Busca avançada
Ano de início
Entree
(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

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

Texto completo
Autor(es):
Furtado, Gilvan Pessoa [1, 2] ; Lourenzoni, Marcos Roberto [2] ; Fuzo, Carlos Alessandro [3] ; Fonseca-Maldonado, Raquel [1] ; Guazzaroni, Maria-Eugenia [4, 3] ; Ribeiro, Lucas Ferreira [1] ; Ward, Richard J. [3]
Número total de Autores: 7
Afiliação do(s) autor(es):
[1] Univ Sao Paulo, FMRP, Dept Bioquim & Imunol, Ribeirao Preto, SP - Brazil
[2] Fiocruz Ceara, Fundacao Oswaldo Cruz, Fortaleza, Ceara - Brazil
[3] Univ Sao Paulo, Dept Quim, FFCLRP, Ave Bandeirantes 3900, BR-14040901 Ribeirao Preto, SP - Brazil
[4] Univ Sao Paulo, Dept Biol, FFCLRP, Ribeirao Preto, SP - Brazil
Número total de Afiliações: 4
Tipo de documento: Artigo Científico
Fonte: International Journal of Biological Macromolecules; v. 120, n. B, p. 2509-2516, DEC 2018.
Citações Web of Science: 5
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
Linha de fomento: Auxílio à Pesquisa - Temático