49NISR Research GRANT
2008 Young Investigator Research Grant

Molecular evolution of Streptomyces serine peptidase forthe synthesis of b -alanyl-histidine
Jiro ARIMA
Faculty of Agriculture, Tottori University

Research aims
Anserine (b -Ala-p -methyl-L-His), carnosine (b -Ala-LHis; Car), and balenine (b -Ala-t -methyl-L-His) have been
known to exist in excitable tissues such as skeletal muscle
and brain. Moreover, b -Ala-branched chain amino acids (b Ala-BCAA) also induced hyperactivity behavior as observed with carnosine.1) Therefore, b -Ala containing dipeptides received special attention as a research target for functional analyses because of the availability of the reagent.
Some serine peptidases exhibit peptide or amino acid ligation (i.e. aminolysis) in accordance with their hydrolytic
activity. Moreover, mutation of catalytic Ser to Cys to engineer the serine peptidase into “transpeptidase” for the peptide ligation has been well characterized.2,3) Our recent approach demonstrates that wide distribution of family S9aminopeptidases was confirmed.4) Among enzymes fromactinomycetes, we obtained S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) that can hydrolyze b -Ala derivatives. In this study, peptide synthetic
activity of S9AP-St and its engineered variant, S502C
S9AP-St was evaluated.
Methods
The engineered S9AP-St variant, S502C S9AP-St, wasconstructed on the basis of mutagenesis technique for
QuickChange site directed mutagenesis kit (Stratagene).
Wild-type and S502C S9AP-Sts were expressed in E. coli
BL21 (DE3) harboring the expression vector for wild-typeor S502C S9AP-St production. The enzymes were purified
with anion exchange chromatography.
Peptide synthesis by aminolysis reaction was performed
as follows: 10 m l of acyl donor substrate and 10 m l of acyl
accepter substrate (they are amino acid derivative solution(in DMSO) at an appropriate concentration (0.5 M)) wereadded into 75 m l of 0.25 M Tris-HCl (pH 8.0). The reaction
was initiated by adding 5 m l of enzyme solution (2 mg/mL).
The reaction was then continued at 25°C for an appropriate
time (20 min to 50 h). After the reaction was terminated byadding 0.1 ml of 3% formic acid, the reaction mixture wasanalyzed by UPLC-ESI-TOF-MS or amino acid analyzer.
Results
S9AP-St displayed efficient hydrolytic activity toward L-Phe-pNA and slight hydrolytic activity toward b -Ala-pNA.
Its hydrolytic activity was abolished by the site-directedmutagenesis of catalytic Ser502 into Cys. We evaluated theaminolytic activity of wild-type and S502C S9AP-Sts using
various aminoacyl derivatives. When hydrophobic aminoacyl derivatives were used as substrates, S502C S9AP-St exhibit high peptide synthetic activity, as expected. However,the enzyme showed no aminolytic activity toward b -Ala-derivatives. In contrast, b -alanyl homopeptides were synthesized by wild-type S9AP-St whereas the enzyme showed noaminolytic activity toward hydrophobic aminoacyl derivatives (Table 1). We next evaluated the synthetic ability ofheteropeptides containing b -Ala by using b -Ala-OBzl andvarious aminoacyl-derivatives (OMe or OEt) as substrates. Wild-type S9AP-St could synthesize a wide varietyof b -Ala peptides (Table 1). Interestingly, S9AP-St preferentially synthesized b -Ala-Xaa derivatives. In contrast,
S502C S9AP-St could synthesize only Xaa-b -Ala derivatives with the specificity toward hydrophobic aminoacyl derivatives. The results indicated that the substrate specificityof aminolysis reaction was changed by the substitution ofcatalytic Ser502 with Cys.
Because S9AP-St had synthetic ability of b -Ala-L-HisOMe (Car-OMe) and b -Ala-BCAA-OMes, we next investigated the optimization of production of such biologicallyactive peptides using S9AP-St. For this investivation, CarOMe was selected as a main product. As shown in Fig. 1A,
the Car-OMe was steadily produced at the b -Ala-OBzl concentration of 1040 mM when L-His-OMe concentration
was maintained at a steady level of 20 mM. In contrast, theproductivity of b -Ala-b -Ala-OBzl was increased following
the increase of b -Ala-OBzl concentration. On the otherhand, the productivity of Car-OMe was increased and thatof b -Ala-b -Ala-OBzl was decreased following the increaseof L-His-OMe concentration (Fig. 1A). As a result of the investigation on the effect of pH, the pH dependence curve
for Car-OMe production was similar with that for the b Ala-pNA hydrolytic activity (Fig. 1B). The Car-OMe wassynthesized at this pH range with maximum production at
pH 8.5. However, the productivity of byproduct, b -Ala-b Ala-OBzl, was increased following the increase of pH (Fig.
1B). The production of another undesirable product, b -Alab -Ala-b -Ala-OBzl, hardly observed at these investigations.
As shown in Fig. 1C, when 20 mM b -Ala-OBzl and 20mM L-His-OMe were used as substrates, Car-OMe was efficiently synthesized until 5 h, and the quantity of Car-OMe

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Table 1. Tested chemicals and peptides synthesized by both wild-type and S502C S9AP-Sts.
Products
wild-type S9AP-St
Substrate

G-OMe
LV-OMeLL-OEtLI-OMeLM-OMeLF-OEt
LW-OMeLY-OMe
LP-OMeLS-OMeLT-OMeLN-OMeLH-OMe
LK-OMeLR-OMeLD-OMeLE-OMebA-OMebA-OBzlbA-OtBu

S502C S9AP-St

w/b A-OBzl

n.d.
(LV)2-OMe
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
(LT)2-OMe
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
(b A)2-OBzl(b A)2-OtBub A-Xaa, Xaa-b A

Byproductsn.d.
b A-LV-OMeb A-LL-OEt
b A-LI-OMeb A-LM-OMeb A-LF-OEt
b A-LW-OMeb A-LY-OMe
LP-b A-OBzl
n.d.
b A-LT-OMeb A-LN-OMeb A-LH-OMeb A-LK-OMeb A-LR-OMe
n.d.
n.d.
———(b A)2-OBzl(b A)2-OBzl, (b A)2-LV-OMe(b A)2-OBzl(b A)2-OBzl(b A)2-OBzl, (b A)2-LM-OMe etc(b A)2-OBzl, (b A, LF)-b A-OBzl etc(b A)2-OBzl(b A)2-OBzl, (b A)2-LY-OMe etc(b A)2-OBzl, LP-(b A)2-OBzl etc(b A)2-OBzl(b A)2-OBzl, (LT)2-OMe(b A)2-OBzl(b A)2-OBzl(b A)2-OBzl(b A)2-OBzl(b A)2-OBzl(b A)2-OBzl———

w/b A-OBzl

G3-OMen.d.
(LL)2-OMe, (LL)3-OMe etcn.d.
(LM)2-OMe(LF)2-OMe, (LF)3-OMe etc(LW)2-OMe(LY)2-OMe
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

b A-Xaa, Xaa-b A

Byproducts

G-b A-OBzln.d.
LL-b A-OBzln.d.
LM-b A-OBzl
LF-b A-OBzl
LW-b A-OBzl
LY-b A-OBzl
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
———

G3-OMe, G2-b A-OBzl
n.d.
(LL)2-OMe, (LL)3-OMe etcn.d.
(LM)2-OMe, (LM)2-b A-OBzl(LF)2-OMe, (LF)3-OMe etc(LW)2-OMe(LY)2-OMe, (b A)2-LY-OMe
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
———

Fig. 1. (A) Effect of the substrate concentration on the production of Car-OMe and b -Ala-b -Ala-OBzl. Upper panel: Effect of b -Ala-OBzl concentration.
L-His-OMe at 20 mM and b -Ala-OBzl at 0 to 40 mM were used as the acyl acceptor and donor, respectively. Lower panel: Effect of L-His-OMe concentration. L-His-OMe at 0 to 30 mM and b -Ala-OBzl at 20 mM were used as the acyl acceptor and donor, respectively. (B) Effect of pH on production of CarOMe and b -Ala-b -Ala-OBzl. (C) Time dependence and yield evaluation of Car-OMe synthesis. The upper panel shows the time dependence in the production of Car-OMe and b -Ala-b -Ala-OBzl. L-His-OMe at 20 mM and b -Ala-OBzl at 20 mM were used as the acyl acceptor and donor, respectively. The lowerpanel shows a chromatogram of amino acid analyzer. L-His-OMe at 20 mM and b -Ala-OBzl at 20 mM were used as the acyl acceptor and donor, respectively.

hardly increased after that. Similarly, byproduct, b -Ala-b Ala-OBzl, was also produced until 5 h. After 24 h reaction,
20 mM b -Ala-OBzl was completely consumed and con-verted into Car-OMe, b -Ala-b -Ala-OBzl, and free b -Ala.
To evaluate the conversion rate of 20 mM b -Ala-OBzl toCar-OMe and the synthesis of undesirable products such as

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b -Ala-b -Ala-OBzl and free b -Ala, we quantified Car, freeb -Ala, and L-His in the alkaline-treated reaction mixture of24 h reaction by amino acid analyzer. The concentration ofCar in the reaction mixture is approximately 6.4 mM, andthose of b -Ala and L-His were respectively approximately
8.0 mM and 9.3 mM (Fig. 1C). From these values, the conversion rate of b -Ala-OBzl to Car-OMe was estimated to beover 30%.

OBzl to Car-OMe being over 30% by the optimization ofproduction of Car-OMe using S9AP-St.
References
1)

2)

Conclusion
We have found that Car-OMe could be produced from b Ala-OBzl and L-His-OMe by aminolysis reaction of S9APSt. To engineer the serine peptidase into “transpeptidase”
for tailoring highly capable biocatalyst, we constructed
S9AP-St variant, S502C S9AP-St. However, the substratespecificity of aminolysis reaction was changed by the substitution of catalytic Ser502 with Cys. Instead, it has successfully produced Car-OMe with a conversion rate of b -Ala-3)4)

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