We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency.
- Authors
Rubio, Marcelo Ventura; Terrasan, César Rafael Fanchini; Contesini, Fabiano Jares; Zubieta, Mariane Paludetti; Gerhardt, Jaqueline Aline; Oliveira, Leandro Cristante; de Souza Schmidt Gonçalves, Any Elisa; Almeida, Fausto; Smith, Bradley Joseph; de Souza, Gustavo Henrique Martins Ferreira; Dias, Artur Hermano Sampaio; Skaf, Munir; Damasio, André
- Abstract
Background: β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. Results: In this study, BxlB—a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites—was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. Conclusions: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.
- Subjects
FUNGAL proteins; GLYCOSIDASES; ASPERGILLUS nidulans; MOLECULAR dynamics; FUNGAL enzymes; GENETIC models
- Publication
Biotechnology for Biofuels, 2019, Vol 12, Issue 1, pN.PAG
- ISSN
1754-6834
- Publication type
Article
- DOI
10.1186/s13068-019-1609-2