Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase
| Autor: | Timo Nuijens, Claudia Poloni, Muhammad I. Arif, Ben L. Feringa, Peter J. L. M. Quaedflieg, Bian Wu, Hein J. Wijma, Yu’e Tian, Henriëtte J. Rozeboom, Lu Song, Wiktor Szymanski, Dick B. Janssen |
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| Přispěvatelé: | Biotechnology, Synthetic Organic Chemistry, Basic and Translational Research and Imaging Methodology Development in Groningen (BRIDGE) |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
STABILIZATION enzymatic catalysis PROTEINS computational protein engineering THERMAL-STABILITY Peptide Catalysis VALIDATION Enzyme catalysis Amidase 03 medical and health sciences STENOTROPHOMONAS-MALTOPHILIA peptide modification Thermostability chemistry.chemical_classification Peptide modification Chemistry SALT BRIDGE MD simulation General Chemistry Protein engineering BIOACTIVE PEPTIDES Combinatorial chemistry 030104 developmental biology Biochemistry protein stability THERMOSTABILITY THERAPEUTICS Surface modification Salt bridge ENZYMES |
| Zdroj: | ACS Catalysis, 6(8), 5405-5414. AMER CHEMICAL SOC |
| ISSN: | 2155-5435 |
| Popis: | The properties 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. Because the scope of application 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 (Delta T-m = 23 degrees 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 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 nonaqueous environment allowed modification of peptide free acids with >85% product yield under thermodynamic control. On the basis of the crystal structure, further mutagenesis gave a biocatalyst that favors introduction of larger functional groups. Thus, the use of computational and rational protein design provided a tool for diverse enzymatic peptide modification. |
| Databáze: | OpenAIRE |
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