| Autor: |
Prabhadevi, Venkataramani, Zhao-Xun, Liang |
| Rok vydání: |
2017 |
| Předmět: |
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| Zdroj: |
Methods in molecular biology (Clifton, N.J.). 1657 |
| ISSN: |
1940-6029 |
| Popis: |
C-di-GMP has emerged as a prevalent bacterial messenger that controls a multitude of bacterial behaviors. Having access to milligram or gram quantities of c-di-GMP is essential for the biochemical and structural characterization of enzymes and effectors involved in c-di-GMP signaling. Although c-di-GMP can be synthesized using chemical methods, diguanylate cyclases (DGC)-based enzymatic synthesis is the most efficient method of preparing c-di-GMP today. Many DGCs are not suitable for c-di-GMP production because of poor protein stability and the presence of a c-di-GMP-binding inhibitory site (I-site) in most DGCs. We have identified and engineered a thermophilic DGC for efficient production of c-di-GMP for characterizing c-di-GMP signaling proteins and riboswitches. Importantly, residue replacement in the inhibitory I-site of the thermophilic DGC drastically relieved product inhibition to enable the production of hundreds of milligrams of c-di-GMP using 5-10 mg of this robust biocatalyst. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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