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 ?- 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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et al. 2007), Alcaligenes denitrificans (AD) (Yun et al. 2004b), Vibrio fluvialis (VF) (Shin et al. 2003), and Caulobacter crescentus (CC) (Hwang et al. 2008). Bold letters mark key active site residues that are flanked by three contiguous residues. Numbers refer to the position of the amino acid at the beginning of each line 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.
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology