30[Noda Institute for Scientific Research GRANT]2006 Research Grant
Outline of Research Result

Peptide Synthesis by Streptomyces Aminopeptidase
Tadashi HATANAKA
Research Institute for Biological Sciences (RIBS), Okayama

Research aims
Some dipeptides exhibit biological activity; for example,
aspartyl-phenylalanine methyl ester (AspPhe-OMe) is ahigh-intensity sweetener, tyrosyl-arginine (TyrArg) is an
opioid dipeptide (1), and valyl-tyrosine (ValTyr) inhibits anangiotensin I-converting enzyme’s activity (2). In addition,some amino acids such as tyrosine (Tyr), which is considered as an essential amino acid for some premature infants,
have a low solubility. Because these immiscible aminoacids become soluble by ligation to another amino acid,alanyl-tyrosine (AlaTyr) is a potential source of tyrosine (3,4).
Several metallopeptidases, such as thermolysin, synthesize peptides in organic solvents (5). Thermolysin prefers
chemically N-protected peptides as substrates for hydrolysis, and therefore N-protected amino acids are used as acyl
donors in dipeptide synthesis by thermolysin. Hence, deprotection is required to obtain a biologically active dipeptide. In addition, chemically N-protected amino acids aremore expensive than free amino acids and their esters.
Thus, from an economical point of view, N-protected aminoacids are undesirable for use in the chemoenzymatic synthesis of biologically active dipeptides.
Recently, we have identified a thermostable aminopeptidase, which contains cocatalytic metallo-active sites, secreted by Streptomyces septatus TH-2 (SSAP), and succeeded in overproducing it using recombinant E. coli (6).
SSAP has a broad specificity toward peptides (7); however,this enzyme cannot hydrolyze N-protected peptides. Thus,
we postulate that SSAP synthesizes a wide variety of dipeptides using non-N-protected amino acids if SSAP has afunction of reverse reaction.
Methods
SSAP was expressed using E. coli and purified as following our previous investigation (6). Dipeptide synthesis by
SSAP was performed as follows: 20 m l of SSAP solution (1mg/ml) was suspended in 10 m l of 1 M Tris-HCl (pH 8.0)
containing 200 mM amino acid. This suspension wasplaced in a 1.5-ml microtube, frozen by immersion in liquidnitrogen, and lyophilized using FreeZone Freeze-Dry Systems Model 7679520 (Labconco) at a vacuum of 0.3 hPa
for 2 h. This lyophilized enzyme was used for dipeptidesynthesis. The activity remained at 78% after lyophiliza-tion. The reaction was initiated by adding 100 m l of 98%
methanol (MeOH) containing 50 mM aminoacyl-OMe tothe lyophilized enzyme. Then, the reaction was continued
with vigorous shaking (300 rpm) at 25°C for an appropriate
time (20 min50 h). Because SSAP could not synthesize
dipeptides in 99.8% MeOH solution, the reaction was terminated by adding 1.9 ml of 100% MeOH. After centrifugation, the reaction mixture was analyzed by thin layer
chromatography or high-performance liquid chromatography.
Results
Substrate specificity. In our previous study, we found that
SSAP could hydrolyze a wide variety of dipeptides containing hydrophobic residues (7). Thus, we postulate that SSAP
can synthesize a wide variety of dipeptides containing hydrophobic residues. To investigate the synthesis of various
dipeptides by SSAP, we examined acyl donor specificity byreacting Phe-OMe with 20 different free amino acids for 3h, and acyl acceptor specificity by reacting free phenylalanine with 17 different kinds of aminoacyl-OMe for 3 h. As
shown in Fig. 1, a high reaction rate is biased toward synthesis using acyl donors and acceptors that have bulky sidechains. In addition, there was no synthesis when free cysteine was used as an acyl donor, and His-OMe and ProOMe were used as acyl acceptors.
Synthesis of biologically active dipeptides by SSAP. We
further examined the application of SSAP to the productionof several biologically active dipeptides, such as AspPheOMe, ValTyr-OMe, AlaTyr-OMe and Ala-3,4-dihydroxyphenylalanine(DOPA) -OMe. We surmised that other insoluble amino acids, such as DOPA, a therapeutic agent for
Parkinson’s disease, may increase their solubilities by conversion to dipeptides. Because DOPA has a similar structure to tyrosine, we predict that DOPA-OMe is a good acylacceptor. All the dipeptides were synthesized by SSAP in
98% MeOH. In these dipeptide syntheses, the conversion
rates of free amino acids to dipeptides were above 25%(data not shown). We further tried to synthesize esterformed TyrArg using free tyrosine and Arg-OMe; however,this opioid dipeptide could not be obtained (data notshown). We surmise that tyrosine and Arg-OMe cannot behave as a good acyl donor and a good acyl acceptor, respectively.

31Fig. 1. Substrate specificity of dipeptide synthesis by SSAP.
A: Specificity of SSAP toward acyl donor. B: Specificity of SSAP toward acyl acceptor.

Conclusion2)

In this study, we first demonstrated that SSAP can synthesize dipeptides using a free amino acid (acyl donor) andaminoacyl-OMe (acyl acceptor) in 98% MeOH. Further, weshowed that SSAP can be applied to the synthesis of several
biologically active peptides.

3)
4)
5)

References

7)1)

Satoh, M., Kawajiri, S., Yamamoto, M., Akaike, A., Ukai, Y. and6)

Takagi, H. (1980) Neurosci. Lett., 16, 319–322
Tokunaga, K. H., Yoshida, C., Suzuki, K. M., Maruyama, H., Futamura, Y., Araki, Y. and Mishima. S. (2004) Biol. Pharm. Bull., 27,189–192
Daabees, T. T., and Stegink, L. D. (1978) J. Nutr., 108, 1104–1113
Daabees, T. T., and Stegink, L. D. (1979) Pediatr. Res., 13, 894–899
Nakanishi, K., and Matsuno, R. (1990) Ann. N.Y. Acad. Sci., 613,652–655
Arima, J., Iwabuchi, M. and Hatanaka, T. (2004) Biochem. Biophys.
Res. Commun., 317, 531–538
Arima, J., Uesugi, Y., Iwabuchi, M. and Hatanaka, T. (2006) Appl.
Microb. Biotechnol., 70, 541–547