Abstract
Laccases are blue multicopper oxidases that can oxidize various organic and inorganic compounds by reducing O2 to water. Despite the decades’ research, the roles of protein glycosylation in laccases are still unclear. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated lcc4) from a white‐rot fungus Lentinus sp. in which two N‐glycosylated sites at Asn75 and Asn458 were identified. We observed the activity of native laccase was decreased significantly after α‐mannosidase digestion, suggesting the importance of glycosylation in laccase activity. To address the structural and functional role of glycosylation, we cloned laccase genes and created a number of mutant enzymes with the Asn→Asp mutation on predicted glycosylation residues, Asn75, Asn236, Asn399, and Asn458. The recombinant mutant enzymes expressed in Pichia pastoris all showed much lower activities compared to wild‐type counterpart. On the other hand, hyperglycosylation observed in the yeast expressed recombinant laccases may interfere the protein folding giving a decrease in the enzyme catalytic activity. This study provides some possible structural and functional roles of glycosylation in laccase enzymes. NSC‐97–3114‐P‐001–001, Taiwan
Published Version
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