Abstract
Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa, which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding.
Highlights
Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides
Substrate binding and regioselectivity of LPMO arise from interactions of aromatic and hydrophilic amino acids located on distinct loops flanking the active site, which position the copper center onto the substrate [12, 25, 26]
In a first attempt to increase thermal stability by enzyme engineering, additional disulfide bridges were introduced into Streptomyces coelicolor LPMO, which increased the Tm value of the enzyme from 51 to 63 °C [31]
Summary
We used a fluorescence-based unfolding assay to analyze the stability of NcLPMO9C. Initial attempts to measure thermal unfolding via LPMO’s intrinsic protein fluorescence showed only a minor intensity difference between the folded and the unfolded state, even at a high protein concentration of 10 M (Fig. 1A). Measurement of the oxygen-reducing activity of LPMO incubated with ascorbic acid after reverting the temperature ramp from 70 to 30 °C at a rate of 1 °C minϪ1 showed that the enzyme retained only ϳ33% of its initial activity (Fig. 1D). This is considerably lower than observed for the refolding of the oxidized enzyme (ϳ90%) and corroborates the previous finding that reducing conditions exert a destabilizing effect on the LPMO structure [22]. Tm,app values were measured in the presence of ANS, as outlined under “Experimental procedures.” Data are expressed as mean values Ϯ S.D. from three independent repeats
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