Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates.
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.
Laboratory of Biochemistry, Bioengineering Department, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.
Trevor
T
Addington
Barbara
B
Calisto
Mercedes
M
Alfonso-Prieto
Carme
C
Rovira
Ignasi
I
Fita
Antoni
A
Planas
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001407:Bacillus; D018360:Crystallography, X-Ray; D057075:Enzyme Assays; D005936:Glucans / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000235:genetics*; D016695:Glycosyltransferases / Q000235:genetics*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D032107:Populus; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry*; D047071:beta-Glucans / Q000737:chemistry
D005936:Glucans; D011993:Recombinant Fusion Proteins; D014990:Xylans; D047071:beta-Glucans; C029353:xyloglucan; D016695:Glycosyltransferases; C473049:xyloglucan - xyloglucosyltransferase; D006026:Glycoside Hydrolases; C028020:licheninase