The Role of the Residue at Position 2 in the Catalytic Activity of AA9 Lytic Polysaccharide Monooxygenases

AA9 lytic polysaccharide monooxygenases (LPMOs) are copper-dependent metalloenzymes that play a major role in cellulose degradation and plant infection. Understanding the AA9 LPMO mechanism would facilitate the improvement of plant pathogen control and the industrial application of LPMOs. Herein, via point mutation, we investigated the role of glycine 2 residue in cellulose degradation by Thermoascus aurantiacus AA9 LPMOs (TaAA9). A computational simulation showed that increasing the steric properties of this residue by replacing glycine with threonine or tyrosine altered the H-bonding network of the copper center and copper coordination geometry, decreased the surface charge of the catalytic center, weakened the TaAA9-substrate interaction, and enhanced TaAA9-product binding. Compared with wild-type TaAA9, G2T-TaAA9 and G2Y-TaAA9 variants showed attenuated copper affinity, reduced oxidative product diversity and decreased substrate Avicel binding, as determined using ITC, MALDI-TOF/TOF MS and cellulose binding analyses, respectively. Consistently, the enzymatic activity and synergy with cellulase of the G2T-TaAA9 and G2Y-TaAA9 variants were lower than those of TaAA9. Hence, the investigated residue crucially affects the catalytic activity of AA9 LPMOs, and we propose that the electropositivity of copper may correlate with AA9 LPMO activity. Thus, the relationship among the amino acid at position 2, surface charge and catalytic activity may facilitate an understanding of the proteins in AA9 LPMOs.

Automated summary

In this study, the role of glycine 2 residue in cellulose degradation by TaAA9 LPMOs was investigated using point mutation. The results showed that replacing glycine with threonine or tyrosine changed the H-bonding network and copper coordination geometry, decreased surface charge, weakened TaAA9-substrate interaction, and enhanced TaAA9-product binding. The G2T-TaAA9 and G2Y-TaAA9 variants exhibited reduced copper affinity, oxidative product diversity, substrate binding, enzymatic activity, and synergy with cellulase compared to wild-type TaAA9. This study highlights the crucial role of the investigated residue in AA9 LPMO catalytic activity and suggests a correlation between the electropositivity of copper and AA9 LPMO activity.