Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization. Issue 3 (13th December 2019)

• Record Type: Journal Article
• Authors: Minges, Hannah; Schnepel, Christian; Böttcher, Dominique; Weiß, Martin S.; Sproß, Jens; Bornscheuer, Uwe T.; Sewald, Norbert
• Is Part Of: ChemCatChem. Volume 12:Issue 3(2020)
• Page Start: 818
• Page End: 831
• Publication Date: 2019-12-13
• Subjects: directed evolution -- enzyme catalysis -- enzyme stability -- rational mutagenesis -- tryptophan halogenase; Catalysis -- Periodicals; 541.39505
• DOI: 10.1002/cctc.201901827
• Languages: English
• ISSNs: 1867-3880

Abstract

Abstract: Halogenases are valuable biocatalysts for selective C−H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavin‐dependent tryptophan 6‐halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi‐rational mutagenesis leading to a triple mutant (Thal‐GLV) with substantially increased thermostability (▵TM =23.5 K) and higher activity at 25 °C than the wild type enzyme. Moreover, an active‐site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts. Abstract : What's hot & what's not : A comprehensive directed evolution campaign led to a more thermostable tryptophan 6‐halogenase. Successive rounds of random evolution were combined with semi‐rational mutagenesis that generated a more robust biocatalyst. Potential hotspots influencing thermostability, activity and substrate selectivity were combined to deduce a rationale for halogenase stabilization. An enhanced tendency of dimerization probably contributing to higher stability was observed for the thermostable mutants by MS studies.