Publication Abstract from PubMed
Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-beta-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-beta-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-beta-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some beta-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-beta-glucanase beta-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 beta-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. Proteins 2010. (c) 2010 Wiley-Liss, Inc.
Re-engineering specificity in 1,3-1, 4-beta-glucanase to accept branched xyloglucan substrates.,Addington T, Calisto B, Alfonso-Prieto M, Rovira C, Fita I, Planas A Proteins. 2010 Sep 22. PMID:21069723[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
