Mutational study of a lytic polysaccharide monooxygenase from Myceliophthora thermophila (MtLPMO9F): Structural insights into substrate specificity and regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the biotechnological exploitation of lignocellulosic biomass, yet their efficient application depends on the in-depth understanding of their mechanism of action. Here, we describe the structural and mutational characterization of a C4-active LPMO from Myceliophthora thermophila, MtLPMO9F, that belongs to auxiliary activity family 9 (AA9). MtLPMO9F is active on cellulose, cello-oligosaccharides and xyloglucan. The crystal structure of MtLPMO9F catalytic domain, determined at 2.3 Å resolution, revealed a double conformation for loop L3 with a potential implication in the formation of aglycon subsites. Product analysis of reactions with cello-oligosaccharides showed a prevalent -4 to +2 binding mode. Subsequent biochemical characterization of 4 MtLPMO9F point mutants further provided insights in LPMO structure-function relationships regarding both substrate binding and the role of second-coordination sphere residues.