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Title: Versatile peptide C-terminal functionalization via a computationally engineered peptide amidase
Unit code: 153212
First author: Wu Bian*, Wijma Hein J., Song Lu, Rozeboom Henriette J., Poloni Claudia, Tian Yue, Arif Muhammad I., Nuijens Timo, Quaedflieg Peter J. L. M., Szymanski Wiktor, Feringa Ben L., and Janssen Dick B.*
Abstract: The bioactivity of synthetic peptides, including potency, stability and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explored the peptide amidase from Stenotrophomonas maltophilia as a versatile catalyst for diverse carboxy-terminal peptide modification reactions. Since the application scope of the enzyme is hampered by its mediocre stability, we used computational protein engineering supported by energy calculations and molecular dynamics simulations to discover a number of stabilizing mutations. Twelve mutations were combined to yield a highly thermostable (ΔTm = 23°C) and solvent-compatible enzyme. Protein crystallography and molecular dynamics simulations revealed the biophysical effects of mutations contributing to the enhanced robustness. The resulting enzyme selectively catalyzed the selective C-terminal modification of synthetic peptides with small nucleophiles such as ammonia, methylamine and hydroxylamine in various organic (co-)solvents. The use of a non-aqueous environment allowed modification of peptide free acids with >85% product yield under thermodynamic control. Based on the crystal structure, further mutagenesis gave a biocatalyst which favors introduction of larger functional groups. Thus, the use of computational and rational protein design provided a tool for diverse enzymatic peptide modification.
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PubYear: 2016
Publication name: ACS Catalysis
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IF: 9.874
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