Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43

Autor(es)

Zanphorlin, Leticia Maria ^[1] ; Bueno de Morais, Mariana Abrahao ^[1] ; Diogo, Jose Alberto ^[1] ; Domingues, Mariane Noronha ^[1] ; Moreira de Souza, Flavio Henrique ^[1] ; Ruller, Roberto ^[1] ; Murakami, Mario Tyago ^[1]

Afiliação do(s) autor(es)

^[1] Natl Ctr Res Energy & Mat, Brazilian Bioethanol Sci & Technol Lab, Giuseppe Maximo Scolfaro 10000, BR-13083100 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Biotechnology and Bioengineering; v. 116, n. 4, p. 734-744, APR 2019.
• Citações Web of Science: 0

Resumo

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-beta-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys(247)) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-beta-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related beta-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the beta-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms. (AU)

• Processo FAPESP: 16/19995-0 - Análise da diversidade estrutural e funcional das enzimas pertencentes à família GH43 em Xanthomonas axonopodis pv. citri: implicações biológicas e possíveis aplicações biotecnológicas
• Beneficiário: Mariana Abrahão Bueno de Morais
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 13/24622-0 - Elucidação da base mecanística da família GH62 presente em organismos especializados em degradação de biomassa vegetal
• Beneficiário: Flavio Henrique Moreira de Souza
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 15/26982-0 - Explorando novas estratégias para a despolimerização de polissacarídeos da parede celular vegetal: da estrutura, função e desenho racional de hidrolases glicosídicas às implicações biológicas e potenciais aplicações biotecnológicas
• Beneficiário: Mário Tyago Murakami
• Modalidade de apoio: Auxílio à Pesquisa - Temático