Engineered RebH Halogenase Variants Demonstrating a Specificity Switch from Tryptophan towards Novel Indole Compounds
| Autor: | Chandra S. Verma, Eunice H. Y. Li, Timothy Ho, Hung A. Duong, Farid J. Ghadessy, Ding Ke, Srinivasaraghavan Kannan, Jayasree Seayad, Barindra Sana |
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| Rok vydání: | 2021 |
| Předmět: |
Indoles
Halogenation biocatalysis Mutagenesis (molecular biology technique) Molecular Dynamics Simulation Biochemistry Substrate Specificity chemistry.chemical_compound Bacterial Proteins directed evolution Molecular Biology Indole test Binding Sites Full Paper Chemistry Organic Chemistry Tryptophan Protein engineering Full Papers Directed evolution Combinatorial chemistry Actinobacteria halogenase enzyme engineering indole Biocatalysis Mutagenesis Site-Directed Molecular Medicine Organic synthesis RebH Oxidoreductases |
| Zdroj: | Chembiochem |
| ISSN: | 1439-7633 1439-4227 |
| Popis: | Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure‐guided semi‐rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated. Two engineered halogenase enzymes were developed by switching substrate specificity of the parental enzyme from tryptophan to indole compounds. They can halogenate several industrially important indole derivatives more efficiently than the parental enzyme; this includes enzymatic chlorination/bromination of novel substrates that were not reported previously. The engineered enzymes could be useful in regioselective synthesis of drug intermediates. |
| Databáze: | OpenAIRE |
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