Biochemical characterization of the α-1,3-mannosidase AnGH92A from Aspergillus nidulans.

α-Mannan is a structurally diverse polysaccharide widely distributed in fungi, yet the eukaryotic enzymes responsible for its degradation remain poorly understood. Glycoside hydrolase family 92 (GH92) enzymes, well characterized in bacteria as Ca2⁺-dependent exo-α-mannosidases, have largely uncharacterized biochemical roles in fungi. Here, we characterized two GH92 enzymes from the filamentous fungus Aspergillus nidulans, designated AnGH92A and AnGH92B. Both recombinant enzymes hydrolyzed 4-nitrophenyl α-D-mannopyranoside (4NP-Man), but only AnGH92A displayed activity toward natural substrates. AnGH92A exhibited high specificity for α-1,3-linked mannooligosaccharides, weak activity toward α-1,4-linkages, and no detectable activity toward α-1,2- or α-1,6-linkages. It also cleaved α-1,3-linked side chains in branched oligosaccharides and released mannose from yeast α-mannan. By contrast, recombinant AnGH92B showed no detectable hydrolytic activity toward α-manno-oligosaccharides and yeast α-mannan. Structural modeling revealed pronounced differences in the substrate-binding pockets of AnGH92A and AnGH92B. Site-directed mutagenesis confirmed that Glu598 and Asp685 serve as the general acid and general base catalysts of AnGH92A, respectively, and that Tyr588 is essential for substrate recognition. Together, these findings indicate that AnGH92A functions as a eukaryotic GH92 α-1,3-mannosidase with weak activity toward α-1,4-linkages and provide the first experimental evidence linking sequence motifs and active-site architecture to catalytic function in fungal GH92 enzymes.