Publication details

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

Authors

BABKOVÁ Petra ŠEBESTOVÁ Eva BREZOVSKÝ Jan CHALOUPKOVÁ Radka DAMBORSKÝ Jiří

Year of publication 2017
Type Article in Periodical
Magazine / Source CHEMBIOCHEM
MU Faculty or unit

Faculty of Science

Citation
Web https://loschmidt.chemi.muni.cz/peg/publications/ancestral-haloalkane-dehalogenases-show-robustness-and-unique-substrate-specificity/
Doi http://dx.doi.org/10.1002/cbic.201700197
Keywords ancestral sequence reconstruction; haloalkane dehalogenase; protein engineering; robustness; substrate specificity
Description Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1–5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (DTm up to 24 8C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.
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