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Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity

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. While 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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Category

Academic article

Language

English

Author(s)

  • Marianne Slang Jensen
  • Geir Klinkenberg
  • Bastien Bissaro
  • Piotr Chylenski
  • Gustav Vaaje-Kolstad
  • Hans Fredrik Nyvold Kvitvang
  • Guro Kruge Nærdal
  • Håvard Sletta
  • Zarah Forsberg
  • Vincent Eijsink

Affiliation

  • SINTEF Industry / Biotechnology and Nanomedicine
  • Norwegian University of Life Sciences

Date

27.10.2019

Year

2019

Published in

Journal of Biological Chemistry

ISSN

0021-9258

Volume

294

Issue

50

Page(s)

19349 - 19364

View this publication at Norwegian Research Information Repository