Atomic resolution of the crystal structure of the hyperthermophilic family 12 endocellulase and stabilizing role of the DxDxDG calcium-binding motif in Pyrococcus furiosus

Hyperthermophilic glycoside hydrolase family 12 endocellulase (EGPf) from the archaeon Pyrococcus furiosus catalyzes the hydrolytic cleavage of β-1,4-glucosidic linkage in β-glucan cellulose. A truncated EGPf (EGPfΔN30) mutant lacking the proline and hydroxyl-residue rich region at the N terminus was constructed, and its crystal structure was resolved at an atomic resolution of 1.07 Å. Our results indicate that the structure of EGPf, which consists of a β-jelly roll, exhibits structural similarity with the endocellulase of Thermotoga maritima  . Additionally, we further determined that the thermostability of EGPf is maintained in part by the binding of Ca2+Ca2+ in a DxDxDG Ca2+Ca2+-binding motif, atypical of most archaeal proteins.

► The crystal structure of Pyrococcus furiosus EGPf was solved at atomic resolution. ► EGPf exhibits structural similarity with the endocellulase of Thermotoga maritima. ► The N terminus of EGPf does not contribute to thermostability. ► EGPf contains a DxDxDG Ca2+-binding motif, atypical of most archaeal proteins. ► The DxDxDG Ca2+-binding motif in EGPf plays a functional role in thermostability.