title: Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase.
abstract: The cyclic dinucleotide c-di-GMP is a widespread bacterial messenger molecule with potential application as a therapeutic agent for treating bacterial infection. Current enzymatic synthesis of c-di-GMP using mesophilic diguanylate cyclase (DGC) proteins suffers from low production yield due to protein instability and strong product inhibition. Here we report the overexpression and characterization of a stand-alone thermophilic diguanylate cyclase domain (tDGC) protein with enhanced thermostability. The product inhibition that severely limited production yield was significantly alleviated by mutation of a conserved residue in the putative regulatory I-site. With the mutant tDGC, we demonstrated that hundreds of milligrams of c-di-GMP can be readily prepared by using the optimized procedures for enzymatic reaction and product purification. The thermophilic enzyme will be a valuable tool for other research laboratories for c-di-GMP synthesis as well as the preparation of c-di-GMP derivatives.
authors.affiliation: Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
authors.forename: Feng
authors.initials: F
authors.lastname: Rao
authors.forename: Swathi
authors.initials: S
authors.lastname: Pasunooti
authors.forename: Yinglu
authors.initials: Y
authors.lastname: Ng
authors.forename: Weichao
authors.initials: W
authors.lastname: Zhuo
authors.forename: Lishi
authors.initials: L
authors.lastname: Lim
authors.forename: Angeline Weixian
authors.initials: AW
authors.lastname: Liu
authors.forename: Zhao-Xun
authors.initials: ZX
authors.lastname: Liang