3D6E

Crystal structure of the engineered 1,3-1,4-beta-glucanase protein from Bacillus licheniformis

Summary for 3D6E

• Entry DOI: 10.2210/pdb3d6e/pdb
• Related: 1gbg
• Descriptor: Beta-glucanase, CALCIUM ION (3 entities in total)
• Functional Keywords: beta-glucan hydrolysis, calcium binding motif, protein engineering, glycosidase, hydrolase
• Biological source: Bacillus licheniformis
• Total number of polymer chains: 2
• Total formula weight: 45586.23

Authors

Fita, I.,Planas, A.,Calisto, B.M.,Addington, T. (deposition date: 2008-05-19, release date: 2009-05-19, Last modification date: 2023-11-01)

Primary citation

Addington, T.,Calisto, B.,Alfonso-Prieto, M.,Rovira, C.,Fita, I.,Planas, A.
Re-engineering specificity in 1,3-1,4-beta-glucanase to accept branched xyloglucan substrates
Proteins, 79:365-375, 2011

PubMed Abstract: 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.

PubMed: 21069723
DOI: 10.1002/prot.22884

Experimental method

X-RAY DIFFRACTION (2.4 Å)