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Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity.
Jensen, Marianne Slang; Klinkenberg, Geir; Bissaro, Bastien; Chylenski, Piotr; Vaaje-Kolstad, Gustav; Kvitvang, Hans Fredrik; Nærdal, Guro Kruge; Sletta, Håvard; Forsberg, Zarah; Eijsink, Vincent G H.
Afiliación
  • Jensen MS; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, NO-1432 Ås, Norway.
  • Klinkenberg G; SINTEF Industry, Department of Biotechnology and Nanomedicine, NO-7465 Trondheim, Norway.
  • Bissaro B; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, NO-1432 Ås, Norway.
  • Chylenski P; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, NO-1432 Ås, Norway.
  • Vaaje-Kolstad G; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, NO-1432 Ås, Norway.
  • Kvitvang HF; SINTEF Industry, Department of Biotechnology and Nanomedicine, NO-7465 Trondheim, Norway.
  • Nærdal GK; SINTEF Industry, Department of Biotechnology and Nanomedicine, NO-7465 Trondheim, Norway.
  • Sletta H; SINTEF Industry, Department of Biotechnology and Nanomedicine, NO-7465 Trondheim, Norway.
  • Forsberg Z; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, NO-1432 Ås, Norway zarah.forsberg@nmbu.no.
  • Eijsink VGH; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, NO-1432 Ås, Norway vincent.eijsink@nmbu.no.
J Biol Chem ; 294(50): 19349-19364, 2019 12 13.
Article en En | MEDLINE | ID: mdl-31656228
ABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.
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Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Polisacáridos / Ingeniería de Proteínas / Celulosa / Quitina / Streptomyces coelicolor / Oxigenasas de Función Mixta Idioma: En Revista: J Biol Chem Año: 2019 Tipo del documento: Article País de afiliación: Noruega

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Polisacáridos / Ingeniería de Proteínas / Celulosa / Quitina / Streptomyces coelicolor / Oxigenasas de Función Mixta Idioma: En Revista: J Biol Chem Año: 2019 Tipo del documento: Article País de afiliación: Noruega