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

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 × 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.

Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature

“…The tyrosine hydroxy group is in the second axial coordination site of the copper, but the Cu-O Tyr distance is too long to form a Cu-O Tyr bond. Recent analysis of X-ray structures at low X-ray dose have confirmed that on binding oligosaccharide the distance to Tyr164 is shortened (Tandrup et al, 2022). In the structure presented here, Tyr164 is positioned in the axial position of the copper with a Cu-O Tyr distance of 2.80 A ˚.…”

“…5). The distances from H 2 O-eq and H 2 O-ax to Cu 2+ are 2.1 and 2.7 A ˚, respectively, confirming that the copper ion is in the resting oxidation state, copper(II), and validating the use of the X-ray data for joint refinement (Tandrup et al, 2022).…”

“…The heterologous expression of LsAA9_A was recently optimized in Escherichia coli, which opens the opportunity to perdeuterate the protein (Herna ´ndez-Rolla ´n et al, 2021). LsAA9_A produced in E. coli was crystallized and high-quality X-ray structures were obtained, including a complex with cellotriose (Banerjee et al, 2022;Tandrup et al, 2022). Future neutron structural investigation of LsAA9_A in complex with carbohydrate ligands will be greatly enhanced by the use of perdeuterated protein.…”

“…Later, LsAA9_A was demonstrated to be active towards soluble oligosaccharides and the first structures of an LPMO enzyme in complex with short soluble carbohydrates were reported (Frandsen et al, 2016;Simmons et al, 2017). LsAA9_A has now been used extensively as a model LPMO, including high-resolution crystallographic studies to determine the protonation state of key histidine residues (Banerjee et al, 2022) and detailed photoreduction studies (Tandrup et al, 2022). Furthermore, the dependence of LsAA9_A on H 2 O 2 has been established and a novel twist on the LPMO mechanism has been suggested, at least for this specific enzyme (Brander et al, 2021).…”

Structural perturbations of substrate binding and oxidation state changes in a lytic polysaccharide monooxygenase

“…A new paper on the analysis of structural differences of the same AA9 LPMOs described in this paper [ 24 ] appeared during a review of this manuscript. Amongst its main conclusions, the authors of this study used global estimates of the error to assess the significance of an ostensible shortening of the Cu…Tyr164 distance upon binding of substrate.…”