Oxidative cleavage of methyl-esterified pectic homogalacturonan by an AA17 lytic polysaccharide monooxygenase from Phytophthora infestans

Lytic polysaccharide monooxygenases (LPMOs) are mono‑copper-dependent enzymes that catalyze the oxidative breakdown of polysaccharides. The recently discovered AA17 family, exclusively found in oomycete genomes, plays a critical role in plant-pathogen interactions, with Phytophthora infestans AA17 (PiAA17) LPMOs shown to degrade homogalacturonan (HG). In plant cell walls, HG is methyl-esterified, but the influence of this modification on the mode-of-action of AA17 LPMOs is unknown. In this study, we established the phylogenetic distribution of the AA17 family, which diverged into six clades. Three AA17 enzymes from distinct clades were successfully heterologously produced. PiAA17C, the only one of three tested AA17 enzymes active on HG substrates, oxidatively cleaved HGs with degrees of methyl-esterification (DM) of 0, 20, and 60, but not highly methyl-esterified HG (DM92). Advanced liquid chromatographic and mass spectrometric analysis demonstrated that PiAA17C generated C4-oxidized and decarboxylated C4-oxidized oligogalacturonides. Among these products, those having internal methyl-esterified galacturonic acid (MeGalA) residues were more abundant than those with reducing end MeGalA residues. PiAA17C preferably cleaved HG between two non-methylated GalA residues, followed by cleavages involving one GalA and one MeGalA residue, with the lowest preference for cleavage between two MeGalA residues.