Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans
Abstract
:1. Introduction
2. Results and Discussion
2.1. Candidate Selection and Recombinant Protein Production
2.2. General Biochemical Properties
2.3. Activity on Acetylated Oligosaccharides
2.4. Complementation of a-Glucuronidase Activity
2.5. Structural Determinants of the New CE Family
2.6. Sequence Determinants of the New CE Family
2.7. Five Conserved Loops Shape the Active Site
2.8. Substrate Binding at the Active Site
3. Materials and Methods
3.1. Materials
3.2. Candidate Selection
3.3. Protein Expression and Purification
3.4. Enzyme Activity Screens
3.5. Optimum Reaction Conditions
3.6. Activity on Acetylated Xylooligosaccharides
3.7. Complementation Assay Using AcXE and α-Glucuronidase
3.8. Protein Crystallization, Data Collection, and Structure Determination
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Substrate | pH | FspAcXE | FspF52AcXE | CspAcXE | PbeAcXE | FreAcXE | AimAcXE |
---|---|---|---|---|---|---|---|
pNPA | 5.5 | 23 | 27 | 52 | 42 | 47 | 24 |
7 | 47 | 47 | 57 | 55 | 50 | 53 | |
4-MUA | 5.5 | 35 | 32 | 33 | 31 | 25 | 25 |
7 | 45 | 42 | 48 | 47 | 40 | 54 |
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Penttinen, L.; Kouhi, V.; Fauré, R.; Skarina, T.; Stogios, P.; Master, E.; Jurak, E. Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans. Molecules 2022, 27, 2655. https://doi.org/10.3390/molecules27092655
Penttinen L, Kouhi V, Fauré R, Skarina T, Stogios P, Master E, Jurak E. Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans. Molecules. 2022; 27(9):2655. https://doi.org/10.3390/molecules27092655
Chicago/Turabian StylePenttinen, Leena, Vera Kouhi, Régis Fauré, Tatiana Skarina, Peter Stogios, Emma Master, and Edita Jurak. 2022. "Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans" Molecules 27, no. 9: 2655. https://doi.org/10.3390/molecules27092655