Elucidation of molecular mechanisms underpinning substrate recognition and catalytic reaction of endoglucanases from the recently characterized families GH128 and GH158: an experimental and computational approach

• Grant number: 22/06298-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: October 01, 2022
• End date: September 30, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Carlos Henrique Inacio Ramos
• Grantee: João Paulo Menezes Spadeto
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

²-1,3-glucans consist of a class of diverse polysaccharides widespread in nature, which have notorious environmental, biotechnological and medical relevance. However, the enzymatic systems acting on these polysaccharides are yet poorly comprehended compared to other glycosidic enzymes active on cellulose, xylans, mannans, for instance. The lack of mechanistic data is more evident when considering recently discovered glycoside hydrolases (GH) families such as GH128 and GH158. Despite the increasing number of Carbohydrate-Active enZymes (CAZymes), mainly boosted by omics approaches, only a few CAZy families have their molecular mechanisms fully elucidated. This current limitation about molecular mechanisms underlying substrate recognition and catalytic reaction of CAZymes is mostly due to the need of the combined use of sophisticated and time-consuming experimental and computational methods, such as quantum mechanics/molecular mechanics (QM/MM) simulations to unveil conformational itineraries and free-energy profiles. The families GH128 and GH158 harbor essentially enzymes for ²-1,3-glucan degradation and had their first crystallographic structures recently published (2020). Although both families belong to GH-A clan, there are important structural divergences between them, including distinct active-site architectures. GH128 and GH158 catalytic properties, such as conformational itineraries, molecular mechanisms underpinning catalysis and possible differences among GH128 subgroups remain elusive. In this project, we will apply experimental techniques (including synchrotron-based experiments), and computational simulations based on classical, QM/MM and enhancing sampling techniques. Using this hybrid approach, we expect to provide unprecedented mechanistic data regarding the processing of this important class of carbohydrates by novel microbial enzymatic systems, advancing our fundamental knowledge in the glycobiology field. (AU)