Perturbed heme binding is responsible for the blistering phenotype associated with mutations in the Caenorhabditis elegans dual oxidase 1 (DUOX1) peroxidase domain.
Dual oxidase (DUOX) enzymes support a wide variety of essential reactions, from cellular signaling to thyroid hormone biosynthesis. In Caenorhabditis elegans, the DUOX system (CeDUOX1/2) plays a crucial role in innate immunity and in stabilizing the cuticle by forming tyrosine cross-links. The current model suggests that superoxide generated by CeDUOX1 at the C-terminal NADPH oxidase domain is rapidly converted to H(2)O(2). The H(2)O(2) is then utilized by the N-terminal peroxidase-like domain to cross-link tyrosines. We have now created a series of mutations in the isolated peroxidase domain, CeDUOX1(1-589). One set of mutations investigate the roles of a putative distal tyrosine (Tyr(105)) and Glu(238), a proposed covalent heme-binding residue. The results confirm that Glu(238) covalently binds to the heme group. A second set of mutations (G246D and D392N) responsible for a C. elegans blistering cuticle phenotype was also investigated. Surprisingly, although not among the catalytic residues, both mutations affected heme co-factor binding. The G246D mutant bound less total heme than the wild type, but a higher fraction of it was covalently bound. In contrast, the D392N mutant appears to fold normally but does not bind heme. Molecular dynamics simulations of a CeDUOX1(1-589) homology model implicate displacements of the proximal histidine residue as the likely cause. Both enzymes are structurally stable and through altered heme interactions exhibit partial or complete loss of tyrosine cross-linking activity, explaining the blistering phenotype. This result argues that the CeDUOX peroxidase domain is primarily responsible for tyrosine cross-linking.
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94518-2517, USA.
Jennifer L
JL
Meitzler
Relly
R
Brandman
Paul R
PR
Ortiz de Montellano
D019943:Amino Acid Substitution; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074623:Dual Oxidases; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D014443:Tyrosine; D006418:Heme; C544706:Bli-3 protein, C elegans; D010088:Oxidoreductases; D000074623:Dual Oxidases; D019255:NADPH Oxidases