Chapter title |
Enzymatic Production of c-di-GMP Using a Thermophilic Diguanylate Cyclase.
|
---|---|
Chapter number | 2 |
Book title |
c-di-GMP Signaling
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Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7240-1_2 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7239-5, 978-1-4939-7240-1
|
Authors |
Venkataramani, Prabhadevi, Liang, Zhao-Xun, Prabhadevi Venkataramani, Zhao-Xun Liang |
Abstract |
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. |
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