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

Author(s)

Pimentel, Agnes C. ; Liberato, Marcelo V. ; Cairo, Joao Paulo L. Franco ; Tomazetto, Geizecler ; Gandin, Cesar A. ; Neto, Mario de Oliveira ; Alvarez, Thabata M. ; Squina, Fabio M.

• Document type: Journal article
• Source: Enzyme and Microbial Technology; v. 165, p. 10-pg., 2023-04-01.

Abstract

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. (AU)

• FAPESP's process: 16/01926-2 - Influence of an accessory domain in biochemical and structural characteristics of the cellulase CelE2
• Grantee: Agnes Cristina Pimentel
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 15/50590-4 - Lignin valorization in cellulosic ethanol plants: biocatalytic conversion via ferulic acid to high value chemicals
• Grantee: Fábio Márcio Squina
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 20/05784-3 - EMU approved in grant 15 / 50590-4: chromatographic system and detectors for analysis of sugars and lignocellulosic monolignols
• Grantee: Fábio Márcio Squina
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 14/04105-4 - Structural and functional characterization of new cellulases, focusing in the relation between catalytic domains and CBMs
• Grantee: Marcelo Vizoná Liberato
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 10/11469-1 - Library generation for biomass-conversion enzymes from soil metagenome
• Grantee: Thabata Maria Alvarez
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 16/09950-0 - Functional, structural characterizations and biotechnological application of lytic polysaccharide monooxygenases from the lower termite Coptotermes gestroi
• Grantee: João Paulo Lourenço Franco Cairo
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/23279-6 - Metagenomic and metasecretomic analyses of an enrichment microbial consortium of anaerobic fungi to the degradation of sugarcane bagasse: prospection of cellulosomas and lignocellolytic enzymes
• Grantee: Geizecler Tomazetto
• Support Opportunities: Scholarships in Brazil - Post-Doctoral