Pectin fine structure modulates the catalytic performance and regioselectivity of AA17 lytic polysaccharide monooxygenases from Phytophthora infestans

Lytic polysaccharide monooxygenases (LPMOs) catalyze the peroxidative cleavage of complex cell wall polysaccharides, yet how polysaccharide fine structure modulates their catalytic performance remains poorly understood. Here, we quantitatively investigate the catalytic performance of the recently established Auxiliary Activity family 17 (AA17) LPMOs using real-time electrochemical monitoring of H 2 O 2 consumption. Two AA17 enzymes from the plant-pathogenic oomycete Phytophthora infestans , Pi AA17C and Pi AA17D, were analyzed on pectic homogalacturonan (HG) substrates differing in degrees (DM 0, 20, 60 and 92) and pattern of methyl-esterification. Both enzymes exhibited DM-dependent catalytic behavior on HG. Detailed characterization of Pi AA17C identified moderately methyl-esterified HG (DM20) as providing the highest catalytic efficiency and operational stability, reflecting an optimal balance between turnover rate and hydrogen peroxide sensitivity. In addition, HG with randomly distributed methyl esters, characteristic of pathogenic pectin methylesterase (PME) activity, supported better catalytic performance than blockwise-esterified HG. Beyond HG, both enzymes were found to oxidatively cleave β-(1 → 4)-galactan side chains of rhamnogalacturonan I, identifying galactan as a previously unrecognized substrate class. In-depth product analysis revealed C1-oxidation on galactan, in contrast to the C4-oxidation observed on HG, demonstrating a substrate-dependent switch in oxidative regioselectivity. Co-expression analysis showed that Pi AA17 genes are upregulated together with putative PMEs and galactan-active enzymes during plant infection, supporting their key roles in breaking down pectic polysaccharides.