APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2010, p. 4109–4112 Vol. 76, No. 120099-2240/10/$12.00 doi:10.1128/AEM.00577-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotesthe Synthesis of Diverse Prolyl Dipeptides�†

Jiro Arima,1* Masazumi Morimoto,1 Hirokazu Usuki,2 Nobuhiro Mori,1and Tadashi Hatanaka2

Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University,Tottori 680-8553, Japan,1 and Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho,

Kaga-gun, Okayama 716-1241, Japan2

Received 4 March 2010/Accepted 16 April 2010

Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has beendemonstrated. In the synthesis, S9AP-St preferentially used L-Pro-OBzl as the acyl donor, yielding synthesizeddipeptides having an L-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acylacceptor. Furthermore, S9AP-St produced cyclo (L-Pro-L-His) with a conversion ratio of substrate to cyclo(L-Pro-L-His) higher than 40%.

Some proline-containing dipeptides and their cyclic analogsexhibit biological activity. For example, cyclo (L-arginyl-D-pro-line) [c(LR-DP)] is known to act as a specific inhibitor of family18 chitinase (4, 10). A cyclic peptide, c(LP-LH), produced bythe cleavage of thyrotropin-releasing hormone protects againstoxidative stress, promotes cytoprotection (6, 7), and exhibitsantihyperglycemic activity (11).

Some serine peptidases exhibit peptide bond formation (i.e.,aminolysis of esters, thioesters, and amides) in accordance withtheir hydrolytic activity (2, 14). The exchange of catalytic Serfor Cys to engineer the serine endopeptidase into “transpep-tidase” for peptide bond formation has been well characterized(3, 5). Our recent approach confirmed the wide distribution offamily S9 aminopeptidases that have catalytic Ser in actinomy-cetes (12). Of them, we obtained S9 aminopeptidase fromStreptomyces thermocyaneoviolaceus NBRC14271 (S9AP-St).The enzyme was engineered into “transaminopeptidase” byexchange of catalytic Ser for Cys, and its aminolytic activity wasevaluated (13). The engineered enzyme, designated as amino-lysin-S, can synthesize hydrophobic dipeptides through an ami-nolysis reaction. However, aminolysin-S was unable to synthe-size peptides containing proline. Although the report ofaminolysin-S demonstrated that S9AP-St shows no aminolysisreaction toward limited substrates, details of its characteristicsremain unknown. This study verified the peptide synthetic ac-tivity of S9AP-St, demonstrating that S9AP-St can synthesizewidely varied prolyl dipeptides through an aminolysis reaction.The report also shows that S9AP-St is applicable to the syn-thesis of a biologically active peptide—c(LP-LH).

Homopeptide synthetic activity of S9AP-St. RecombinantS9AP-St was purified from cultivated cells of Escherichia coliBL21(DE3) harboring pET28-His6-S9AP-ST, the expression

vector for S9AP-St production, as described by Usuki et al.(13). The assay for peptide bond formation by aminolysis re-action was conducted using various aminoacyl derivatives un-der the conditions described in the supplemental material. Thesynthesized peptides were analyzed using electrospray ioniza-tion-time of flight mass spectrometry (ESI-TOF MS) (LCTPremier XE; Waters Corp.). Among 32 aminoacyl derivatives,homopeptide was detected when using L-Val-OBzl, L-Thr-OMe, D-Val-OBzl, or D-Leu-OBzl as the substrate (Table 1).The result indicates that S9AP-St has peptide synthetic activitythrough its aminolysis reaction.

Synthesis of prolyl peptides using S9AP-St. We next inves-tigated whether S9AP-St possesses the ability to synthesizeprolyl heteropeptides. The assay was conducted as described inthe supplemental material. It is particularly interesting thatS9AP-St can use L-Pro-OBzl only as an acyl donor, with theresult that all synthetic peptides have an LP-X structure (Table1). The investigation shows that almost all aminoacyl deriva-tives are useful as acyl acceptors, which was independent ofenantiospecificity. It is especially noteworthy that by-productcyclic dipeptides were observed.

When the reaction time was extended, it was observed thatdipeptidyl derivatives were converted into cyclic dipeptides. Aspresented in Fig. 1, LP-LH-OMe and c(LP-LH) were detected atalmost mutually identical levels after a 3-h reaction. In con-trast, only the peak of product c(LP-LH) was detected after a24-h reaction. The cyclization of LP-LH-OMe might have oc-curred nonenzymatically because the c(LP-LH) production wascontinued when LP-LH-OMe was exposed at neutral pH (datanot shown). The proposed mechanism for c(LP-LH) productionis presented in Fig. 2.

We further evaluated the D-prolyl peptide synthetic activityusing D-Pro-OBzl (Table 1). Although S9AP-St only showedhydrolytic activity toward L-aminoacyl-pNA (13), it was sur-prising that S9AP-St can use D-Pro-OBzl as an acyl donor.When using D-Pro-OBzl as acyl donor, some acidic and basicaminoacyl-OMe’s, L-Ala, D-Val, and L-Ser-OMe’s could not beused as acyl acceptors whereas they acted as acyl acceptorwhen using L-Pro-OBzl as acyl donor. Therefore, specificity

* Corresponding author. Mailing address: Department of Agricul-tural, Biological, and Environmental Sciences, Faculty of Agriculture,Tottori University, Tottori 680-8553, Japan. Phone: 81-857-31-5363.Fax: 81-857-31-5363. E-mail: arima@muses.tottori-u.ac.jp.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 23 April 2010.

4109

on January 3, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

toward the acyl acceptor is narrower than that when usingL-Pro-OBzl as the acyl donor.

Effects of substrate concentration and pH on c(LP-LH) syn-thesis. We next investigated the reaction condition for theproduction of c(LP-LH), a cyclic dipeptide with antihyper-glycemic activity. The production level of products was de-termined using ultraperformance liquid chromatography(UPLC)-ESI-TOF MS with a C18 reverse-phase system (Ac-quity UPLC; Waters Corp.). Under the UPLC conditions de-scribed in the supplemental material, two products, LP-LH-OMe and c(LP-LH), were detected at different retention times(see Fig. S1 in the supplemental material).

We first investigated the effect of the substrate concentra-tion on synthesis. As depicted in Fig. 3A, the production ofLP-LH-OMe and c(LP-LH) was increased following the in-crease of L-Pro-OBzl concentration. On the other hand, whenthe L-Pro-OBzl concentration was maintained at a steady levelof 20 mM, the c(LP-LH) concentration was lowered when theL-His-OMe concentration was higher than 20 mM (Fig. 3B).We next examined the effect of pH on synthesis. As portrayedin Fig. 3C, the production of LP-LH-OMe was decreased at apH higher than 8.5. An enzymatic product, LP-LH-OMe, mightbe liable to cause cyclization at high pH. Consequently, thec(LP-LH) productivity was increased following the increaseof pH.

Time dependence of prolyl dipeptide synthesis. Extension ofthe reaction time engenders the emergence of a cyclicdipeptide. As presented in Fig. 4, LP-LH-OMe was synthe-sized efficiently until 30 min, and then the product was de-creased gradually because of conversion into c(LP-LH). After24 h, LP-LH-OMe was converted completely into c(LP-LH). Interms of the substrate consumption, L-Pro-OBzl was com-pletely converted into LP-LH-OMe or free L-Pro after 120 min.In contrast, L-His-OMe decreased only slightly during 24-hreaction, indicating that S9AP-St uses L-His-OMe only as anacyl acceptor; it has no hydrolytic activity toward L-His-OMe.

Yield of prolyl dipeptide. To evaluate the conversion ratio ofthe substrate to c(LP-LH), we quantified free L-His in thealkaline-treated reaction mixture of a 24-h reaction with orwithout enzyme using an amino acid analyzer (JLC-500/V2;JEOL). Using the method described in the supplemental ma-terial, the concentrations of L-His in the reaction mixture withand without enzyme were estimated, respectively, as 19.2 � 0.4and 11.0 � 0.5 mM (see Fig. S2 in the supplemental material).The data indicate that 8.2 mM L-His-OMe was used as an acylacceptor. From these values, the conversion ratio of substrateto LP-LH-OMe was estimated as higher than 40%.

In enzymatic peptide synthesis by reverse reaction, not bythe aminolysis reaction, peptides that act as good substrates inhydrolysis are appropriate targets of synthesis (1, 8, 9). In

TABLE 1. Tested chemicals and peptides synthesized using S9AP-St

Substrate(aminoacyl derivative) Source

Product(s) (area of ion intensity)a

Homopeptide With L-Pro-OBzl With D-Pro-OBzl

Gly-OMe Aldrich ND ND NDL-Ala-OMe Aldrich ND LP-LA-OMe (181) NDD-Ala-OMe Bachem ND ND NDL-Val-OMe Aldrich ND LP-LV-OMe (1,300) DP-LV-OMe (342)L-Val-OBzl Bachem (LV)2-OBzl (98)D-Val-OMe Bachem ND LP-DV-OMe (186) NDD-Val-OBzl Bachem (DV)2-OBzl (295)L-Leu-OEt Bachem ND LP-LL-OEt (1,487) DP-LL-OEt (544)D-Leu-OMe Bachem ND LP-DL-OMe (1,065), (DL)2-OMe (328) DP-DL-OMe (1,212), (DL)2-OMe (1,161)D-Leu-OBzl Bachem (DL)2-OBzl (2,427)L-Ile-OMe Sigma ND LP-LI-OMe (1,369) DP-LI-OMe (443)L-Met-OMe Aldrich ND LP-LM-OMe (1,473) DP-LM-OMe (365)L-Phe-OEt Aldrich ND LP-LF-OEt (1,494) DP-LF-OMe (633)D-Phe-OMe Aldrich ND LP-DF-OMe (199) DP-DF-OMe (224)L-Trp-OMe Aldrich ND LP-LW-OMe (1,524) DP-LW-OMe (544)D-Trp-OMe Bachem ND LP-DW-OMe (398) DP-DW-OMe (1,092)L-Tyr-OMe Wako ND LP-LY-OMe (1,502) DP-LY-OMe (1,005)D-Tyr-OMe Bachem ND LP-DY-OMe (210) DP-DY-OMe (133)L-Pro-OMe Bachem ND NDD-Pro-OMe Bachem ND NDL-Ser-OMe Aldrich ND LP-LS-OMe (236) NDD-Ser-OMe Bachem ND ND NDL-Thr-OMe Aldrich (LT)2-OMe (148) LP-LT-OMe (495) (LT)2-OMe (67)L-Asn-OMe Bachem ND ND NDL-Lys-OMe Sigma ND LP-LK-OMe (823) NDL-Arg-OMe Bachem ND LP-LR-OMe (904), c(LP-LR) (60) NDD-Arg-OMe Bachem ND LP-DR-OMe (289), c(LP-DR) (239) NDL-His-OMe Aldrich ND LP-LH-OMe (373), c(LP-LH) (1,295) DP-LH-OMe (409), c(DP-LH) (155)D-His-OMe Bachem ND ND NDL-Asp-OMe Bachem ND LP-L-OMe (135) NDD-Asp-OMe Bachem ND ND NDL-Glu-OMe Bachem ND LP-LE-OMe (298) ND

a Products with areas of ion intensity greater than 1,000 have been highlighted in boldface. ND, not detectable (the area of ion intensity was less than 30).

4110 ARIMA ET AL. APPL. ENVIRON. MICROBIOL.

on January 3, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

contrast, the investigation of S9AP-St shows that free L-Proincreased only slightly after L-Pro-OBzl was completely con-sumed (Fig. 4), indicating that the LP-LH-OMe product washydrolyzed only slightly by S9AP-St itself. We infer that prolylderivatives are good substrates for hydrolytic or aminolyticactivity of S9AP-St. However, S9AP-St recognizes prolyldipeptides as a substrate only to a slight degree.

The results of this study demonstrated that S9AP-St is ap-plicable for syntheses of various prolyl dipeptides, includingc(LP-LH). However, in the synthesis using S9AP-St, some ami-

FIG. 1. MS of product synthesized with L-Pro-OBzl and L-His-OMe. The reaction was performed for 3 h (upper panel) or 24 h(middle panel). The lower panel portrays the spectrum of the reactionmixture without the enzyme (w/o S9AP-St) for comparison of synthesisof the unknown product by S9AP-St with a negative control.

FIG. 2. Proposed mechanism for c(LP-LH) production by S9AP-St.

FIG. 3. Effect of substrate concentration and pH on the productionof LP-LH-OMe and c(LP-LH). (A) Effect of L-Pro-OBzl concentration.L-His-OMe at 20 mM and L-Pro-OBzl at 0 to 40 mM were used.(B) Effect of L-His-OMe concentration. L-His-OMe at 0 to 30 mM andL-Pro-OBzl at 20 mM were used. (C) Effects of pH on production ofLP-LH-OMe and c(LP-LH). For these investigations, each value is theaverage of three independent experiments � the standard deviation.

VOL. 76, 2010 SYNTHESIS OF DIVERSE PROLYL DIPEPTIDES 4111

on January 3, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

noacyl derivates cannot be used as acyl acceptors. To performmore convenient dipeptide synthesis, it is crucial to alter thespecificity of S9AP-St toward acyl acceptors.

This work was supported by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Culture, Sports, Science and Tech-nology, Japan.

REFERENCES

1. Arima, J., Y. Uesugi, M. Uraji, M. Iwabuchi, and T. Hatanaka. 2006. Dipep-tide synthesis by aminopeptidase from Streptomyces septatus TH-2 and itsapplication to synthesis of biologically active peptides. Appl. Environ. Mi-crobiol. 72:4225–4231.

2. Bratovanova, E. K., and D. D. Petkov. 1987. Glycine flanked by hydrophobicbulky amino acid residues as minimal sequence for effective subtilisin catal-ysis. Biochem. J. 248:957–960.

3. Elliott, R. J., A. J. Bennet, C. A. Braun, A. M. MacLeod, and T. J. Borgford.2000. Active-site variants of Streptomyces griseus protease B with peptide-ligation activity. Chem. Biol. 7:163–171.

4. Houston, D. R., B. Synstad, V. G. Eijsink, M. J. Stark, I. M. Eggleston, andD. M. J. van Aalten. 2004. Structure-based exploration of cyclic dipeptidechitinase inhibitors. Med. Chem. 47:5713–5720.

5. Joe, K., T. J. Borgford, and T. J. Bennet. 2004. Generation of a thermostableand denaturant-resistant peptide ligase. Biochemistry 43:7672–7677.

6. Minelli, A., C. Conte, S. Grottelli, I. Bellezza, I. Cacciatore, and J. Bolanos.29 March 2008, posting date. Cyclo(His-Pro) promotes cytoprotection byactivating Nrf2-mediated up regulation of antioxidant defence. J. Cell. Mol.Med. [Epub ahead of print.]

7. Minelli, A., I. Bellezza, S. Grottelli, and F. Galli. 2008. Focus on cyclo(His-Pro): history and perspectives as antioxidant peptide. Amino Acids 35:283–289.

8. Morihara, K., and H. Tsuzuki. 1970. Thermolysin: kinetic study with oli-gopeptides. Eur. J. Biochem. 15:374–380.

9. Oka, T., and K. Morihara. 1980. Peptide bond synthesis catalyzed by ther-molysin. J. Biochem. 88:807–813.

10. Prasad, C. 1995. Bioactive cyclic dipeptides. Peptides 16:151–164.11. Song, M. K., I. K. Hwang, M. M. Rosenthal, D. M. Harris, D. T. Yamaguchi,

I. Yip, and V. L. Go. 2003. Anti-hyperglycemic activity of zinc plus cyclo(His-Pro) in genetically diabetic Goto-Kakizaki and aged rats. Exp. Biol.Med. (Maywood) 228:1338–1345.

12. Usuki, H., Y. Uesugi, M. Iwabuchi, and T. Hatanaka. 2009. Putative“acylaminoacyl” peptidases from Streptomyces griseus and S. coelicolor dis-play “aminopeptidase” activities with distinct substrate specificities and sen-sitivities to reducing reagent. Biochim. Biophys. Acta 1794:468–475.

13. Usuki, H., Y. Uesugi, J. Arima, Y. Yamamoto, M. Iwabuchi, and T. Ha-tanaka. 2010. Engineered transaminopeptidase, aminolysin-S for catalysis ofpeptide bond formation to give linear and cyclic dipeptides by one-potreaction. Chem. Commun. (Camb.) 46:580–582.

14. Yokozeki, K., and S. Hara. 2005. A novel and efficient enzymatic method forthe production of peptides from unprotected starting materials. J. Biotech-nol. 115:211–220.

FIG. 4. Time dependence of LP-LH-OMe and c(LP-LH) synthe-sis. The upper panel shows the time course for a 24-h reaction. Thelower panel shows the time course for a 2-h reaction. Each value isthe average of three independent experiments � the standard de-viation.

4112 ARIMA ET AL. APPL. ENVIRON. MICROBIOL.

on January 3, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from