Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes able to cleave glycosidic bonds in a range of recalcitrant polysaccharides including cellulose and chitin. This activity makes these enzymes attractive candidates for engineering, utilising their powerful monocopper active sites to evolve new activities. To-date, most research has focused on LPMOs from the auxiliary activity (AA)9 and AA10 families, while LPMOs from the AA11 family have not received the same attention since their classification almost a decade ago, despite their wide abundance in fungi. Previous studies of two AA11 LPMOs from Aspergillus fumigatus, AfAA11A and AfAA11B, show the former behaves like chitin-active LPMOs in the AA10 family, while the stands out for its exceptionally high oxidase activity, low reduction potential and the ability to degrade soluble chito-oligomers.
To elucidate the unique catalytic features of AfAA11B, its crystal structure was solved, and we performed site directed mutagenesis on a key active-site glutamate known to modulate copper reactivity. Structural analysis and molecular dynamics simulations revealed distinctive features that may explain the activity on soluble substrates. Mutations of the glutamate showed its crucial role in controlling the low reduction potential and high oxidase activity of AfAA11B. The impact of these mutations on copper reactivity was consistent with findings for an AA9 LPMO from Neurospora crassa (NcAA9C)1, which naturally has a glutamine in this position. This highlights how the nature and distance of the headgroup to the copper fine tune LPMO functionality and copper reactivity. However, electrochemical and UV-vis measurements revealed differences in productive catalysis and in a protective hole-hopping mechanism, suggesting the effect of this glutamate/glutamine residue is dependent on additional structural or dynamic factors.