Characterization by Mutagenesis of Heme Binding Pocket Interactions in Caenorhabditis elegans Dual Oxidase 1 (DUOX1)

Abstract

The DUOX members of the NOX protein family, which are found throughout eukaryotic species, including Caenorhabditis elegans and sea urchins, have garnered much attention for their unique N‐terminal peroxidase‐like domain(s) and their ability to produce hydrogen peroxide. Two highly conserved DUOX isoforms are encoded in the C. elegans genome; CeDUOX1/BLI‐3 and CeDUOX2. RNAi experiments in C. elegans to knock out the expression of nematode DUOX1 have found that this protein participates in the biogenesis of the nematode cuticle through formation of di‐ and trityrosine linkages. Peroxidases, including mammalian peroxidases and ovoperoxidases, have been identified as enzymes capable of catalyzing such cross‐linking in the presence of hydrogen peroxide. However, the absence of key active site residues within the N‐terminal domain of DUOX(s) makes their heme binding and catalytic activity uncertain. To further characterize the function and mechanism of action of the DUOX peroxidase‐like domain, the N‐terminal region of C. elegans DUOX1 was expressed and purified from a baculovirus system. Evaluation of this protein demonstrated that it binds heme covalently and exhibits a modest peroxidase activity. Mutation studies of this protein conducted to investigate the role of residues interacting with the co‐factor in the heme binding pocket will be presented. This work was partially supported by NIH Grant GM32488.