A glycerol oxidase from Norway spruce (Picea abies L. Karst.) expands biochemical and structural attributes of the GMC oxidoreductase superfamily

O_LIInvestigations to uncover the versatility of carbohydrate active enzymes belonging to the auxiliary activity family 3 (AA3) have focused on microbial enzymes. Plant genomes also harbor AA3 encoding genes, and reverse genetics approaches have shown their importance in plant development. To date, however, detailed biochemical characterizations and structures of plant AA3 enzymes have not been reported. C_LIO_LIHere, we describe screening AA3 encoding genes from Norway spruce and the first in-depth biochemical and structural characterization of a plant AA3 oxidase, PaAOX1. PaAOX1 was crystallized, and the structure was solved by X-ray crystallography. C_LIO_LIPaAOX1 demonstrated highest oxidase activity against glycerol. It oxidized glycerol to glyceraldehyde, then further to glyceric acid, and showed preference for L-glyceraldehyde over D-glyceraldehyde. Despite the low sequence similarity with fungal alcohol oxidases, it had all the structural features of the typical AA3 enzyme. C_LIO_LIPaAOX1 has a novel activity among the characterized AA3s, and it showed a new structural arrangement of the region surrounding the catalytic center. Our findings contribute to a deeper understanding of the structural and functional aspects of enzymes in the GMC superfamily, and especially of alcohol oxidases, expanding the knowledge from fungi to plants. C_LI