Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43

Author(s)

Zanphorlin, Leticia Maria ^[1] ; Bueno de Morais, Mariana Abrahao ^[1] ; Diogo, Jose Alberto ^[1] ; Domingues, Mariane Noronha ^[1] ; Moreira de Souza, Flavio Henrique ^[1] ; Ruller, Roberto ^[1] ; Murakami, Mario Tyago ^[1]

Affiliation

^[1] Natl Ctr Res Energy & Mat, Brazilian Bioethanol Sci & Technol Lab, Giuseppe Maximo Scolfaro 10000, BR-13083100 Campinas, SP - Brazil

• Document type: Journal article
• Source: Biotechnology and Bioengineering; v. 116, n. 4, p. 734-744, APR 2019.
• Web of Science Citations: 0

Abstract

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-beta-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys(247)) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-beta-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related beta-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the beta-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms. (AU)

• FAPESP's process: 16/19995-0 - Analysis of structural and functional diversity of GH43 enzymes from Xanthomonas axonopodis pv. citri: biological implications and potential biotechnological applications
• Grantee: Mariana Abrahão Bueno de Morais
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 13/24622-0 - Elucidation of the mechanistic basis of family GH62 present in organisms specialized in the degradation of plant biomass
• Grantee: Flavio Henrique Moreira de Souza
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/26982-0 - Exploring novel strategies for depolymerization of plant cell-wall polysaccharides: from structure, function and rational design of glycosyl hydrolases to biological implications and potential biotechnological applications
• Grantee: Mário Tyago Murakami
• Support Opportunities: Research Projects - Thematic Grants