Format

Send to

Choose Destination
Anal Biochem. 2009 Jun 15;389(2):138-42. doi: 10.1016/j.ab.2009.03.031. Epub 2009 Mar 27.

Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase.

Author information

1
Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.

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.

PMID:
19328769
DOI:
10.1016/j.ab.2009.03.031
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center