Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction
| Autor: | Mark van der Giezen, Rhys Cutlan, Nicholas J. Harmer, William Finnigan, Adam Thomas |
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| Rok vydání: | 2019 |
| Předmět: |
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Molecular 0301 basic medicine Ancestral reconstruction Protein Conformation Carboxylic acid Medicine (miscellaneous) Article General Biochemistry Genetics and Molecular Biology Substrate Specificity Evolution Molecular Structure-Activity Relationship 03 medical and health sciences 0302 clinical medicine Molecular evolution Enzyme Stability Matematikk og Naturvitenskap: 400 [VDP] lcsh:QH301-705.5 Phylogeny Thermostability chemistry.chemical_classification Sequence reconstruction Protein engineering Enzyme Activation Kinetics 030104 developmental biology lcsh:Biology (General) chemistry Biocatalysis Evolutionary biology Thermodynamics Oxidoreductases General Agricultural and Biological Sciences 030217 neurology & neurosurgery Neutral mutation |
| Zdroj: | Communications Biology Communications Biology, Vol 2, Iss 1, Pp 1-12 (2019) |
| ISSN: | 2399-3642 |
| DOI: | 10.1038/s42003-019-0677-y |
| Popis: | Carboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis. Thomas et al. uses ancestral sequence reconstruction (ASR) tool to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues, representing one of the largest reconstructed proteins to date. These ancestral CARs display varied tolerances to solvents, pH and in vivo-like salt concentrations along with high thermostability compared to well-studied extant CARs. |
| Databáze: | OpenAIRE |
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