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

IUCrJ, 2022-09, Vol.9 (5), p.666-681

Tobias Tandrup et al. 2022 2022 ;ISSN: 2052-2525 ;EISSN: 2052-2525 ;DOI: 10.1107/S2052252522007175 ;PMID: 36071795

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  • Title:
    Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding
  • Author: Tandrup, Tobias ; Muderspach, Sebastian J. ; Banerjee, Sanchari ; Santoni, Gianluca ; Ipsen, Johan Ø. ; Hernández-Rollán, Cristina ; Nørholm, Morten H. H. ; Johansen, Katja S. ; Meilleur, Flora ; Lo Leggio, Leila
  • Subjects: active-site copper ; active-site geometry ; lentinus similis ; lpmo ; lytic polysaccharide monooxygenases ; Research Papers ; saccharide binding ; thermoascus aurantiacus ; x-ray photoreduction
  • Is Part Of: IUCrJ, 2022-09, Vol.9 (5), p.666-681
  • Description: 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 Cu 2+ to Cu + . Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis ( Ls AA9_A) and one from Thermoascus aurantiacus ( Ta AA9_A), as the active-site Cu is photoreduced in the X-ray beam. For Ls AA9_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 Cu 2+ form use a total X-ray dose below 3 × 10 4  Gy, while the Cu + form can be attained using 1 × 10 6  Gy. 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 θ 2 parameter and a decrease of the θ 3 parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θ T . Thus, the θ 2 and θ 3 parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to Ls AA9_A, regardless of the production source, the θ T parameter increases, making the Cu site less planar, while the active-site Tyr—Cu distance decreases reproducibly for the Cu 2+ form. Thus, the θ T increase found on copper reduction may bring Ls AA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced Ls AA9_A for cellulosic substrates.
  • Publisher: International Union of Crystallography
  • Language: English
  • Identifier: ISSN: 2052-2525
    EISSN: 2052-2525
    DOI: 10.1107/S2052252522007175
    PMID: 36071795
  • Source: PubMed Central
    ROAD: Directory of Open Access Scholarly Resources
    ProQuest Central
    DOAJ Directory of Open Access Journals
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