Abstract
The direct biochemical synthesis of tertiary α-amino acids with a wide range of diversity was recently reported using natural threonine aldolases LTA from Aeromonas jandei and DTA from Pseudomonas sp. Here, we describe the identification of five novel threonine aldolases which accept alanine and serine as amino acid donors. The enzymes were found by sequence database analysis using known aldolases as template. All enzymes were overexpressed in Escherichia coli and purified, and their biochemical properties were characterized. The new enantiocomplementary l- and d-threonine aldolases catalyze the asymmetric synthesis of β-hydroxy α-methyl- and α-hydroxymethyl-α-amino acids with good conversion and perfect enantioselectivity at α-carbon of the products (e.e. >99 %). The structural basis for the broad donor specificity of these threonine aldolases is analyzed based on crystal structure alignments and amino acid sequences comparison.
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Acknowledgments
The activity leading to the present results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution for the Innovative Medicine Initiative under Grant Agreement No. 115360 (Chemical manufacturing methods for the 21st century pharmaceutical industries, CHEM21). We would like to thank Dr. Martina Geier for the support with genetic experiments.
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This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.
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The authors declare that they have no competing interests.
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Fesko, K., Strohmeier, G.A. & Breinbauer, R. Expanding the threonine aldolase toolbox for the asymmetric synthesis of tertiary α-amino acids. Appl Microbiol Biotechnol 99, 9651–9661 (2015). https://doi.org/10.1007/s00253-015-6803-y
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DOI: https://doi.org/10.1007/s00253-015-6803-y