Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

Tobias Tandrup, Sebastian J. Muderspach, Sanchari Banerjee, Gianluca Santoni, Johan Ipsen, Cristina Hernández-Rollán, Morten H.H. Nørholm, Katja S. Johansen, Flora Meilleur, Leila Lo Leggio*, K. Moffat

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading polysaccharide substrates oxidatively. The generally accepted first step in the LPMO reaction is the reduction of the active-site metal ion from Cu2+to Cu+. Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis (LsAA9_A) and one from Thermoascus aurantiacus (TaAA9_A), as the active-site Cu is photoreduced in the X-ray beam. For LsAA9_A, the protein produced in two different recombinant systems was crystallized to probe the effect of post-translational modifications and different crystallization conditions on the active site and metal photoreduction. We can recommend that crystallographic studies of AA9 LPMOs wishing to address the Cu2+form use a total X-ray dose below 3 × 104Gy, while the Cu+form can be attained using 1 × 106Gy. In all cases, we observe the transition from a hexacoordinated Cu site with two solvent-facing ligands to a T-shaped geometry with no exogenous ligands, and a clear increase of the θ2 parameter and a decrease of the θ3 parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θT. Thus, the θ2 and θ3 parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to LsAA9_A, regardless of the production source, the θT parameter increases, making the Cu site less planar, while the active-site Tyr - Cu distance decreases reproducibly for the Cu2+form. Thus, the θT increase found on copper reduction may bring LsAA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced LsAA9_A for cellulosic substrates.

Original languageEnglish
JournalIUCrJ
Volume9
Pages (from-to)666-681
Number of pages16
ISSN2052-2525
DOIs
Publication statusPublished - 2022

Keywords

  • active-site copper
  • active-site geometry
  • Lentinus similis
  • LPMO
  • lytic polysaccharide monooxygenases
  • saccharide binding
  • Thermoascus aurantiacus
  • X-ray photoreduction

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