig. S5 of ESIw). It remains to
be clarified which substrate—LF-O-Et or the benzyl ester
derivatives of the amino acids—acts as an acyl donor or
acceptor. The following are important aspects of the
aminolysin-S catalyzed peptide synthesis. It has broad
substrate specificity. Moreover, the enzymatic reaction used
to synthesize linear dipeptides or cyclic dipeptides can be
controlled. Furthermore, deprotection procedures are
unnecessary for obtaining the produced peptides. Biocatalysts
possessing the characteristics described above have never
before been reported except for the case of peptidic catalysts.25
In that report, Huang et al. designed a short peptide catalyst
mimicking the reaction of NRPS to synthesise cyclic dipeptides.
Nevertheless, the method requires a preloading procedure
involving peptidic catalysts with specific substrates for the
efficient synthesis of cyclic dipeptides. Furthermore, directly
obtaining a good yield of a linear dipeptide should be difficult
because the proposed catalyst mechanism was as follows: the
produced dipeptide in the reaction was linked to the
Cys residue of the peptidic catalyst by thioester bonding
followed by intramolecular aminolysis to yield cyclic
dipeptides. Therefore, our enzymatic aminolysis reaction is
expected to be a beneficial tool to synthesize diverse dipeptides
and cyclic dipeptides from cost-effective substrates such as the
simple ester derivatives of amino acids. Furthermore, our
method requires only a single step to obtain diverse dipeptides,
including cyclic dipeptides.
as acceptors gives the linear dipeptides as the major products.
When cyclic dipeptides are desired, using the benzyl ester
derivatives of amino acids as substrates is an efficient way to
obtain the cyclized peptides. Aminolysin-S catalyzed peptide
synthesis will be a beneficial and cost-effective method for the
production of diverse dipeptides and their analogues.
The unnatural amino acids were a gift from Nagase and
Co., Ltd. This research was partially supported by a Grant-
in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology.
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This study provided a method for the enzymatic synthesis of
diverse dipeptides and cyclic dipeptides. The novel family S9
aminopeptidase from actinomycetes was engineered into
transaminopeptidase using the site-directed mutagenesis of
catalytic Ser502 to Cys. The biocatalyst aminolysin-S can
produce a linear dipeptide and cyclic dipeptide using a
one-pot reaction. Its reaction mechanism was clarified as
aminolysis—not a reverse reaction of hydrolysis—because
peptides were not produced in the absence of the ester
derivatives of amino acids as acyl donor substrates. In this
reaction, cost-effective substrates such as the ester derivatives
of amino acids were converted into corresponding linear
dipeptides and cyclic dipeptides. Furthermore, the distinct
production of linear or cyclic dipeptides can be controlled
using appropriate substrates as follows. Using the ester
derivatives of amino acids as acyl donors and free amino acids
25 Z. Z. Huang, L. J. Leman and M. R. Ghadiri, Angew. Chem., Int.
Ed., 2008, 47, 1758–1761.
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582 | Chem. Commun., 2010, 46, 580–582