Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2

Cellulose is the most abundant natural polymer on Earth, representing an attractive feedstock for bioproducts and biofuel production. Cellulases promote the depolymerization of cellulose, generating short oligosaccharides and glucose, which are useful in biotechnological applications. Among the classical cellulases, those from glycoside hydrolase family 5 (GH5) are one of the most abundant in Nature, displaying several modular archi-tectures with other accessory domains attached to its catalytic core, such as carbohydrate-binding modules (CBMs), Ig-like, FN3-like, and Calx-beta domains, which can influence the enzyme activity. The metagenome-derived endoglucanase CelE2 has in its modular architecture an N-terminal domain belonging to the GH5 family and a C-terminal domain with a high identity to the Calx-beta domain. In this study, the GH5 and the Calx-beta domains were subcloned and heterologously expressed in E. coli, to evaluate the structural and functional properties of the individualized domains of CelE2. Thermostability analysis by circular dichroism (CD) revealed a decrease in the denaturation temperature values around 4.6 degrees C for the catalytic domain (CelE21-381) compared to CelE2 full-length. The CD analyses revealed that the Calx-beta domain (CelE2382-477) was unfolded, suggesting that this domain requires to be attached to the catalytic core to become structurally stable. The three-dimensional structure of the catalytic domain CelE21-381 was determined at 2.1 angstrom resolution, showing a typical (alpha/beta)8-bar-rel fold and a narrow active site compared to other cellulases from the same family. The biochemical charac-terization showed that the deletion of the Calx-beta domain increased more than 3-fold the activity of the catalytic domain CelE21-381 towards the insoluble substrate Avicel. The main functional properties of CelE2, such as substrate specificity, optimal pH and temperature, thermal stability, and activation by CaCl2, were not altered after the deletion of the accessory domain. Furthermore, the Small Angle X-ray Scattering (SAXS) analyses showed that the addition of CaCl2 was beneficial CelE21-381 protein solvency. This work contributed to funda-mental concepts about the structure and function of cellulases, which are useful in applications involving lignocellulosic materials degradation into food and feedstuffs and biofuel production.

Automated summary

This study evaluated the structural and functional properties of the individualized GH5 and Calx-beta domains of CelE2 by subcloning and heterologously expressing them in E. coli. The results showed that the denaturation temperature of the catalytic domain (CelE21-381) decreased compared to the full-length CelE2, and the Calx-beta domain (CelE2382-477) was unfolded without attachment to the catalytic core. The deletion of the Calx-beta domain increased the activity of the catalytic domain towards the insoluble substrate Avicel, without altering other important functional properties.