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. Here we have monitored the Cu-site geometry 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. In addition, we have characterized in detail the structural effects of oligosaccharide binding in differently photoreduced states of LsAA9_A.

. With increasing X-ray dose both the equatorial and axial water molecule increase their distance to Cu. Most notably the axial water molecule, as large distances at the equatorial position is consistently prevented by polyacrylic acid, modeled as acrylic acid. Close to the axial position is also a HEPES molecule with 60 % occupancy, though not shown here for clarity. For all panels the 2F O -F C map (blue mesh) is shown at 1.0 σ contour level, and difference map in green/red mesh at +/-3.0 σ contour level. An animation of the transition between these structures is available as Supplementary Movie 3. PDB codes: 7PZ3 (a), 7PZ4 (b), 7PZ5 (c), 7PZ6 (d), 7PZ7 (e), 7PZ8 (f).

Figure S12
Active site of LsAA9_A structure solved by serial synchrotron crystallography. The structure was solved from 13 crystals, each estimated to have received an X-ray dose of 7.02 × 10 4 Gy. Active site distances are to some extent comparable to the 5.99 × 10 4 Gy native LsAA9_A(f) structure presented in Figure 2b. Active site distances listed in Table 3 and Supplementary Table 7 Table 3 and Supplementary Table 7. At increasing X-ray dose, the equatorial Cl-ion (green spheres, present from crystallization conditions) increase its distance to the Cu. Distances in Å are measured between the Cl-ion and the Cu. In panels c) and e) the equatorial water molecule has been modeled in a double conformation. The Tyr164-O to Cu distance is here in all cases shorter than for the substrate free structures presented in Supplementary Figure 7. This distance increases slightly with increasing X-ray dose (see Figure 6). For all panels the 2F O -F C map (blue mesh) is shown at 1.0 σ contour level and the difference map in green/red mesh at +/-3.0 σ contour level. An animation of the transition between these structures is available as Supplementary Movie 4. PDB codes:   Table 7. Similar to LsAA9_A(f)-Cell 4 no axial water molecule is present with cello-oligosaccharide bound. Cell 3 binds from subsite -1 to +2, as has been described previously for the fungal expressed LsAA9_A (Frandsen et al., 2016;Tandrup et al., 2020).
All panels show a well-defined Clion at the equatorial position. This Clion increase its distance to Cu with increasing X-ray dose. Distances in Å are measured between the Cl-ion and the Cu. For all panels the 2F O -F C map (blue mesh) is shown at 1.0 σ contour level, and difference map in green mesh at 3.0 σ contour level.
An animation of the transition between these structures is available as Supplementary Movie 5. PDB codes:

Figure S15
Difference maps demonstrating shortening of the Tyr-OH Cu distance on saccharide binding.
To avoid refinement bias, difference maps were calculated using the low dose saccharide-bound data, LsAA9_A(Ec)-Cell 3 (1.49 × 10 4 Gy) after rigid body refinement using the low dose saccharide-free model. A single rigid body including the protein and Cu (at fixed distance) were used for refinement and phasing (a).
The difference density clearly indicate that Cu is closer to the Tyr-OH in the saccharide-bound structure.

Table S3
Crystallographic data and refinement statistics.