Abstract
Compared to (S)-selective amine transaminase ((S)-AT), the (R)-selective counterpart ((R)-AT) has been less studied. As such, a simplified “Motif Sequence Blast” search (Höhne et al. Nat Chem Biol 6:807–813, 2010) was carried out to identify new (R)-ATs from the protein databases. The combined conserved sequence motifs of (R)-ATs based on the previous in silico method of predicting (R)-selective amine transaminase were used as the template sequence for BLASTP search at default settings in NCBI, and six candidate sequences were identified. These putative (R)-AT genes were synthesized and overexpressed in Escherichia coli. Among them, five new (R)-ATs were expressed as soluble protein and showed unusual substrate specificity and high stereoselectivity. Furthermore, several unnatural amino acids, such as d-alanine, d-2-aminobutyric acid, and d-norvaline, were synthesized via the (R)-AT-catalyzed amino transfer reaction to the corresponding keto acids. Optically pure (S)-amines were also obtained by kinetic resolution of racemic amines catalyzed with these new (R)-ATs. Therefore, the Motif Sequence Blast search offers a quick and effective method for in silico identification of new (R)-ATs, and the newly identified (R)-ATs are attractive additions to the toolbox of (R)-ATs for further study and industrial application.
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Acknowledgments
This work was financially supported by National Basic Research Program of China (973 Program, no. 2011CB710801), the National Natural Science Foundation of China (grant no. 21072151), and the CAS Special Grant for Postgraduate Research, Innovation and Practice (grant no. Y2J8041021). We thank Professor Xiaoping Yi (Chinese Academy of Tropical Agricultural Sciences) for supplying the strain Fusarium oxysporum Fo5176.
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Jiang, J., Chen, X., Zhang, D. et al. Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast. Appl Microbiol Biotechnol 99, 2613–2621 (2015). https://doi.org/10.1007/s00253-014-6056-1
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DOI: https://doi.org/10.1007/s00253-014-6056-1