TY - JOUR
T1 - Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding
AU - Tandrup, Tobias
AU - Muderspach, Sebastian J.
AU - Banerjee, Sanchari
AU - Santoni, Gianluca
AU - Ipsen, Johan
AU - Hernández-Rollán, Cristina
AU - Nørholm, Morten H.H.
AU - Johansen, Katja S.
AU - Meilleur, Flora
AU - Lo Leggio, Leila
AU - Moffat, K.
N1 - Publisher Copyright:
© 2022 International Union of Crystallography. All rights reserved.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
KW - active-site copper
KW - active-site geometry
KW - Lentinus similis
KW - LPMO
KW - lytic polysaccharide monooxygenases
KW - saccharide binding
KW - Thermoascus aurantiacus
KW - X-ray photoreduction
U2 - 10.1107/S2052252522007175
DO - 10.1107/S2052252522007175
M3 - Journal article
C2 - 36071795
AN - SCOPUS:85139211626
SN - 2052-2525
VL - 9
SP - 666
EP - 681
JO - IUCrJ
JF - IUCrJ
ER -