Functional and structural characterization of an alpha-L-arabinofuranosidase from Thermothielavioides terrestris and its exquisite domain-swapped beta-propeller fold crystal packing

Author(s)

Camargo, Suelen ; Mulinari, Evandro J. ; de Almeida, Leonardo R. ; Bernardes, Amanda ; Prade, Rolf A. ; Garcia, Wanius ; Segato, Fernando ; Muniz, Joao R. C.

• Document type: Journal article
• Source: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1868, n. 12, p. 12-pg., 2020-12-01.

Abstract

The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an alpha-L-arabinofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric substrates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 degrees C and 4.5-5.0, respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity for pNP-Araf (K-M: 4 +/- 1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high efficiency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a crystal-induced artifact. Structural information will form the basis for further studies aiming the development of optimized enzymatic properties to be used in biotechnological applications. (AU)

• FAPESP's process: 17/16291-5 - Structural and biophysical characterization of thermophilic enzymes prospected from the fungus Thielavia terrestris for biomass degradation and biotechnological products generation
• Grantee: João Renato Carvalho Muniz
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/09152-6 - Structural and functional studies of cellulosomal thermophilic glycoside hydrolases involved on lignocellulosic biomass hydrolysis
• Grantee: Leonardo Rodrigues de Almeida
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/18714-2 - Enzymatic oxidation of sugarcane bagasse: discovery, characterization and new application of oxidative enzymes active in carbohydrates, applied to the enhancement of a fungal cell factory
• Grantee: Fernando Segato
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants
• FAPESP's process: 14/50897-0 - INCT 2014: Open-acess Medicinal Chemistry Centre (OpenMedChem)
• Grantee: Katlin Brauer Massirer
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/22284-7 - Biochemical, functional characterization and application of polysaccharide lytic monoxygenases identified in transcriptome and secretoma studies of filamentous fungi cultivated in sugarcane bagasse
• Grantee: Fernando Segato
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Regular Program Grants
• FAPESP's process: 17/50025-0 - Development of lignin active enzyme mixture to produce sugars from lignocellulosic biomass for production of fuels, chemicals and bio based materials
• Grantee: Fernando Segato
• Support Opportunities: Regular Research Grants