Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides

Autor(es)

Furtado, Gilvan Pessoa ; Lourenzoni, Marcos Roberto ; Fuzo, Carlos Alessandro ; Fonseca-Maldonado, Raquel ; Guazzaroni, Maria-Eugenia ; Ribeiro, Lucas Ferreira ; Ward, Richard J.

• Tipo de documento: Artigo Científico
• Fonte: International Journal of Biological Macromolecules; v. 120, p. 8-pg., 2018-12-01.

Resumo

Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CeIH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide beta-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 mutagenesis generated 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 calorimetry demonstrated increased xyloglucan affinity for both mutants and reduced affinity for beta-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. (C) 2018 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 10/18850-2 - Identificação, caracterização e engenharia de enzimas que degradam a parede celular das plantas
• Beneficiário: Richard John Ward
• Modalidade de apoio: Auxílio à Pesquisa - Temático