BACKGROUND: NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play 
varied biological and pathological roles via regulated generation of reactive 
oxygen species (ROS). Members of the Nox/Duox family have been identified in a 
wide variety of organisms, including mammals, nematodes, fruit fly, green 
plants, fungi, and slime molds; however, little is known about the molecular 
evolutionary history of these enzymes.
RESULTS: We assembled and analyzed the deduced amino acid sequences of 101 
Nox/Duox orthologs from 25 species, including vertebrates, urochordates, 
echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In 
contrast to ROS defense enzymes, such as superoxide dismutase and catalase that 
are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared 
later in evolution. Molecular taxonomy revealed seven distinct subfamilies of 
Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies 
diverged early and are the most widely distributed in biology. Subunit-regulated 
Noxes represent a second major subdivision, and appeared first in fungi and 
amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in 
gravity perception, emerged the most recently, corresponding to full-time 
adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus 
possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 
first appeared somewhat later in urochordates. Comparison of evolutionary 
substitution rates demonstrates that Nox2, the regulatory subunits p47phox and 
p67phox, and Duox are more stringently conserved in vertebrates than other Noxes 
and Nox regulatory subunits. Amino acid sequence comparisons identified key 
catalytic or regulatory regions, as 68 residues were highly conserved among all 
Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in 
variants of X-linked chronic granulomatous disease. In addition to canonical 
motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were 
identified as important for Nox activity, as verified by mutational analysis. 
The presence of these non-canonical, but highly conserved regions suggests that 
all Nox/Duox may possess a common biological function remained in a long history 
of Nox/Duox evolution.
CONCLUSION: This report provides the first comprehensive analysis of the 
evolution and conserved functions of Nox and Duox family members, including 
identification of conserved amino acid residues. These results provide a guide 
for future structure-function studies and for understanding the evolution of 
biological functions of these enzymes.