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Applied and Environmental Microbiology, November 2005, p. 7229-7235, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7229-7235.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Alteration of Leucine Aminopeptidase from Streptomyces septatus TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis

Jiro Arima, Yoshiko Uesugi, Masaki Iwabuchi, and Tadashi Hatanaka*

Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan

Received 4 April 2005/ Accepted 16 June 2005

To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed L-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with L-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.

* Corresponding author. Mailing address: Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan. Phone: 81-866-56-9452. Fax: 81-866-56-9454. E-mail: hatanaka{at}bio-ribs.com.

Applied and Environmental Microbiology, November 2005, p. 7229-7235, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7229-7235.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Arima, J., Uesugi, Y., Iwabuchi, M., Hatanaka, T. (2008). Streptomyces aminopeptidase P: biochemical characterization and insight into the roles of its N-terminal domain. Protein Eng Des Sel 21: 45-53 [Abstract] [Full Text]  
  • Arima, J., Uesugi, Y., Iwabuchi, M., Hatanaka, T. (2006). Change in Substrate Preference of Streptomyces Aminopeptidase through Modification of the Environment around the Substrate Binding Site. Appl. Environ. Microbiol. 72: 7962-7967 [Abstract] [Full Text]  
  • Arima, J., Uesugi, Y., Uraji, M., Iwabuchi, M., Hatanaka, T. (2006). Dipeptide Synthesis by an Aminopeptidase from Streptomyces septatus TH-2 and Its Application to Synthesis of Biologically Active Peptides.. Appl. Environ. Microbiol. 72: 4225-4231 [Abstract] [Full Text]  
  • Arima, J., Uesugi, Y., Uraji, M., Yatsushiro, S., Tsuboi, S., Iwabuchi, M., Hatanaka, T. (2006). Modulation of Streptomyces Leucine Aminopeptidase by Calcium: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF KEY RESIDUES IN ACTIVATION AND STABILIZATION BY CALCIUM. J. Biol. Chem. 281: 5885-5894 [Abstract] [Full Text]  


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