Probing impact of active site residue mutations on stability and activity ofNeisseria polysacchareaamylosucrase
| Autor: | David Daudé, Christopher M. Topham, Isabelle André, Magali Remaud-Simeon |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
0303 health sciences
FoldX biology 010405 organic chemistry Chemistry Stereochemistry Mutant Mutagenesis Substrate (chemistry) Active site Protein engineering 01 natural sciences Biochemistry 0104 chemical sciences 03 medical and health sciences Amylosucrase Molecular evolution biology.protein Molecular Biology 030304 developmental biology |
| Zdroj: | Protein Science. 22:1754-1765 |
| ISSN: | 0961-8368 |
| Popis: | The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite −1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite −1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8°C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite −1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes. |
| Databáze: | OpenAIRE |
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