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. Despite rapid expansion in LPMO research, the mechanism of these enzymes remains largely unknown, with few mutagenesis studies performed on residues in the catalytic centre. A previous study identified a residue in SmAA10A (an AA10 family LPMO from Serratia marcescens), as playing a pivotal role in the peroxygenase reaction through positioning the oxygen co-substrate and the hydroxyl radical intermediate. This residue is universally conserved in all LPMOs, as either a negatively charged glutamate (typical for AA10 LPMOs) or a neutral glutamine (typical for AA9 LPMOs).
We investigated the role of this residue in NcAA9C, an AA9 family LPMO from Neurospora crassa. Using site directed mutagenesis, the conserved glutamine residue in NcAA9C was mutated to three different amino acids (glutamate, aspartate or asparagine). Our results show that the nature and distance of the headgroup to the copper fine tune LPMO functionality and copper reactivity1. The presence of a glutamate or aspartate close to the copper lowered the redox potential and decreased the ratio between reduction and reoxidation rates by up to 500-fold. The glutamate mutant also exhibited a lower redox potential and higher oxidase activity. All mutants showed increased enzyme inactivation, likely due to changes in confinement of radical intermediates and displayed changes in a protective hole-hopping pathway. Taken together, these results provide insights into how second sphere residues fine-tune the functionality of the LPMO copper site.