Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability

Daniel Kracher*, Martina Andlar, Paul G. Furtmüller, Roland Ludwig

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa, which is active toward cellulose and soluble -glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding.

Original languageEnglish
Pages (from-to)1676-1687
Number of pages12
JournalJournal of Biological Chemistry
Volume293
Issue number5
DOIs
Publication statusPublished - 2 Feb 2018
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability'. Together they form a unique fingerprint.

Cite this