Kawahara Tsukasa, Quinn Mark T, Lambeth J David
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
BMC Evol Biol. 2007 Jul 6;7:109. doi: 10.1186/1471-2148-7-109.
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.
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.
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.
NADPH氧化酶（Nox）和相关的双氧化酶（Duox）通过调节活性氧（ROS）的产生发挥多种生物学和病理学作用。Nox/Duox家族的成员已在多种生物中被鉴定出来，包括哺乳动物、线虫、果蝇、绿色植物、真菌和黏菌；然而，关于这些酶的分子进化历史知之甚少。
我们组装并分析了来自25个物种的101个Nox/Duox直系同源物的推导氨基酸序列，这些物种包括脊椎动物、尾索动物、棘皮动物、昆虫、线虫、真菌、黏菌变形虫、藻类和植物。与原核生物中存在的超氧化物歧化酶和过氧化氢酶等ROS防御酶不同，产生ROS的Nox/Duox直系同源物在进化过程中出现得较晚。分子分类学揭示了Noxes和Duoxes的七个不同亚家族。代表4个亚家族的钙调节直系同源物早期分化，在生物学中分布最广泛。亚基调节的Noxes代表第二个主要分支，最早出现在真菌和变形虫中。Nox5在啮齿动物中丢失，在内耳重力感知中起作用的Nox3最近才出现，这与脊椎动物完全适应陆地相对应。海胆紫球海胆拥有脊椎动物Nox1、2和3最早的Nox2共同直系同源物，而Nox4最早出现在尾索动物中。进化替代率的比较表明，Nox2、调节亚基p47phox和p67phox以及Duox在脊椎动物中比其他Noxes和Nox调节亚基更严格地保守。氨基酸序列比较确定了关键的催化或调节区域，因为68个残基在所有Nox/Duox直系同源物中高度保守，其中14个与X连锁慢性肉芽肿病变体中Nox2突变的残基相同。除了典型基序外，B环、TM6-FAD、VXGPFG基序和极端C末端区域被确定对Nox活性很重要，这通过突变分析得到了验证。这些非典型但高度保守区域的存在表明，所有Nox/Duox可能具有在Nox/Duox漫长进化历史中保留下来的共同生物学功能。
本报告首次对Nox和Duox家族成员的进化和保守功能进行了全面分析，包括保守氨基酸残基的鉴定。这些结果为未来的结构-功能研究以及理解这些酶的生物学功能进化提供了指导。
