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Molecular determinants for substrate selectivity of ω-transaminases

Applied Microbiology and Biotechnology volume 93, pages 2425–2435 (2012)Cite this article

Abstract

ω-Transaminase (ω-TA) is an industrially important enzyme for production of chiral amines. About 20 (S)-specific ω-TAs known to date show remarkably similar substrate selectivity characterized by stringent steric constraint precluding entry of a substituent larger than an ethyl group in the small binding pocket (S) and dual recognition of an aromatic substituent as well as a carboxylate group in the large pocket (L). The strictly defined substrate selectivity of the available ω-TAs remains a limiting factor in the production of structurally diverse chiral amines. In this work, we cloned, purified, and characterized three new ω-TAs from Ochrobactrum anthropi, Acinetobacter baumannii, and Acetobacter pasteurianus that were identified by a BLASTP search using the previously studied ω-TA from Paracoccus denitrificans. All the new ω-TAs exhibited similar substrate specificity, which led us to explore whether the molecular determinants for the substrate specificity are conserved among the ω-TAs. To this end, key active site residues were identified by docking simulation using the X-ray structure of the ω-TA from Pseudomonas putida. We found that the dual recognition in the L pocket is ascribed to Tyr23, Phe88*, and Tyr152 for hydrophobic interaction and Arg414 for recognition of a carboxylate group. In addition, the docking simulation indicates that Trp60 and Ile262 form the S pocket where the substituent size up to an ethyl group turns out to be sterically allowed. The six key residues were found to be essentially conserved among nine ω-TA sequences, underlying the molecular basis for the high similarity in the substrate selectivity.

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Acknowledgments

This work was supported by the Advanced Biomass R&D Center (ABC-2010-0029737) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology, and Seoul R&BD Program (KU080657). The authors are grateful to Dr. Malik for helpful advice on the manuscript.

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Affiliations

  1. Department of Biotechnology, Yonsei University, Shinchon-Dong 134, Seodaemun-Gu, Seoul, 120-749, South Korea

    Eul-Soo Park, Minji Kim & Jong-Shik Shin

  2. Engineering Building 2 (Rm 507), Yonsei University, Shinchon-Dong 134, Seodaemun-Gu, Seoul, 120-749, South Korea

    Jong-Shik Shin

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  1. Eul-Soo Park
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  2. Minji Kim
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  3. Jong-Shik Shin
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Correspondence to Jong-Shik Shin.

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Park, ES., Kim, M. & Shin, JS. Molecular determinants for substrate selectivity of ω-transaminases. Appl Microbiol Biotechnol 93, 2425–2435 (2012). https://doi.org/10.1007/s00253-011-3584-9

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  • DOI: https://doi.org/10.1007/s00253-011-3584-9

Keywords

  • Transaminase
  • Active site
  • Substrate specificity
  • Docking simulation
  • Chiral amine