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Appl Environ Microbiol. 2010 June; 76(12): 4109–4112.
Published online 2010 April 23. doi:  10.1128/AEM.00577-10
PMCID: PMC2893465

The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides[down-pointing small open triangle]


Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. 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 analogs exhibit biological activity. For example, cyclo (l-arginyl-d-proline) [c(lR-dP)] is known to act as a specific inhibitor of family 18 chitinase (4, 10). A cyclic peptide, c(lP-lH), produced by the cleavage of thyrotropin-releasing hormone protects against oxidative stress, promotes cytoprotection (6, 7), and exhibits antihyperglycemic activity (11).

Some serine peptidases exhibit peptide bond formation (i.e., aminolysis of esters, thioesters, and amides) in accordance with their hydrolytic activity (2, 14). The exchange of catalytic Ser for Cys to engineer the serine endopeptidase into “transpeptidase” for peptide bond formation has been well characterized (3, 5). Our recent approach confirmed the wide distribution of family S9 aminopeptidases that have catalytic Ser in actinomycetes (12). Of them, we obtained S9 aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 (S9AP-St). The enzyme was engineered into “transaminopeptidase” by exchange of catalytic Ser for Cys, and its aminolytic activity was evaluated (13). The engineered enzyme, designated as aminolysin-S, can synthesize hydrophobic dipeptides through an aminolysis reaction. However, aminolysin-S was unable to synthesize peptides containing proline. Although the report of aminolysin-S demonstrated that S9AP-St shows no aminolysis reaction toward limited substrates, details of its characteristics remain unknown. This study verified the peptide synthetic activity of S9AP-St, demonstrating that S9AP-St can synthesize widely varied prolyl dipeptides through an aminolysis reaction. The report also shows that S9AP-St is applicable to the synthesis of a biologically active peptide—c(lP-lH).

Homopeptide synthetic activity of S9AP-St.

Recombinant S9AP-St was purified from cultivated cells of Escherichia coli BL21(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 reaction was conducted using various aminoacyl derivatives under the conditions described in the supplemental material. The synthesized peptides were analyzed using electrospray ionization-time of flight mass spectrometry (ESI-TOF MS) (LCT Premier 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 (Table1).1). The result indicates that S9AP-St has peptide synthetic activity through its aminolysis reaction.

Tested chemicals and peptides synthesized using S9AP-St

Synthesis of prolyl peptides using S9AP-St.

We next investigated whether S9AP-St possesses the ability to synthesize prolyl heteropeptides. The assay was conducted as described in the supplemental material. It is particularly interesting that S9AP-St can use l-Pro-OBzl only as an acyl donor, with the result that all synthetic peptides have an lP-X structure (Table (Table1).1). The investigation shows that almost all aminoacyl derivatives are useful as acyl acceptors, which was independent of enantiospecificity. It is especially noteworthy that by-product cyclic dipeptides were observed.

When the reaction time was extended, it was observed that dipeptidyl derivatives were converted into cyclic dipeptides. As presented in Fig. Fig.1,1, lP-lH-OMe and c(lP-lH) were detected at almost mutually identical levels after a 3-h reaction. In contrast, only the peak of product c(lP-lH) was detected after a 24-h reaction. The cyclization of lP-lH-OMe might have occurred nonenzymatically because the c(lP-lH) production was continued when lP-lH-OMe was exposed at neutral pH (data not shown). The proposed mechanism for c(lP-lH) production is presented in Fig. Fig.22.

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 reaction mixture without the enzyme (w/o S9AP-St) for comparison of synthesis ...
FIG. 2.
Proposed mechanism for c(lP-lH) production by S9AP-St.

We further evaluated the d-prolyl peptide synthetic activity using d-Pro-OBzl (Table (Table1).1). Although S9AP-St only showed hydrolytic activity toward l-aminoacyl-pNA (13), it was surprising that S9AP-St can use d-Pro-OBzl as an acyl donor. When using d-Pro-OBzl as acyl donor, some acidic and basic aminoacyl-OMe's, l-Ala, d-Val, and l-Ser-OMe's could not be used as acyl acceptors whereas they acted as acyl acceptor when using l-Pro-OBzl as acyl donor. Therefore, specificity toward the acyl acceptor is narrower than that when using l-Pro-OBzl as the acyl donor.

Effects of substrate concentration and pH on c(lP-lH) synthesis.

We next investigated the reaction condition for the production of c(lP-lH), a cyclic dipeptide with antihyperglycemic activity. The production level of products was determined using ultraperformance liquid chromatography (UPLC)-ESI-TOF MS with a C18 reverse-phase system (Acquity UPLC; Waters Corp.). Under the UPLC conditions described 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 concentration on synthesis. As depicted in Fig. Fig.3A,3A, the production of lP-lH-OMe and c(lP-lH) was increased following the increase of l-Pro-OBzl concentration. On the other hand, when the l-Pro-OBzl concentration was maintained at a steady level of 20 mM, the c(lP-lH) concentration was lowered when the l-His-OMe concentration was higher than 20 mM (Fig. (Fig.3B).3B). We next examined the effect of pH on synthesis. As portrayed in Fig. Fig.3C,3C, the production of lP-lH-OMe was decreased at a pH higher than 8.5. An enzymatic product, lP-lH-OMe, might be liable to cause cyclization at high pH. Consequently, the c(lP-lH) productivity was increased following the increase of pH.

FIG. 3.
Effect of substrate concentration and pH on the production of 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 and ...

Time dependence of prolyl dipeptide synthesis.

Extension of the reaction time engenders the emergence of a cyclic dipeptide. As presented in Fig. Fig.4,4, lP-lH-OMe was synthesized efficiently until 30 min, and then the product was decreased gradually because of conversion into c(lP-lH). After 24 h, lP-lH-OMe was converted completely into c(lP-lH). In terms of the substrate consumption, l-Pro-OBzl was completely converted into lP-lH-OMe or free l-Pro after 120 min. In contrast, l-His-OMe decreased only slightly during 24-h reaction, indicating that S9AP-St uses l-His-OMe only as an acyl acceptor; it has no hydrolytic activity toward l-His-OMe.

FIG. 4.
Time dependence of lP-lH-OMe and c(lP-lH) synthesis. The upper panel shows the time course for a 24-h reaction. The lower panel shows the time course for a 2-h reaction. Each value is the average of three independent experiments ± the standard ...

Yield of prolyl dipeptide.

To evaluate the conversion ratio of the substrate to c(lP-lH), we quantified free l-His in the alkaline-treated reaction mixture of a 24-h reaction with or without enzyme using an amino acid analyzer (JLC-500/V2; JEOL). Using the method described in the supplemental material, the concentrations of l-His in the reaction mixture with and without enzyme were estimated, respectively, as 19.2 ± 0.4 and 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 acyl acceptor. From these values, the conversion ratio of substrate to lP-lH-OMe was estimated as higher than 40%.

In enzymatic peptide synthesis by reverse reaction, not by the aminolysis reaction, peptides that act as good substrates in hydrolysis are appropriate targets of synthesis (1, 8, 9). In contrast, the investigation of S9AP-St shows that free l-Pro increased only slightly after l-Pro-OBzl was completely consumed (Fig. (Fig.4),4), indicating that the lP-lH-OMe product was hydrolyzed only slightly by S9AP-St itself. We infer that prolyl derivatives are good substrates for hydrolytic or aminolytic activity of S9AP-St. However, S9AP-St recognizes prolyl dipeptides as a substrate only to a slight degree.

The results of this study demonstrated that S9AP-St is applicable for syntheses of various prolyl dipeptides, including c(lP-lH). However, in the synthesis using S9AP-St, some aminoacyl derivates cannot be used as acyl acceptors. To perform more convenient dipeptide synthesis, it is crucial to alter the specificity of S9AP-St toward acyl acceptors.

Supplementary Material

[Supplemental material]


This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


[down-pointing small open triangle]Published ahead of print on 23 April 2010.

Supplemental material for this article may be found at


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