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Literature DB >> 24849696

Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution.

Catherine B Poor¹, Mary C Andorfer, Jared C Lewis.

Abstract

We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3-LR, with a Topt over 5 °C higher than that of wild-type, and 3-LSR, with a Tm 18 °C higher than that of wild-type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non-natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.

Entities: Chemical Disease Mutation Species

Keywords: RebH; biocatalysis; directed evolution; halogenases; thermostability

Mesh：

Substances：
Oxidoreductases

Year: 2014 PMID： 24849696 PMCID： PMC4124618 DOI： 10.1002/cbic.201300780

Source DB: PubMed Journal: Chembiochem ISSN： 1439-4227 Impact factor: 3.164

28 in total

1. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair.

Authors: Magnus Wolf-Watz; Vu Thai; Katherine Henzler-Wildman; Georgia Hadjipavlou; Elan Z Eisenmesser; Dorothee Kern
Journal: Nat Struct Mol Biol Date: 2004-08-29 Impact factor: 15.369

2. The structure of flavin-dependent tryptophan 7-halogenase RebH.

Authors: Eduard Bitto; Yu Huang; Craig A Bingman; Shanteri Singh; Jon S Thorson; George N Phillips
Journal: Proteins Date: 2008-01-01

3. Supercharging proteins can impart unusual resilience.

Authors: Michael S Lawrence; Kevin J Phillips; David R Liu
Journal: J Am Chem Soc Date: 2007-08-01 Impact factor: 15.419

Review 4. The unique role of halogen substituents in the design of modern agrochemicals.

Authors: Peter Jeschke
Journal: Pest Manag Sci Date: 2010-01 Impact factor: 4.845

5. Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability.

Authors: Rakesh Kumar; Monika Sharma; Ranvir Singh; Jagdeep Kaur
Journal: Mol Cell Biochem Date: 2012-10-27 Impact factor: 3.396

6. Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor.

Authors: Weslee S Glenn; Ezekiel Nims; Sarah E O'Connor
Journal: J Am Chem Soc Date: 2011-11-11 Impact factor: 15.419

Review 7. Halogen atoms in the modern medicinal chemistry: hints for the drug design.

Authors: Marcelo Zaldini Hernandes; Suellen Melo T Cavalcanti; Diogo Rodrigo M Moreira; Walter Filgueira de Azevedo Junior; Ana Cristina Lima Leite
Journal: Curr Drug Targets Date: 2010-03 Impact factor: 3.465

8. Directed evolution converts subtilisin E into a functional equivalent of thermitase.

Authors: H Zhao; F H Arnold
Journal: Protein Eng Date: 1999-01

9. Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis.

Authors: Ellen Yeh; Sylvie Garneau; Christopher T Walsh
Journal: Proc Natl Acad Sci U S A Date: 2005-03-02 Impact factor: 11.205

10. Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures.

Authors: Indira Wu; Frances H Arnold
Journal: Biotechnol Bioeng Date: 2013-03-01 Impact factor: 4.530

20 in total

Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution.

1. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair.

2. The structure of flavin-dependent tryptophan 7-halogenase RebH.

3. Supercharging proteins can impart unusual resilience.

Review 4. The unique role of halogen substituents in the design of modern agrochemicals.

5. Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability.

6. Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor.

Review 7. Halogen atoms in the modern medicinal chemistry: hints for the drug design.

8. Directed evolution converts subtilisin E into a functional equivalent of thermitase.

9. Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis.

10. Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures.

Review 1. Halogenase engineering and its utility in medicinal chemistry.

2. Late-Stage Diversification of Biologically Active Molecules via Chemoenzymatic C-H Functionalization.

3. Enantioselective Desymmetrization of Methylenedianilines via Enzyme-Catalyzed Remote Halogenation.

4. Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal.

Review 5. Protein Engineering for Improving and Diversifying Natural Product Biosynthesis.

6. Aromatic Halogenation by Using Bifunctional Flavin Reductase-Halogenase Fusion Enzymes.

Review 7. Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse.

8. Directed evolution of RebH for site-selective halogenation of large biologically active molecules.

Review 9. Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis.

10. Flavin Adenine Dinucleotide-Dependent Halogenase XanH and Engineering of Multifunctional Fusion Halogenases.