Title
Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides
Author
Gilvan Pessoa Furtado, Marcos Roberto Lourenzoni, Carlos Alessandro Fuzo, Raquel Fonseca-Maldonado, María-Eugenia Guazzaroni, Lucas Ferreira Ribeiro, Richard J. Ward
Year
2018
Journal
International Journal of Biological Macromolecules
Abstract
Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide β-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage library created by error prone PCR for xyloglucan binding identified RtCBM11 mutants with enhanced xyloglucan affinity. Subsequent recombination of the selected variants by site-directed mutagenesisgenerated the H102L/Y152F and Y46N/G52D/H102L/Y152F mutants. Fusion of the quadruple RtCBM11 mutant with the xyloglucanase from Aspergillus niveus increased the catalytic efficiency of the enzyme by 38%. Isothermal titration calorimetrydemonstrated increased xyloglucan affinity for both mutants and reduced affinity for β-glucan in the H102L/Y152F mutant. Molecular dynamics simulations indicated that the increased xyloglucan specificity results both from formation of a xylosyl binding pocket in the carbohydrate binding cleft, and via modulation of a hydrogen bond network between the oligosaccharide ligand and the protein. These results explain the improved xyloglucan binding in the RtCBM11 H102L/Y152F mutant and advance the understanding of the structural determinants of CBMs binding that discriminate between branched and unbranched polysaccharides.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry