Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding
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 systema...
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| Format: | Article |
| Language: | English |
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International Union of Crystallography
2022-09-01
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| Series: | IUCrJ |
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| Online Access: | http://scripts.iucr.org/cgi-bin/paper?S2052252522007175 |
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| author | Tobias Tandrup Sebastian J. Muderspach Sanchari Banerjee Gianluca Santoni Johan Ø. Ipsen Cristina Hernández-Rollán Morten H. H. Nørholm Katja S. Johansen Flora Meilleur Leila Lo Leggio |
| author_facet | Tobias Tandrup Sebastian J. Muderspach Sanchari Banerjee Gianluca Santoni Johan Ø. Ipsen Cristina Hernández-Rollán Morten H. H. Nørholm Katja S. Johansen Flora Meilleur Leila Lo Leggio |
| author_sort | Tobias Tandrup |
| collection | DOAJ |
| 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 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 × 104 Gy, while the Cu+ form can be attained using 1 × 106 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 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. |
| first_indexed | 2024-04-11T13:05:30Z |
| format | Article |
| id | doaj.art-f55e8c4da97646a8aef9109ae3ae787c |
| institution | Directory Open Access Journal |
| issn | 2052-2525 |
| language | English |
| last_indexed | 2024-04-11T13:05:30Z |
| publishDate | 2022-09-01 |
| publisher | International Union of Crystallography |
| record_format | Article |
| series | IUCrJ |
| spelling | doaj.art-f55e8c4da97646a8aef9109ae3ae787c2022-12-22T04:22:47ZengInternational Union of CrystallographyIUCrJ2052-25252022-09-019566668110.1107/S2052252522007175mf5061Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide bindingTobias Tandrup0Sebastian J. Muderspach1Sanchari Banerjee2Gianluca Santoni3Johan Ø. Ipsen4Cristina Hernández-Rollán5Morten H. H. Nørholm6Katja S. Johansen7Flora Meilleur8Leila Lo Leggio9Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100-DK, Copenhagen, DenmarkDepartment of Chemistry, University of Copenhagen, Universitetsparken 5, 2100-DK, Copenhagen, DenmarkDepartment of Chemistry, University of Copenhagen, Universitetsparken 5, 2100-DK, Copenhagen, DenmarkESRF, Structural Biology Group, 71 avenue des Martyrs, 38027 Grenoble cedex, FranceDepartment of Geosciences and Natural Resource Management, University of Copenhagen, 1958-DK, Frederiksberg, DenmarkNovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800-DK, Kgs. Lyngby, DenmarkNovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800-DK, Kgs. Lyngby, DenmarkDepartment of Geosciences and Natural Resource Management, University of Copenhagen, 1958-DK, Frederiksberg, DenmarkDepartment of Molecular and Structural Biochemistry, North Carolina State University, Campus Box 7622, Raleigh, NC 27695, USADepartment of Chemistry, University of Copenhagen, Universitetsparken 5, 2100-DK, Copenhagen, DenmarkThe 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 × 104 Gy, while the Cu+ form can be attained using 1 × 106 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 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.http://scripts.iucr.org/cgi-bin/paper?S2052252522007175active-site geometryx-ray photoreductionlytic polysaccharide monooxygenaseslpmolentinus similisthermoascus aurantiacusactive-site coppersaccharide binding |
| spellingShingle | Tobias Tandrup Sebastian J. Muderspach Sanchari Banerjee Gianluca Santoni Johan Ø. Ipsen Cristina Hernández-Rollán Morten H. H. Nørholm Katja S. Johansen Flora Meilleur Leila Lo Leggio Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding IUCrJ active-site geometry x-ray photoreduction lytic polysaccharide monooxygenases lpmo lentinus similis thermoascus aurantiacus active-site copper saccharide binding |
| title | Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding |
| title_full | Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding |
| title_fullStr | Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding |
| title_full_unstemmed | Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding |
| title_short | Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding |
| title_sort | changes in active site geometry on x ray photoreduction of a lytic polysaccharide monooxygenase active site copper and saccharide binding |
| topic | active-site geometry x-ray photoreduction lytic polysaccharide monooxygenases lpmo lentinus similis thermoascus aurantiacus active-site copper saccharide binding |
| url | http://scripts.iucr.org/cgi-bin/paper?S2052252522007175 |
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