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.
CITATION STYLE
Venkataramani, P., & Liang, Z. X. (2017). Enzymatic production of c-di-GMP using a thermophilic diguanylate cyclase. In Methods in Molecular Biology (Vol. 1657, pp. 11–22). Humana Press Inc. https://doi.org/10.1007/978-1-4939-7240-1_2
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