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Enzymatic Production of c-di-GMP Using a Thermophilic Diguanylate Cyclase

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c-di-GMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1657))

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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Acknowledgment

The work on c-di-GMP in the lab of ZXL is supported by a Tier II ARC grant from the Ministry of Education of Singapore and a Tier I ARC grant from Nanyang Technological University (Singapore).

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Correspondence to Zhao-Xun Liang .

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Venkataramani, P., Liang, ZX. (2017). Enzymatic Production of c-di-GMP Using a Thermophilic Diguanylate Cyclase. In: Sauer, K. (eds) c-di-GMP Signaling. Methods in Molecular Biology, vol 1657. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7240-1_2

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  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7239-5

  • Online ISBN: 978-1-4939-7240-1

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