Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity
Language English Country Germany Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- ancestral sequence reconstruction, haloalkane dehalogenase, protein engineering, robustness, substrate specificity,
- MeSH
- Genetic Code MeSH
- Hydrolases chemistry genetics metabolism MeSH
- Multivariate Analysis MeSH
- Protein Engineering MeSH
- Directed Molecular Evolution MeSH
- Substrate Specificity MeSH
- Thermodynamics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- haloalkane dehalogenase MeSH Browser
- Hydrolases MeSH
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 (ΔTm up to 24 °C), 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.
References provided by Crossref.org
Machine Learning-Guided Protein Engineering
Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA
Structures of hyperstable ancestral haloalkane dehalogenases show restricted conformational dynamics
