Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

A multicopper oxidase-1 (MCO1) ortholog has been identified in all of the insect genomes analyzed to date; therefore, we predict that MCO1 orthologs have an important, conserved function in insects. MCO1 is expressed in most tissues in all developmental stages. It is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. The physiological functions of multicopper oxidases are dictated by their substrate specificity. Most of the well-studied multicopper oxidases are laccases, which oxidize diphenols and aromatic diamines. A smaller number of carefully analyzed multicopper oxidases are ferroxidases, which oxidize ferrous iron, or ascorbate oxidases, which oxidize ascorbate. Our kinetic analysis of recombinant MCO1 from Drosophila melanogaster, Anopheles gambiae, Tribolium castaneum and Manduca sexta demonstrated that MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing diphenols or ferrous iron. These results suggest that MCO1 orthologs function as ascorbate oxidases. This outcome is somewhat surprising because, until now, ascorbate oxidases had been identified only in plants. An analysis of putative substrate binding residues identified a conserved histidine that is required for oxidation of ascorbate. RNAi-mediated knockdown experiments suggest that MCO1 activity is required for iron homeostasis, presumably because of complex interactions that occur between ascorbate metabolism and iron metabolism. Further studies are needed to understand the precise functions of MCO1 orthologs in insects.