title: Structure of a diguanylate cyclase from Thermotoga maritima: insights into activation, feedback inhibition and thermostability.
abstract: Large-scale production of bis-3'-5'-cyclic-di-GMP (c-di-GMP) would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Angeline
authors.initials: A
authors.lastname: Deepthi
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Chong Wai
authors.initials: CW
authors.lastname: Liew
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Zhao-Xun
authors.initials: ZX
authors.lastname: Liang
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore; Centre d' Immunologie et des Maladies Infectieuses, Centre Hospitalier Universitaire Pitié-Salpêtrière Faculté de Médecine Pierre et Marie Curie, Paris, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
mesh_terms: D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D009154:Mutation; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D029968:Escherichia coli Proteins; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase