Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates
| Autor: | Antoni Planas, Carme Rovira, Bárbara M. Calisto, Mercedes Alfonso-Prieto, Ignacio Fita, Trevor Addington |
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| Rok vydání: | 2010 |
| Předmět: |
beta-Glucans
Glycoside Hydrolases Recombinant Fusion Proteins Amino Acid Motifs Molecular Sequence Data Chimeric enzyme Library science Bacillus Molecular Dynamics Simulation Crystallography X-Ray Protein Engineering Xyloglucan endotransglycosylase Biochemistry S-glucanase Substrate Specificity chemistry.chemical_compound Structural Biology Political science Botany Engineering specificity European commission Amino Acid Sequence Re engineering Glucans Molecular Biology Enzyme Assays Glycosyltransferases Glucanase ß-glucanase Protein Structure Tertiary Xyloglucan Populus chemistry Structural Homology Protein Mutagenesis Site-Directed Xylans Christian ministry Sequence Alignment Protein Binding |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 0887-3585 2001-0044 |
| DOI: | 10.1002/prot.22884 |
| Popis: | Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity. © 2010 Wiley-Liss, Inc. Grant sponsor: European Commission; Grant numbers: QLK5-CT-2001-00443; Grant sponsors: Ministry of Science and Innovation, Spain; Grant numbers: BIO2007-67904-C02-02, FIS2008-03845; Grant sponsors: Institut Químic de Sarrià , Ministry of Science and Innovation, the Generalitat de Catalunya. |
| Databáze: | OpenAIRE |
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