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Molecular determinants for substrate selectivity of ω-transaminases
- Park, Eul-Soo, Kim, Minji, Shin, Jong-Shik
- Applied microbiology and biotechnology 2012 v.93 no.6 pp. 2425-2435
- Acetobacter pasteurianus, Acinetobacter baumannii, Ochrobactrum anthropi, Paracoccus denitrificans, Pseudomonas putida, active sites, amines, hydrophobic bonding, production economics, substrate specificity
- 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.