Polysaccharide Lyase family 3 subfamily 2 pectin is specific for highly methyl-esterified homogalacturonan characterized for two Aspergillus nidulans lyases

Fungal pectin-active lyases degrade homogalacturonan (HG) and are classified as pectate or pectin lyases based on their preference for non-methyl-esterified or methyl-esterified HG, respectively. Polysaccharide Lyase family 3 subfamily 2 (PL3_2) has been reported to comprise exclusively fungal pectate lyases, yet biochemical data remain scarce. We characterized two Aspergillus nidulans PL3_2 lyases, AnPel3_2H and AnPel3_2G, and one PL1_4 lyase (AnPel1_4B) for comparison. Typical for pectin lyase, AnPel1_4B exhibited calcium-independent activity on highly methyl-esterified HG. Interestingly, AnPel3_2H and AnPel3_2G also displayed activity towards highly methyl-esterified HG at both low (0–7.5 μM) and high (7500 μM) calcium levels. Both PL3_2 lyases cleaved bonds adjacent to methyl-esterified residues. At high calcium levels (750–7500 μM), both PL3_2 lyases showed activity towards non-methyl-esterified HG‑calcium complexes, unlike AnPel1_4B, but only limited degradation (17 %) was reached, yielding mainly unsaturated dimers. The latter observation is proposed to underly the earlier reported ‘pectate’ lyase activity in the PL3_2 family, and should be interpreted with caution. Our results demonstrate that the PL3_2 family members investigated are specific for highly methyl-esterified HG and induce an extensive depolymerization into oligosaccharides, providing compelling evidence that these PL3_2 members function primarily as pectin lyases rather than pectate lyases.