Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave many of nature's most recalcitrant polysaccharides by acting on the C1-and/or C4-carbon of the glycosidic bond. Here, the results of an extensive mutagenesis study on three LPMO representatives, Phanerochaete chrysosporium LPMO9D (C1-oxidizer), Neurospora crassa LPMO9C (C4), and Hypocrea jecorina LPMO9A (C1/C4), are reported. Using a previously published indicator diagram, the authors demonstrate that several structural determinants of LPMOs play an important role in their oxidative regioselectivity. N-glycan removal and alterations of the aromatic residues on the substrate-binding surface are shown to alter C1/C4-oxidation ratios. Removing the carbohydrate binding module (CBM) is found not to alter the regioselectivity of HjLPMO9A, although the effect of mutational changes is shown to increase in a CBM-free context. The accessibility to the solvent-exposed axial position of the coppersite reveales not to be a major regioselectivity indicator, at least not in PcLPMO9D. Interestingly, a HjLPMO9A variant lacking two surface exposed aromatic residues combines decreased binding capacity with a 22% increase in synergetic efficiency. Similarly to recent LPMO10 findings, our results suggest a complex matrix of surface-interactions that enables LPMO9s not only to bind their substrate, but also to accurately direct their oxidative force.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity

“…Regioselectivity defines whether LPMOs oxidize glucosyl residues in cellulose at the C1 or C4 position, or both [19]. Studies to link sequences and structural features to the regioselectivity of LPMO have been attempted [19][20][21][22][23][24][25][26][27][28][29][30]. So far, although similarities have occurred, such phylogenetic analysis could not unambiguously predict the three LPMO regioselectivities.…”

Current understanding of substrate specificity and regioselectivity of LPMOs

“…Thus, the accessibility of surface-exposed axial position of Cu, or the ability to bind a ligand in the axial position, could be a determinant of C4-oxidizing activity. However, recent studies suggested that, mutations affecting accessibility of this axial position did not change the regioselectivities of PcLPMO9D and MaLPMO10B (Danneels et al 2019;Forsberg et al 2018).…”

“…Therefore, it is speculated that LPMOs may bind to the substrates in different directions, resulting in different regioselectivity (Li et al 2012). Recently, Danneels et al studied the oxidative regioselectivity of LPMO9s in detail (Danneels et al 2019). One part of the research was the mutation of aromatic amino acids on the substrate binding surfaces of PcLPMO9D, ScLPMO9C and HjLPMO9A.…”

“…The authors speculated that the non-oxidized products are the oligosaccharides derived from C1-oxidation near the reducing end of cellulose, which may be due to the grafted CBM affecting the localization of the enzyme on the substrate . But the impact of CBMs on the regioselectivity of LPMOs is also controversial, e.g., removing the CBM domains did not significantly change the regioselectivity of MaLPMO10B, NcLPMO9C and HjLPMO9A (Danneels et al 2019;Forsberg et al 2018;Laurent et al 2019).…”

“…Mutation studies showed that removing this N-glycan can alter the C1/C4-oxidation ratios of HjLP-MO9A. The authors suggested that this is because N-glycan affects the structural features of the substrate binding surface, which in turn affects the substrate binding and oxidative force accurate directions (Danneels et al 2019).…”