REVIEW ARTICLE Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species Hideki Sumimoto Medical Institute of Bioregulation, Kyushu University, Fukuoka CREST, Japan Science and Technology Agency, Tokyo, Japan Keywords Duox; Nox; Noxa1; Noxo1; p22phox; p40phox; p47phox; p67phox; Rac; Rboh Correspondence H Sumimoto, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Fax: +81 92 642 6807 Tel: +81 92 642 6806 E-mail: hsumi@bioreg.kyushu-u.ac.jp (Received January 2008, revised April 2008, accepted 30 April 2008) doi:10.1111/j.1742-4658.2008.06488.x NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc1 complex During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit For example, one to four Ca2+-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin–NADP+ reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata Members of the Nox1–4 subfamily in animals form a stable heterodimer with the membrane protein p22phox, which functions as a docking site for the SH3 domain-containing regulatory proteins p47phox, p67phox, and p40phox; the small GTPase Rac binds to p67phox (or its homologous protein), which serves as a switch for Nox activation Similarly, Rac activates the fungal NoxA via binding to the p67phox-like protein Nox regulator (NoxR) In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation Abbreviations AIR, autoinhibitory region; Duox, dual oxidase; FNR, ferredoxin–NADP+ reductase; Fre, ferric reductase; FRO, ferric-chelate reductase; Noxa1, Nox activator 1; Noxo1, Nox organizer 1; NoxR, Nox regulator; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PMA, 4b-phorbol 12-myristate 13-acetate; PPII, polyproline II; PRR, proline-rich region; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PX domain, phagocyte oxidase domain; Rboh, respiratory burst oxidase homolog; ROS, reactive oxygen species; TPR, tetratricopeptide repeat FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3249 Structure, regulation and evolution of Nox H Sumimoto Introduction Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious byproducts of aerobic metabolism On the other hand, there exist enzymes dedicated to ROS production The first example of such enzymes is an NADPH oxidase expressed in mammalian professional phagocytes [1–10] During engulfment of invading microbes, the phagocyte NADPH oxidase becomes activated to reduce molecular oxygen to superoxide anion (O2)), a precursor of microbicidal ROS, in conjunction with oxidation of NADPH As the rapid increase in oxygen consumption during phagocytosis is known as the respiratory burst, this enzyme is also called respiratory burst oxidase The significance of the phagocyte oxidase in host defense is exemplified by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease, whose phagocytes genetically lack the superoxide-producing activity [11,12] The catalytic core of the phagocyte NADPH oxidase (phox) is gp91phox, a membrane-integrated glycoprotein with an apparent molecular mass of about 91 kDa gp91phox contains two hemes in the N-terminal transmembrane region, and NADPH-binding and FADbinding domains in the C-terminal cytoplasmic region (Fig 1A), forming a complete apparatus that transports electrons from NADPH via FAD and two hemes to molecular oxygen In the mid-1990s, homologs of the flavocytochrome gp91phox were discovered in land plants; these have been designated respiratory burst oxidase homolog (Rboh) [13–15] Subsequent searches in genome databases led to the identification of novel homologs of gp91phox in animals, which are presently known as Nox (NADPH oxidase) or Duox (dual oxidase) [1–10] The human genome contains seven genes encoding gp91phox homologs: Nox1–Nox5, where gp91phox is renamed Nox2, and the distantly related oxidases Duox1 and Duox2 It is currently known that a wide variety of eukaryotes express superoxide-producing NADPH oxidases that harbor a gp91phox-like electron-transferring system; the enzymes constitute the Nox family Recent studies on Nox-family enzymes have increasingly clarified the importance of deliberate ROS production in various biological events, including signal transduction, development, and hormone biosynthesis, in addition to well-established roles in host defense [1–10] It is likely that individual Nox enzymes have developed regulatory systems, according to their respective special functions, during the course of eukaryote evolution by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit Regulation by these proteins includes multiple protein– protein and protein–lipid interactions In this review, I describe post-translational regulation of Nox-family enzymes on the basis of three-dimensional structures and evolutionary conservation Structure of Nox-family enzymes Fig (A) A model for the structure of gp91phox Cylinders represent six transmembrane a-helices (B) Bis-heme ligation in gp91phox Heme-coordnating His residues are numbered according to their localization in gp91phox (C) Intramembrane bis-heme motifs in various cytochromes The numbers of intervening amino acids that separate a pair of His residues in a transmembrane segment are indicated 3250 The phagocyte NADPH oxidase gp91phox ⁄ Nox2 (570 amino acid residues) exists in not only the plasma membrane but also the membrane of the specific granule in neutrophils: the latter contains a higher amount of Nox2 [1–10] Although the phagosomal membrane is considered to primarily derive from the plasma membrane, the specific granule is fused to the phagosome during phagocytosis, and thus gp91phox ⁄ Nox2 is further enriched in the phagosomal membrane For killing engulfed microbes, superoxide (and microbicidal ROS derived from superoxide) must be produced from molecular oxygen within the phagosome The intraphagosomal production requires electrons to be transported from the cytoplasmic NADPH across the membrane into the interior of the phagosome Such FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto transmembrane electron transport very often involves di-heme membrane proteins gp91phox ⁄ Nox2 can be divided into two parts of similar size (Fig 1A) The C-terminal half is a cytoplasmic domain homologous to ferredoxin–NADP+ reductase (FNR), bearing the NADPH-binding and FAD-binding sites [16,17], whereas the N-terminal moiety comprises six predicted a-helical transmembrane segments (Fig 1A) The bipartite structure is common not only to all the Nox-family enzymes but also to the family of fungal ferric reductases (Fre) [18] Fre enzymes, which are expressed in the plasma membrane, reduce Fe3+ and Cu2+ for iron and copper uptake, but fail to use molecular oxygen as a substrate [19,20] Among the conserved transmembrane segments of gp91phox ⁄ Nox2, the third and fifth helices each contain two invariant His residues, which are considered to provide the axial and distal ligands for binding to the irons of two nonidentical hemes, thereby placing one heme towards the cytoplasmic face and the other towards the outer face (Fig 1B) As the hemes are oriented perpendicular to the surface of the membrane, electrons are transferred from the cytosolic NADPH, through FAD, and across the membrane via the hemes to molecular oxygen, leading to superoxide production Thus, transmembrane electron transport in gp91phox ⁄ Nox2 is considered to occur in the N-terminal bis-heme-containing region [18,21] This model was initially proposed on the basis of a similar motif consisting of two pairs of spaced His residues that was predicted to be linked to heme coordination in a class of organelle and bacterial b-type cytochromes, such as the bis-heme cytochrome b of the mitochondrial cytochrome bc1 complex (complex III) and cytochrome b6 of the chloroplast cytochrome b6f complex [22–24] (Fig 1C) The model for two bis-histidyl heme ligation was subsequently verified by determination of crystal structures of protein complexes containing these cytochromes [25–28] In cytochrome b of the cytochrome bc1 complex, containing eight a-helical transmembrane segments, the two b-type hemes are bound within a four helix bundle formed by the first four segments: His residues ligated to both hemes are located in the second and fourth helices [25,26,29], and a pair of the His residues in each helix are separated by 13 intervening amino acids (Fig 1C) A similar coordination occurs in cytochrome b6 of the cytochrome b6f complex in cyanobacteria and chloroplasts [27,28]: cytochrome b6 comprises four a-helical transmembrane segments [30,31], and the two hemes are bis-His-coordinated by imidazole side chains separated by 13 and 14 residues in the second and fourth helices, respectively (Fig 1C) In the fungal Fre-family enzymes, both His Structure, regulation and evolution of Nox pairs are separated by 13 amino acids, as in cytochrome b of the cytochrome bc1 complex (Fig 1C) Although gp91phox ⁄ Nox2, as well as other Nox-family enzymes, contains a pair of His residues in the third transmembrane a-helix with 13 intervening amino acids (His101 and His115 in human gp91phox ⁄ Nox2), the other pair in the fifth helix (His209 and His222 in human gp91phox ⁄ Nox2) are separated by 12 amino acids (Fig 1) It should be noted that the imidazoles separated by 12–14 amino acids are likely to face the same side of the helix Substitution of any of these four His residues results in disrupted insertion of hemes into gp91phox ⁄ Nox2 [32], supporting the view that the two bis-histidyl heme ligation also occurs in gp91phox ⁄ Nox2 Reduction of molecular oxygen to superoxide in gp91phox ⁄ Nox2 requires both heme groups to be in the low-spin (hexacoordinate) state, which implies that electron transfer to oxygen occurs via the outer heme in a pocket near the heme edge, rather than through direct coordination of oxygen to the heme iron [33,34] This outer sphere (or peripheral) mechanism is consistent with the ‘two bis-histidyl heme ligation’ structure, and explains well why gp91phox ⁄ Nox2-catalyzed superoxide production is not inhibited by cyanide or carbon monoxide Taken together, electron transfer from NADPH to molecular oxygen occurs in a module designated the Nox superdomain The Nox superdomain comprises two moieties, the N-terminal bis-heme cytochrome b, composed of six a-helical transmembrane segments, and the C-terminal FNR, which contains FAD-binding and NADPH-binding domains Thus, gp91phox ⁄ Nox2 and its relatives (the Nox family) are flavocytochromes [16,17,35] It is known that members of the Duox subfamily in animals, in contrast to oxidases of the other subfamilies, release H2O2 without forming detectable amounts of superoxide [36] However, they are also expected to produce superoxide as an initial product, as Duox has the superoxide-producing Nox superdomain that comprises the bis-heme-containing transmembrane region and the FNR-like moiety Indeed, it has been reported that Duox in an immature form is capable of producing superoxide [37] In mature Duox, superoxide produced by the Nox superdomain may be rapidly converted to H2O2 via intramolecular dismutation, possibly by a peroxidase-like ectodomain; this module is located on the outer surface of the membrane, where superoxide is expected to be released In addition to the Nox and Fre families, both the bis-heme transmembrane segment and FNR-related moiety are present in ferric-chelate reductase (FRO) of land plants [38] To acquire iron from soils of low iron availability, land plants such as Arabidopsis thaliana FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3251 Structure, regulation and evolution of Nox H Sumimoto reduce Fe3+ to Fe2+ by FRO in the plasma membrane of root epithelial cells Four His residues in FRO that lie on two predicted, similarly orientated, transmembrane a-helices are in equivalent locations to the His residues in Nox enzymes that coordinate the two hemes: the 13 and 12 amino acids separating heme-liganding His residues exist in the helices (Fig 1C) The FRO-family enzymes contain eight or 10 transmembrane segments, which is different from the situation in members of the Nox and Fre families; the precise membrane topology of FRO enzymes remains controversial [39,40] Origin of Nox-family enzymes There is no evidence for the presence of Nox, Fre or FRO in prokaryotes: a superfamily of flavocytochromes that transport electrons across membranes On the other hand, members of this superfamily are present in a variety of eukaryotes The shared bis-heme binding motif raises the possibility of an evolutionary and functional relationship between the eukaryotic cell surface membrane proteins Nox, Fre and FRO and the prokaryotic (or organelle) b-type cytochromes On the other hand, the C-terminal moiety of the flavocytochrome superfamily is homologous to FNR, a prokaryotic (organelle) protein that is made up of two structural domains, each containing about 150 amino acids: the C-terminal region includes most of the residues involved in NADPH binding, whereas the large cleft between the two domains accommodates the FAD group [41,42] It is tempting to postulate that a gene encoding a protein containing two di-heme transmembrane helices was fused to an FNR gene in eukaryote evolution, leading to a common ancestor of the Nox and Fre families (Fig 2) In this context, it seems inter- esting that FNR directly interacts with the cytochrome b6f complex of plant chloroplasts, albeit in a noncovalent manner, and participates in electron transfer [43]; cytochrome b6 in the complex has two b-type hemes across the membrane, as expected for Nox A similar fusion of the FNR gene with a gene encoding an electron-transporting protein is also considered to have occurred during evolution: eukaryotic diflavin reductases such as NADPH–cytochrome P450 reductase (CPR or P450R), methionine synthase reductase and novel reductase probably arose from the fusion of the ancestral genes for FNR and flavodoxin, a prokaryotic FMN-containing protein that transfers electrons in a variety of photosynthetic and nonphotosynthetic reactions in prokaryotes [43–48] (Fig 2) In turn, a diflavin reductase is likely to be the precursor of further fusion products such as nitric oxide reductase, which consists of a C-terminal CPR-like domain and an N-terminal, heme-containing oxygenase domain [48,49] Distribution of Nox-family enzymes in eukaryotes In contrast to the absence of Nox in prokaryotes, genes encoding Nox-family enzymes are found in a wide variety of eukaryotes Eukaryotes can be divided into several major supergroups, including the Opisthokonta, the Amoebozoa, the Plantae, the Excavata, the Rhizaria, and the Chromalveolata (the Heterokonta plus the Alveolata): animals and fungi belong to the Opisthokonta; the social amoeba Dictyostelium discoideum is a member of the Amoebozoa; land plants and red algae belong to the Plantae; and diatoms and oomycetes are members of the Heterokonta, a group that belongs to the Chromalveolata (Fig 3) [50–52] Fig A putative common ancestor of the Nox family CPR, NADPH–cytochrome P450 reductase 3252 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto Structure, regulation and evolution of Nox Fig Distribution of Nox-family enzymes in eukaryotes and animals Upper panel: eukaryotes can be divided into six major supergroups, including the Opisthokonta, the Amoebozoa, the Plantae, the Excavata, the Rhizaria, and the Chromalveolata All the supergroups except the Rhizaria are known to contain Nox genes Lower panel: M b., Mo brevicollis; N v., Ne vectensis; L g., Lot gigantea; C sp I, Capitella sp I; C e., Ca elegans; D m., Dr melanogaster; S p., S purpuratus; B f., B floridae; C i., Ci intestinalis; D r., Da rerio; X t., X tropicalis; G g., Ga gallus; H s., Homo sapiens The relationships between these supergroups, however, remain to be determined, and thus the root of eukaryotes is presently uncertain [53,54] Recent expansion of information available in genome databases has revealed that Nox-family enzymes are present in all the eukaryotic supergroups except the Rhizaria (Fig 3) This suggests that a common ancestor of Nox genes emerged at an early stage in the evolution of eukaryotes; it diverged well in some lineages (e.g in the Opisthokonta and Plantae), whereas it was often lost in some other lineages The loss of Nox genes appears to have occurred at multiple stages in eukaryote evolution For example, in the supergroup Amoebozoa, the social amoeba Di discoideum contains three Nox genes [55], whereas they are absent in Entamoeba histolytica [56] In the supergroup Excavata, the heterolobosa Naegleria gruberi has at least two Nox genes, as found by the present search using the database of the DOE’s Joint Genome Institute (http:// genome.jgi/psf.org/euk_home.html) (Fig 3); on the other hand, no Nox gene has been found in the kinetoplastid Leishmania major or Trypanosoma brunei, or the diplomonad Giardia lamblia [56] In the Chromalveolata, Nox genes are present in genomes of the oomycete Phytophthora sojae [56,57] and the diatom Thalassiosira pseudonana [58], although they have not been found in the genomes of Plasmodium falciparum and Theileria parva, both of which belong to the same supergroup [56] Even in the Opisthokonta, Nox genes have been lost independently in several fungal lineages; for example, a Nox gene is absent in budding and fission yeasts [56,57,59] Regulation of Nox-family enzymes by Ca2+ Superoxide production by Nox is regulated by various mechanisms Several subfamilies of Nox enzymes appear to be directly regulated by Ca2+ It is well established that mammalian thyroid oxidase and sea urchin NADPH oxidase, both of which belong to the Duox subfamily, are reversibly activated by Ca2+ In addition to the Nox superdomain comprising the bisheme-containing transmembrane region and the FNRhomologous moiety, Duox-subfamily oxidases feature an N-terminal peroxidase-like ectodomain that is separated from two EF-hands by an additional transmembrane segment (Fig 4) Biosynthesis of thyroid hormones in humans requires Duox2, which is highly expressed in the thyroid gland: mutations in Duox2 are associated with a loss of thyroid hormone synthesis and can lead to permanent and severe congenital hypothyroidism [60] With the H2O2 produced by Doux2, thyroid peroxidase catalyzes conjugation of iodide ions to Tyr residues on thyroglobulin in the thyroid follicles, an essential step for the synthesis of the active hormone [61] On the other hand, during fertilization of sea urchins, a rapid increase in H2O2 generation occurs, which is catalyzed by the sea urchin Duox homolog Udx1, leading to formation of the fertiliza- FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3253 Structure, regulation and evolution of Nox H Sumimoto Fig Models for structures of various subtypes of Nox-family enzymes Cylinders represent six transmembrane a-helices EF, Ca2+-binding EF-hand motif tion envelope as the physical block to polyspermy [62] It is also known that Duox1 of Caenorhabditis elegans in the phylum Nematoda is involved in cross-linking of Tyr residues of extracellular matrix proteins, thereby facilitating cuticle formation [63], and that Duox plays a critical role in innate immunity in the gut of the fruit fly Drosophila melanogaster in the phylum Arthropoda [64] Regulation of Duox by Ca2+ probably occurs via its paired EF-hand motif It has been reported that limited proteolysis with a-chymotrypsin renders thyroid NADPH oxidase fully and irreversibly active independently of Ca2+ [65] This implies that the Ca2+-binding EF-hands of Duox serve as an autoinhibitory domain, whereas those of Nox5 function as an activation domain [66] The inhibition of Duox by the EF-hands might be released reversibly by physiological Ca2+-induced conformational change and irreversibly by proteolytic removal of the autoinhibitory domain [37] A recent study has shown that ectopically expressed Duox produces ROS without cell stimulants, and the production is enhanced two-fold by the addition of ionomycin [67], suggesting that elevation of cytoplasmic concentrations of Ca2+ is dispensable Besides direct regulation by Ca2+, protein kinase C (PKC) may modulate Duox in a Ca2+-independent manner, as ROS production in thyrocytes is triggered by 4b-phorbol 12-myristate 13-acetate (PMA), an agent that activates PKC without elevating the cytoplasmic concentration of Ca2+ [68] A PKC-dependent 3254 pathway may also function in H2O2 generation at fertilization in the sea urchin [62] In addition to the animal Duox subfamily, oxidases of other two subfamilies have been shown to be directly regulated by elevations in cytoplasmic Ca2+ concentrations: the Nox5 subfamily in animals [66,69,70], and the Rboh subfamily in land plants [71,72] (Fig 4) The regulation by Ca2+ appears to be consistent with the presence of the Ca2+-binding EF-hand motif in the cytoplasmic region N-terminal to the Nox superdomain (Fig 4) Human Nox5 is abundantly expressed in T and B cells of spleen and lymph nodes, and also in the sperm precursors of testis [69] Although the role for mammalian Nox5 remains unknown, Drosophila Nox5 has been reported to mediate smooth muscle contraction [70] Oxidases of this subfamily build on the basic structure of the Nox prototype, adding an N-terminal extension that contains four EF-hands: three canonical motifs and one noncanonical motif [63] (Fig 4) Biochemical analysis has shown that activation of Nox5 is directly regulated by Ca2+: superoxide production by Nox5containing membrane fractions is dependent on the presence of Ca2+ [66]; and cells ectopically expressing Nox5 produce superoxide in response to the Ca2+ ionophore ionomycin [69] The Ca2+-binding domain of Nox5, in contrast to that of Duox, may function as an activator module: the binding of Ca2+ causes a conformational change, which leads to intramolecular interaction of the N-terminal Ca2+-binding domain with the FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto C-terminal Nox superdomain, culminating in Nox5 activation [66] On the other hand, the EC50 for calcium of about lm, determined in a cell-free activation system for Nox5 [66], is relatively high and unlikely to be achieved in most cells treated with physiological stimulants Two mechanisms for the elevation of the Ca2+ sensitivity have recently been proposed [73,74] First, PKC phosphorylates Ser ⁄ Thr residues in the FAD-binding domain of Nox5, which increases the Ca2+ sensitivity of the Nox5 activity regulated by the N-terminal Ca2+-binding domain [73] Second, a consensus calmodulin-binding site is present in the NADPH-binding domain of Nox5; calmodulin interacts with the site at a lower concentration of Ca2+, thereby elevating the Ca2+ sensitivity for Nox5 activation [74] The Rboh subfamily of NADPH oxidases is responsible for ROS formation associated with plant defense responses, and also plays a crucial role in plant development [13,14,75] Ten and nine members are present in A thaliana [14] and the rice Oryza sativa [76], respectively The Rboh-subfamily enzymes carry an N-terminal extension with two EF-hand motifs (Fig 4) It has been reported that only about a twofold to three-fold increase in superoxide production occurs in the membrane fraction of tobacco and tomato upon addition of Ca2+; in addition, the effect requires high concentrations of Ca2+ (approximately millimolar) [71] This observation suggests that the direct effect of Ca2+ may not contribute to Rboh activation to a large extent A recent study has shown that elicitor-responsive phosphorylation of the N-terminal region of Rboh is involved in superoxide production [77,78], which is mediated by Ca2+-dependent protein kinases [77] Activation of Rboh is also regulated by plant homologs of the Rho-family small GTPase Rac (also known as Rop for Rho-like protein) [79–81]; Rac in the GTP-bound form functions by directly binding to the N-terminal region of Rboh, and this is probably inhibited by Ca2+ [76] Thus, regulation of Rboh is more complicated than previously expected, as described in detail in a later section As in plant Rboh, two copies of EF-hands are present in the N-terminal cytoplasmic region of the NoxCsubfamily members in the Amoebozoa and Nox enzymes in oomycetes of the eukaryotic supergroup Chromalveolata [57], whereas enzymes in the fungal NoxC subfamily contain a single EF-hand in the N-terminal cytoplasmic region [56,57,59] These subfamilies of Nox enzymes may be regulated by Ca2+; however, no experimental evidence for Ca2+-mediated regulation has been obtained It is presently unknown whether the EF-hand-containing Nox subfamilies originated from a common Structure, regulation and evolution of Nox ancestor gene It seems rather likely that EF-hand motifs have been obtained independently several times during evolution The genomes of Monosiga brevicollis in the choanoflagellates (a sister group of animals) and Nematostella vectensis in the cnidarians (a basal group of animals) contain solely Nox2-like enzymes, and not EF-hand-containing oxidases such as Nox5 and Duox, although these two families are found in a variety of species of protostomes and deuterostomes (Fig 3) Thus, Nox5 and Duox may have evolved from Nox2like prototype oxidases Similarly, in fungi, the NoxC subfamily containing an EF-hand is found solely in more evolved groups, including the Sordariomycetes and Dothideomycetes, whereas the Nox2-like EF-hand-free subfamilies NoxA and NoxB are present also in relatively basal groups such as the Chytridiomyceta and Basidiomycota [57] These features suggest that the NoxC subfamily emerged at a later stage of fungal evolution Thus, the classification of the Nox family in eukaryotes into the two major groups, depending on the presence or absence of the EF-hand motif, does not seem to reflect molecular evolution Regulation of Nox-family enzymes by protein–protein interactions The genome of Na gruberi (http://genome.jgi/psf.org/ euk_home.html), a member of the eukaryotic superfamily Excavata, contains two genes encoding Noxfamily enzymes of 627 and 630 amino acids, both of which have an SH3 domain and thus are tentatively designated as NoxSH3 (Fig 5) The SH3 domain is inserted into a loop region in the NADPH-binding domain of the C-terminal FNR-homologous region (Fig 5) It is tempting to postulate that the NoxSH3 enzymes in Naegleria are regulated by a protein harboring a proline-rich region (PRR); SH3 domains are generally known as modules that recognize a PRR to mediate protein–protein interactions [82,83] Identification of a NoxSH3-binding protein will shed light on our understanding of Nox regulation Nox1–Nox4 in animals form a heterodimer with the nonglycosylated integral membrane protein p22phox, which contains two (or possibly four) putative transmembrane segments (Fig 4) The complex of gp91phox ⁄ Nox2 with p22phox in phagocytes is known as flavocytochrome b558 In the C-terminal cytoplasmic region of p22phox, there exists a PRR, which serves as an anchoring site, thereby juxtaposing the catalytic center gp91phox and the SH3 domain-containing regulatory proteins p47phox, p67phox, and p40phox; on the other hand, the small GTPase Rac functions in Nox activation by interacting with p67phox or its homolo- FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3255 Structure, regulation and evolution of Nox H Sumimoto Fig Structure of the SH3 domain-containing enzyme NoxSH3 in Na gruberi The two NoxSH3 enzymes in Na gruberi, tentatively named NoxSH3-1 and NoxSH3-2, contain 627 and 630 amino acids, respectively The amino acid sequences of NoxSH3-1 and NoxSH3-2 are shown: the heme-coordinated His residues in transmembrane segment (TM3) and TM5 are shown in red; residues of FAD-binding motifs are shown in green; residues of NADPH-binding motifs are shown in blue; and residues of the SH3 domain are shown in magenta A model for the structure of NoxSH3 is also shown The SH3 domain is inserted into the C-terminal NADPH-binding domain Cylinders represent six transmembrane a-helices Fig Models for the structure of Duox1 ⁄ complexed with DuoxA1 ⁄ Cylinders represent six and five transmembrane a-helices of Duox1 ⁄ and DuoxA1 ⁄ 2, respectively EF, Ca2+-binding EF-hand motif ization of each protein [1–10] Formation of the mutually stabilizing complex appears to require the correct folding of Nox, because heme incorporation into gp91phox ⁄ Nox2 is essential for heterodimer formation [84] It is known that human Duox2 associates with a specific maturating protein named DuoxA2, which contains putative five transmembrane helices [67,85] (Fig 6) The membrane protein DuoxA2 allows the transition from the endoplasmic reticulum to the Golgi apparatus, maturation and translocation to the plasma membrane of functional Duox2 [67,85] Interestingly, the DuoxA2 gene is arranged head-to-head and coexpressed with the Duox2 gene; the gene for DuoxA1, a paralog of DuoxA2, is similarly linked to the Duox1 gene [85] Biallelic inactivation of the DuoxA2 gene has recently been reported as a novel cause of congenital hypothyroidism [86], confirming its crucial contribution to function of Duox2, which is directly involved in thyroid hormone synthesis [60] gous proteins Detailed mechanisms for regulation by these proteins will be described below Complex formation of p22phox with Nox also contributes to the stabil3256 The Nox subfamilies in animals The Nox enzymes in animals can be divided into three subfamilies: one containing Nox1–Nox4 (the Nox1–4 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto subgroup), which form a heterodimer with p22phox [87– 93]; the Nox5 subfamily; and the Duox subfamily (Fig 4) Although the Nox5 and Duox subfamilies are not found in the sea anemone Ne vectensis of the Cnidaria (the basal group of animals) (http://genome.jgi/psf.org/euk_home.html) [94] (Fig 3), they were probably present at the protostome–dueterostome divergence This is because the Nox5 and Duox subfamilies exist in both extant protostomes and deuterostomes, with the exception that Nox5 has been lost in the lineage of the phylum Nematoda and in that of the order Rodentia in mammals: Nox5 is absent in the nematode Ca elegans and the rodents mouse and rat (Fig 3) On the other hand, Nox2, a member of the Nox1–4 subgroup, was present before the divergence of the Choanoflagellata and Metazoa (equivalent to animals): Nox2 is found not only in the Cnidaria but also in the choanoflagellate Mo brevicollis (http://genome.jgi/ psf.org/euk_home.html) [94] (Fig 3) During the evolution of protostomes, one of the two major groups of animals, Nox2 has been lost in the lineage of the clade Ecdysozoa, including the phyla Arthropoda and Nematoda: Nox2 is absent in the fruitfly Drosophila and the nematode Ca elegans On the other hand, Nox2 is present in the Lophotrochozoa, another major protostomian clade, including the phyla Mollusca and Annelida: this oxidase exists in the limpet Lottia gigantea (Mollusca) and the leech Capitella species (Annelida) (Fig 3) Thus, it is likely that Nox2 is the closest to the ancestral Nox in animals In contrast, Nox1, Nox3 and Nox4 are not found in protostomes or the Echinodermata, a phylum that belongs to the deuterostomes (a sister group of protostomes) These three Nox enzymes appear to have diverged from Nox2 at distinct stages of evolution of the phylum Chordata in deuterostomes The Chordata is divided into three subphyla: the Vertebrata, Urochordata (also known as the Tunicata), and the Cephalochordata Although tunicates were long considered to be the earliest offshoot of the chordate lineage, and cephalochordates (such as amphioxus) as the closest group to vertebrates, recent analyses have reversed their positions: amphioxus is now viewed as the ‘basal chordate’ [95], and tunicates as the sister group, or closest relatives, of the vertebrates [96] (Fig 3) Thus, our understanding of the evolution of a certain protein in the Chordata requires information on the corresponding protein in the subphylum Cephalochordata The present search for the database of the amphioxus (lancelet) Branchiostroma floridae (http://genome.jgi/psf.org/euk_home.html) revealed that, in addition to Nox2, Nox5, and Duox, Nox4 exists in the Cephalochordata It is known that Structure, regulation and evolution of Nox Nox4 is present in Ciona intestinalis (the Urochordata) [97] but not in the sea urchin Strongylocentrotus purpuratus of the phylum Echinodermata, another major group of the Chordata [90] Therefore, Nox4 appears to have branched from a root close to Nox2 with the emergence of the Chordata Nox1 is found from fishes to mammals, but not in the Urochordata or Cephalochordata, suggesting that this oxidase probably arose with the emergence of the Vertebrata Nox3 has emerged most recently, probably from a common ancestor of birds and mammals, because it is found solely in mammals and birds, but not in fishes or amphibians [98] (Fig 3) The membrane protein p22phox has been demonstrated to be complexed with mammalian Nox1–Nox4 Consistent with this, p22phox is absent in the Ecdysozoa, where the Nox1–4 subfamily is absent, but widely distributed in species that have Nox2, including the choanoflagellate Mo brevicollis and the cnidarian Ne vectensis The known exceptions are the leech Capitella species (the Annelida) and the sea urchin S purpuratus (the Echinodermata) Nox2 of these species or groups might be stable without p22phox, leading to loss of the p22phox gene It may also be possible that the p22phox gene escaped cloning or correct sequencing for unknown reasons, which is known to often occur in various genome projects Regulation of the Nox1–4 subfamily in animals The phagocyte oxidase gp91phox ⁄ Nox2 in mammals is dormant in resting cells, but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal ROS The oxidase activity is spatially and temporally restricted to the phagosome, as inappropriate or excessive production of ROS results in damage to surrounding cells and severe inflammation Activation of gp91phox ⁄ Nox2 requires stimulus-induced membrane translocation of p47phox, p67phox, p40phox, and Rac, i.e formation of the active oxidase complex at the membrane (Fig 7A) The essential role of these regulatory proteins is evident from the following two lines of evidence First, the phagocyte NADPH oxidase activity can be reconstituted in a cell-free system with gp91phox, p22phox, p47phox, p67phox, and Rac, using an anionic amphiphile, e.g arachidonic acid, as an in vitro stimulant Second, defects in any of the four genes encoding gp91phox, p22phox, p47phox and p67phox cause the primary immunodeficiency chronic granulomatous disease [11,12] In the cytoplasm of resting cells, p47phox, p67phox and p40phox form a ternary complex, whereas Rac is FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3257 Structure, regulation and evolution of Nox H Sumimoto Fig Activation of gp91phox ⁄ Nox2 and Nox1 (A) Interactions required for activation of gp91phox ⁄ Nox2 Interactions in a resting state are indicated by blue arrows, stimulus-induced interactions by arrows in magenta, and constitutive interactions by green arrows (B) Interactions required for activation of Nox1 Interactions in a resting state are indicated by blue arrows; and stimulus-induced interactions by arrows in magenta T1, T2, T3 and T4, tetratricopeptide repeats 1, 2, and 4, respectively; AD, activation domain complexed with Rho GDP dissociation inhibitor Upon cell stimulation, the three phox proteins are en bloc recruited to the membrane; on the other hand, Rac translocates independently but without Rho GDP dissociation inhibitor, which remains in the cytoplasm Although activation of gp91phox ⁄ Nox2 complexed with p22phox in cells absolutely requires p47phox, p67phox and Rac as cytosolic regulators, p47phox is dispensable for cell-free activation in the presence of excess amounts of p67phox and Rac [99,100] It is thus considered that p47phox functions as an organizer, whereas p67phox serves as an activator that directly participates in gp91phox ⁄ Nox2 activation Nox1 is abundantly expressed in colon epithelial cells and also in vascular smooth muscle cells [101,102], and seems to be involved in angiotensin IImediated hypertension [103–105] Nox1, as well as Nox2, forms a complex with p22phox [88,106] Although Nox1 is also inactive without an organizer or an activator, it generates superoxide in the presence of the p47phox paralog Noxo1 (Nox organizer 1) and the p67phox paralog Noxa1 (Nox activator 1) [106–109] (Fig 7B) Rac is directly involved in Nox1 activation as well [110–112] In the inner ear of mice, Nox3 plays a crucial role in formation of otoconia, tiny mineralized structures that are required for perception of balance and gravity [113] Although Nox3 also forms a functional heterodimer with p22phox, this oxidase is capable of producing superoxide in the absence of an organizer or an activa3258 tor [88] The superoxide-producing activity can be strongly enhanced by p47phox, Noxo1, and p67phox [89,110,114,115] In the presence of p67phox or Noxa1, Nox3 activity is upregulated by Rac [110,116] Although it is well known that Nox4 is highly expressed in epithelial cells of the adult and fetal kidney [117,118] and vascular endothelial cells [91,119], its function remains to be elucidated Nox4 is complexed with p22phox as well [80,82,84], and constitutively generates superoxide in an NADPH-dependent manner [120] The mechanism of Nox4 regulation is largely unknown at present: Nox4-mediated superoxide generation appears to be independent of p47phox, Noxo1, p67phox, or Noxa1 [89,117,118], whereas the role of Rac remains controversial [79,121] The organizer p47phox is required for Nox2 activation The oxidase organizer p47phox is a 390 amino acid protein that contains, from the N-terminus, a phagocyte oxidase (PX) domain, tandem SH3 domains, and a PRR (Fig 7) The two SH3 domains cooperatively interact with the PRR in the C-terminal cytoplasmic region of p22phox, an interaction that is essential for both membrane translocation of p47phox and oxidase activation [122–124] The tandem SH3 domains sandwich a short PRR of p22phox (amino acid residues 151–160), containing a polyproline II (PPII) helix [125–127] (Fig 8) Pro152, Pro156 and Arg158 in the human p22phox PRR are indispensable for the interaction with p47phox: Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 directly contacts with the C-terminal one [127] (Fig 8) On the other hand, Pro151, Pro155, Pro157 and Pro160 are also involved in binding to p47phox but to a lesser extent [127] The gene encoding p22phox exists in a wide variety of animals and also in the Choanoflagellata, a sister group of the Metazoa (Animalia) [128,129]; the p22phox gene is absent in the Ecdysozoa, which is consistent with the absence of the Nox1–4 subfamily in this clade (Fig 9) The p22phox region comprising the N-terminal cytoplasmic region and the two transmembrane segments is functionally important [130] and well conserved in the Metazoa (animals) and Choanoflagellata, whereas the C-terminal cytoplasmic region is highly variable except for the PRR (Fig 8) The three residues indispensable for binding to p47phox (Pro152, Pro156 and Arg158 in human p22phox) are invariant in all known animal p22phox proteins (Fig 8), although identifiable p47phox exists solely in the phylum Chordata, as described later (Fig 9) FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto Fig 14 The complex of GTP-bound Rac with the N-terminal TPR domain of p67phox (A) The b-hairpin insertion in the TPR domain of p67phox is colored blue Arg102 and Asp67, each involved in binding to Rac, are drawn as blue and green sticks, respectively GTP bound to Rac is shown in magenta The figure was drawn using PYMOL software (http://www.pymol.org) and the Protein Data Bank coordinates 1E96 (B) The amino acid sequences of the b-hairpin insertion and activation domain (AD) in human p67phox are shown; secondary structure elements are indicated below the sequence of the b-hairpin insertion The consensus sequences of the two regions among p67phox proteins derived from various species are also shown side chain atoms of no less than four residues from Rac [168] Asp67 in the loop that connects TPR1 and TPR2 (position 31 of TPR2) also makes direct contacts with Rac (Fig 14) and is completely conserved among p67phox and its related proteins such as vertebrate Noxa1 and fungal NoxR In addition, the two residues involved in binding to Rac, Ser37 in the loop between the a-helices of TPR1 and TPR2 (position of TPR2) and Asn204 in the b-hairpin (position 34 of TPR3), are highly conserved among p67phox and its related proteins (Fig 14) In addition, substitution of Glu for the invariant Arg in human Noxa1 (Arg103) [106] and in fungal NoxR (Arg101) [170] results in a loss of binding to Rac Thus, these proteins probably recognize Rac in the same way as p67phox Structure, regulation and evolution of Nox Between the Rac-binding TPR domain and the N-terminal SH3 domain, human p67phox contains a PRR region, PPPRPKTP (amino acids 227–234), which is well conserved among mammalian, avian and amphibian p67phox proteins, but not among fish p67phox [147] or mammalian Noxa1 [106] Cell-free experiments using a series of C-terminally truncated mutants of p67phox showed that the PRR is dispensable for oxidase activation in vitro [171] Intriguingly, these experiments also revealed that the region of amino acids 200–212, C-terminal to the Rac-binding TPR domain, plays an essential role in cell-free activation of the NADPH oxidase: a truncated p67phox of amino acids 1–212 is fully active, whereas a mutant protein of amino acids 1–199 is completely inactive [171] These results were subsequently confirmed, and the short region (amino acids 201–210) was named the ‘activation domain’ [172] Among amino acids in the activation domain, Ala substitution for Val204 results in an almost complete loss of gp91phox ⁄ Nox2 activation, not only under cell-free conditions [172], but also in intact cells [173] The corresponding mutation in the p67phox homolog Noxa1, the V205A substitution, attenuates Nox1 activation [109] Thus, both the Rac-binding TPR domain and the activation domain are probably minimal prerequisites for p67phox function Careful alignment of regions C-terminal to the TPR domain of various Nox activators reveals the consensus sequence of the activation domain: (V ⁄ L) xxLxxKD(Y ⁄ F)LGKAxVV(A ⁄ S)(S ⁄ A) (amino acids 190–208) (Fig 14) Leu193 (the third residue in the consensus), Asp197, Leu199, Gly200 and Val205 (the amino acid number corresponds to that of human p67phox) are completely conserved in all Nox activator proteins, including p67phox, Noxa1, and NoxR The ninth residue, Tyr (equivalent to Tyr198 of human p67phox), is common to p67phox proteins, but is replaced by Phe in Noxa1 Binding of Rac is believed to induce a conformational change in p67phox, which may allow the activation domain to act on gp91phox ⁄ Nox2 [174–177] However, it is unknown how the activation domain functions at the atomic level The extended activation domain as proposed here (amino acids 190–208) appears to be flexible or disordered, judging from the crystal structure of an isolated p67phox TPR domain (amino acids 1–203) complexed with Rac [168], and that of a TPR domain alone (amino acids 1–213) [169]; in the latter case, the N-terminus of the activation domain is a part of an a-helix (amino acids 187–193) [169] The activation domain in the Rac-bound p67phox might adopt a defined structure upon interaction with the gp91phox–p22phox complex It FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3263 Structure, regulation and evolution of Nox H Sumimoto has been also proposed that GTP-bound Rac by itself induces electron transport from NADPH by directly interacting with gp91phox ⁄ Nox2 [178] In this case, the insert helix of Rac (amino acids 123–135), a region specific for the Rho-family GTPases, is suggested to play a crucial role [174]; however, the importance of this region remains controversial [179] Interaction of p40phox with p67phox p67phox contains a PB1 domain [180] between the two SH3 domains; via PB1 domain-mediated heterodimerization, p67phox stably interacts with p40phox in phagocytes (Fig 15) Human p40phox of 339 amino acids, comprising PX, SH3 and PB1 domains, is not essential for oxidase activation This protein, however, enhances recruitment of p67phox and p47phox to the membrane [181], especially to the phagosomal membrane, and is Fig 15 Upper panel: domain architecture of p40phox and p67phox Middle panel: ribbon diagram of structures of the p40phox–p67phox PB1 complex The notations of these secondary structural elements are indicated, and the OPCA motif p40phox is highlighted Lower panel: basic residues (p67phox) that directly interact with conserved acidic residues of the OPCA motif (p40phox) Secondary structure elements of the OPCA motif are indicated below the sequence The figures were drawn using PYMOL software (http:// www.pymol.org) and the Protein Data Bank coordinates 1OEY 3264 therefore considered to play a crucial role in oxidase assembly at the phagosome [181–183] The PB1 domains, comprising about 80 amino acid residues, adopt a ubiquitin-like b-grasp fold, containing two a-helices and a mixed five-stranded b-sheet [184– 189] This module is classified into groups harboring an acidic OPCA motif (also known as a PC motif) (type I), the invariant Lys residue on the first b-strand (type II), or both (type I ⁄ II) [180] Heterodimeric assembly occurs between type I and II PB1 domains [180] The dimerization involves specific electrostatic interactions involving a conserved acidic Dx(D ⁄ E)GD region of the OPCA motif from a type I PB1 domain and an invariant Lys residue from a type II PB1 domain The type II PB1 domain in human p67phox contains Lys355 on the b1-strand, which directly participates in binding to p40phox, membrane targeting of p67phox, and oxidase activation [181] The crystal structures of the p67phox– PB1 and p40phox–PB1 complexes [187] demonstrate that Lys355 of p67phox forms direct bonds with Asp289, Glu291 and Asp293 in the DxEGD sequence of the OPCA motif, which is presented on the PB1 domain (type I) of human p40phox (Fig 15); this is also consistent with the findings that a mutant p40phox with the D289A substitution neither enhances p67phox translocation nor supports superoxide production at the phagosome [181,182] Importantly, it is evident from the crystal structure that even a basic residue such as Arg is incapable of replacing Lys355 Besides the first electrostatic interaction, a contact of Lys382 (of the p67phox PB1 domain) with Asp302 (of the p40phox OPCA motif) also contributes to p67phox binding to p40phox, but to a much lesser extent (Fig 15) [190] Furthermore, Thr361 and Trp425 of p67phox and Arg296 of p40phox also make intramolecular contacts, enhancing the interaction between p67phox and p40phox [187] In agreement with the presence of the PB1 (type II)-containing p67phox in the amphioxus B floridae of the Cephalochordata (Fig 16), its genome contains the gene for p40phox of 346 amino acids, which comprises PX, SH3 and PB1 (type I) domains (http://genome.jgi/psf.org/euk_home html) Residues that play crucial roles in PB1 heterodimerization, especially the two Lys residues in p67phox and the five OPCA residues (Asp289, Glu291, Asp293, Arg296 and Asp302 in human p40phox), are all conserved in the amphioxus proteins, indicating that the p67phox– p40phox interaction is maintained in the Chordata In contrast, p40phox is absent in other phyla, including the sea urchin S purpuratus of the Echinodermata, another phylum that belongs to the deuterostomes Thus, p40phox appears to have emerged in the Chordata Translocation of p40phox to the phagosome is probably mediated via the N-terminal PX domain This PX FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto Structure, regulation and evolution of Nox euk_home.html) to fishes and mammals [147], suggesting that p40phox plays the conserved role in recruiting the p47phox–p67phox–p40phox complex correctly to the phagosome In contrast to the PX and PB1 domains, the role of the SH3 domain in p40phox remains obscure, although it is capable of binding to p47phox very weakly [158,197] Origin of p67phox-like proteins functioning via interaction with Rac Fig 16 Ribbon diagram of structure of the p40phox PX domain complexed with PtdIns(3)P Residues interacting with PtdIns(3)P via hydrogen bonds (Arg58, Arg60, Lys92, and Arg105) are drawn as blue sticks, and Tyr59, which makes hydrophobic contacts with the inositol ring, is drawn as green sticks PtdIns(3)P is shown in magenta The figure was drawn using PYMOL software (http:// www.pymol.org) and the Protein Data Bank coordinates 1H6H domain is capable of specifically and strongly binding to phosphatidylinositol 3-phosphate [PtdIns(3)P] [142,143,191], a lipid that is produced by type III PI3K and is highly enriched in the phagosomal membrane [192,193] The PtdIns(3)P-binding activity, however, appears to be normally suppressed via an intramolecular interaction with the PB1 domain [194,195] The high affinity and specificity for PtdIns(3)P requires the following three residues in human p40phox [196]: Arg58 undergoes extensive interactions with the 3-phosphate moiety of the bound PtdIns(3)P, NH2 and NE in the side chain of this residue forming hydrogen bonds with oxygens of the 3-phosphate; NH1 and NH2 of Arg105 form hydrogen bonds with the 4-OH and 5-OH of the inositol moiety; and the aromatic ring of Tyr59 stacks against one face of the inositol ring (the face with the axial 2-OH) (Fig 16) In addition, Lys92 and Arg60 also contribute to binding to PtdIns(3)P but to a lesser extent (Fig 16): these residues make hydrogen bonds with the nonbridging oxygens of the 1-phosphoryl group, although the contribution of Arg60 is much less than that of Lys92 [196] The p40phox residues involved in binding to PtdIns(3)P, except Arg60, are completely conserved from amphioxus (http://genome.jgi/psf.org/ Two major subgroups in animals are protostomes and deuterostomes, the latter of which includes the phyla Chordata and Echinodermata (Fig 9) As described above, human p67phox comprises the Rac-binding TPR domain, the activation domain, the N-terminal SH3 domain, the PB1 domain (type II), and the C-terminal SH3 domain (Fig 17) Such a canonical p67phox is found not only in the Vertebrata (from fishes to mammals) but also in the Cephalochordata, a basal group of the Chordata, as shown in the present search for the database of the amphioxus B floridae (http:// genome.jgi/psf.org/euk_home.html) Thus, the domain architecture of p67phox has arisen with the emergence of the Chordata On the other hand, p67phox seems to be absent in the ascidian Ci intestinalis of the Urochordata, a sister group of the Vertebrata, suggesting loss of the p67phox gene in this lineage Instead, an ascidian protein that contains solely a Rac-binding TPR domain but not the other domains of p67phox (Fig 17) has been considered as a functional p67phox homolog [97]; however, its identity as a protein that supports oxidase activation awaits functional analysis Noxa1, a p67phox paralog for Nox1 activation, seems to have emerged in the Vertebrata (Fig 17), which is consistent with the appearance of Nox1 and the Nox1 organizer Noxo1 in this subphylum of the Chordata The coincidence of the emergence of Nox1 and its regulatory proteins may be related to two rounds of whole genome duplication that occurred in early vertebrate evolution [198,199] Noxa1 of the zebrafish Da rerio almost retains the domain architecture of p67phox but harbors only a remnant of the N-terminal (the first) SH3 domain (Fig 17) Its activation domain is of the Noxa1 type: the Tyr residue conserved in p67phox is replaced by Phe as in other Noxa1 proteins (Fig 14) As zebrafish Noxa1 appears to contain an intact PB1 domain, it might function by interacting with p40phox [94] On the other hand, Nox1 of the frog Xenopus tropicalis has an almost complete N-terminal SH3 domain and a nonfunctional PB1 domain (Fig 17): the PB1-like region lacks crucial residues FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3265 Structure, regulation and evolution of Nox H Sumimoto Fig 17 Domain structures of p67phox and its related proteins The total number of amino acid residues of each protein is indicated on the right: human, H sapiens; avian, Ga gallus; amphibian, X tropicalis; zebrafish, Da rerio; ascidian, Ci intestinalis; amphioxus, B floridae; molluscan, Lot gigantea; fungal, Ep festucae; amoebal, Di scoideum T1, tetratricopeptide repeat 1; T2, tetratricopeptide repeat 2; T3, tetratricopeptide repeat 3; T4, tetratricopeptide repeat 4; AD, activation domain such as the irreplaceable Lys (corresponding to Lys355 in human p67phox), indicating that amphibian Noxa1 is not expected to bind to p40phox Human Noxa1 does not contain an N-terminal SH3 domain but has a PB1 domain-like region that lacks the invariant Lys: indeed, human Noxa1 fails to interact with p40phox [106] In addition, it was reported that the irreplaceable Lys is substituted with Arg in Noxa1 of the chicken Gallus gallus [94], and the present analysis has revealed that no identifiable PB1 domain is found in avian (chicken) Noxa1 (Fig 17); these findings indicate that p40phox does not participate in activating Nox1 in the chicken Similar to the tail-to-tail interaction between p67phox and p47phox, Noxa1 is capable of binding to Noxo1 via its C-terminal SH3 domain (Fig 7B), which plays a crucial role in formation of the active Nox1 complex [110–112,200] A protein homologous to p67phox exists in the sea urchin S purpurtatus, a member of the Echinodermata (Genebank accession number XP_781983] (Fig 17) This protein contains two sets of the p67phox core cassette, comprising the Rac-binding TPR domain and activation domain (Fig 17): the sequences of the Racbinding insert region and the activation domain are almost completely fitted to the consensus ones (Fig 14) The echinodermal p67phox harbors an SH3 domain at the C-terminus, like vertebrate homologs, but lacks a p40phox-binding PB1 domain, which seems 3266 to be consistent with the absence of p40phox in this species In animals, a p67phox-like protein is present also in the limpet Lot gigantea of the Mollusca, a member of the protostomes [94] This protein contains a Rac-binding TPR domain and an activation domain, but not other known modular domains or motifs (Fig 17) Although the two species S purpurtatus and Lot gigantea not seem to have a recognizable p47phox homolog, the echinodermal and molluscan p67phox-homologous proteins might participate in oxidase activation without cooperating with p47phox It is known that p47phox is dispensable for gp91phox ⁄ Nox2 activation in the presence of large excess amounts of p67phox and Rac in a cell-free system [99,100], and that, even in a whole cell system, p67phox functions in activation of Nox3 (but not gp91phox ⁄ Nox2) in a manner independent of p47phox: p67phox is capable of activating Nox3 in cells lacking p47phox [88,115] and also in cells expressing a mutant p22phox that is defective in binding to p47phox [88] The fungi, a major group of the eukaryotic supergroup Opisthokonta, have three Nox-family members: NoxA and NoxB are close to animal Nox2; and NoxC has an N-terminal cytoplasmic extension containing a single EF-hand motif [56,57,59] The fungal oxidases are involved in a variety of biological events such as development of sexual fruiting bodies and ascospore germination [59,201], and symbiotic interactions such FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto as that between the clavicipitaceous fungal endophyte Epichloeă festucae and its grass host Lolium perenne [202] It has recently been shown that a p67phox-like oxidase activator, named NoxR (Nox regulator), exists in fungi possessing the NoxA gene [57,170], whereas p47phox or p22phox appears to be absent in fungi or so divergent that it has escaped identification NoxR contains a typical core cassette of p67phox, comprising a Rac-binding TPR domain and an activation domain, suggestive of its role in Nox activation (Fig 17) Indeed, NoxR in Ep festucae is involved in ROS production by NoxA; a noxR deletion mutant confers a defect in fungus–grass mutualistic symbiosis, which is similar to that observed for a noxA mutant; and the phenotype in the noxR deletion mutant is complemented by expression of the wild-type NoxR, but not by a mutant NoxR carrying the R101E substitution, indicative of the crucial role of Rac [170] In addition, NoxR harbors a PB1 domain of type II [170,180]; a fungal protein carrying a type I PB1 domain might be involved in oxidase activation, although a p40phox-like protein is not found in fungi Thus, a Rac-interacting Nox activator appears to have evolved in the supergroup Opisthokonta and possibly also in the supergroup Amoebozoa, as described below The social amoeba Di discoideum, which belongs to the Amoebozoa, is known to have three Nox enzymes [55] Two of them lack an EF-hand and are close to fungal NoxA and NoxB [55,57], which may imply the presence of a Nox activator The Dictyostelium genome contains a gene encoding a protein similar to p67phox, but not one homologous to p47phox, p40phox, or p22phox [55] The p67phox-like protein contains four TPR motifs, a type II PB1 domain, and a WW domain (Fig 17), and is expected to bind to Rac, because amino acids that participate in the interaction with this GTPase (Fig 14) are well conserved in the TPR domain, including Arg101, which is equivalent to Arg102 of human p67phox However, the Dictyostelium protein does not harbor an identifiable activation domain Future studies should be addressed to determine whether this protein indeed serves as an oxidase activator Another mechanism whereby Rac activates Nox: direct binding of Rac to Rboh As described above, the three p67phox-like proteins in the Opisthokonta, namely mammalian p67phox, mammalian Noxa1, and fungal NoxR, have been experimentally proved to collaborate with Rac in activating Structure, regulation and evolution of Nox Fig 18 Mechanisms whereby Rac activates animal Nox2 and plant Rboh Nox enzymes that lack an EF-hand motif; on the other hand, Rac does not seem to participate in regulation of the animal EF-hand-containing oxidases Nox5 and Duox [68,203] In contrast, in the eukaryotic supergroup Plantae, Rac (also known as Rop) plays a crucial role in activation of Rboh [79–81], which harbors two EF-hand motifs that directly bind to Ca2+ [204] Plants not appear to have a gene for p67phox or its homologous protein; although there exist proteins containing TPR motifs and a PB1 domain [94,180], they not retain conserved Rac-binding residues and are therefore not expected to function by interacting with Rac Instead, Rac directly binds to the N-terminal region of Rboh containing two EF-hand motifs [76] (Fig 18) A current model for Rboh activation is as follows [76,77]: the stimulus-elicited initial (weaker) elevation in cytoplasmic Ca2+ may activate Ca2+-dependent protein kinases, which phosphorylate the N-terminal region of Rboh to induce a conformational change; this change may expose the N-terminus for the interaction with Rac; the subsequent binding of Rac activates the superoxide-producing activity of Rboh; and, in a second (more prolonged) phase of cytoplasmic Ca2+ accumulation, which may be induced by ROS and ⁄ or the initial Ca2+ elevation, induced binding of Ca2+ to the EF-hands suppresses the Rac–Rboh interaction Thus, Ca2+ is considered to play a negative role in Rboh regulation via direct interaction with the EF-hands, which is in contrast to the positive roles of Ca2+ –EF-hand interaction in the activation of Nox5 and Duox in animals [65,66] Perspectives About a decade has passed since the discovery of the gp91phox ⁄ Nox2 homologs in a variety of eukaryotes, and increasing attention has been paid to the Nox family However, many unanswered questions still remain The mechanism of electron transfer in Nox FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3267 Structure, regulation and evolution of Nox H Sumimoto molecules is largely unknown at present In particular, there is no information at the atomic level about how electron transfer is induced by interaction of Nox with regulatory proteins such as a p67phox-related protein and Rac Studies of the crystal structures of full-length Nox proteins and their complexes will help immensely to extend our understanding of the molecular mechanisms that govern oxidase activation The Nox family exists in a wide variety of eukaryotes (Fig 3), but oxidases from only a limited number of species have been experimentally investigated As shown in Fig 1, members of the Nox family contain a pair of His residues in the third transmembrane a-helix with 13 intervening amino acids (His101 and His115 in human gp91phox ⁄ Nox2), with the other pair in the fifth helix (His209 and His222 in human gp91phox ⁄ Nox2) being separated by 12 amino acids However, proposed Nox members in the red algae Cyanidioschyzon merolae, Cyanidioschyzon crispus, and Porphyra yezoensis, which belong to the eukaryotic supergroup Plantae [58,98], contain 13 intervening amino acids between His residues in both the third and the fifth transmembrane segments, like the fungal Fre family (Fig 1) It thus seems interesting to test whether these oxidases (called NoxD) indeed reduce molecular oxygen to superoxide, or instead use Fe3+ as a substrate In addition, it was reported that flavocytochromes, carrying 14 and 13 intervening amino acids, are present in the diatom Phaeodactylum tricornutum (supergroup Chromalveolata), although typical Nox enzymes (with 13 and 12 intervening amino acids) exist in another diatom, Tha pseudonana [58] Biochemical studies of Nox enzymes derived from various eukaryotes are now required to establish their identities as real Nox enzymes or other distinct oxidoreductases and to clarify their regulatory mechanisms p22phox, the membrane partner of the Nox1–4 subfamily, is present in various animals and in the choanoflagellate Mo brevicolli, and retains a conserved PRR in the C-terminal cytoplasmic region (Figs and 9) On the other hand, p47phox or Noxo1, a protein that specifically binds via its bis-SH3 domain to the PRR of p22phox, is found solely in an animal group of the Chordata (Figs and 10) Although it was proposed that proteins with single PX domain and multiple SH3 domains might function as p47phox [94,97], experimental evidence supporting this hypothesis has not yet been provided Identification of a novel p22phox-binding protein will improve our understanding of regulation of the Nox1–4 subfamily enzymes In contrast to the finding that the presence of p47phox or its homolog is restricted to the Chordata, proteins homologous to p67phox exist in a variety of 3268 eukaryotes (Figs and 17) However, ta role in Nox activation has been shown for only three types of p67phox homolog: p67phox and Noxa1 in mammals, and NoxR in fungi Thus, it is worth testing whether proteins carrying a Rac-binding TPR domain but not an activation domain, like those found in the ascidian Ci intestinalis and the social amoeba Di discoideum (Fig 17), are indeed involved in Nox regulation It would be also interesting to know how a p67phox-like protein of the mollusc Lot gigantea functions; this protein contains both the Rac-binding TPR domain and the activation domain, but lacks other known modular interaction domains (Fig 17) As discussed in the present review, it remains unknown whether the EF-hand-containing Nox subfamilies are descended from a single ancestor oxidase that initially obtained EF-hand motifs It seems more likely that an EF-hand motif has been obtained independently several times during evolution Nox2, but not Nox5 or Duox, has been found in the Choanoflagellata (a sister group of animals) and Cnidaria (a basal group of animals), whereas these three oxidases are widely distributed in protostomes and deuterostomes (Fig 3), suggesting that Nox5 and Duox, containing four and two EF-hands, respectively, diverged after the emergence of a p22phox-binding Nox2-like oxidase in animal evolution Similarly, in fungi, the NoxC subfamily containing a single EF-hand is found solely in more evolved groups, such as the Sordariomycetes and Dothideomycetes, whereas the Nox2-like EF-hand-free subfamilies NoxA and NoxB are present also in relatively basal groups such as the Chytridiomyceta and Basidiomycota [57], suggesting that the NoxC subfamily has emerged at a later stage of fungal evolution Thus, the classification of the Nox family in eukaryotes into the two major groups, depending on the presence or absence of the EF-hand motif, does not seem to reflect molecular evolution Although many studies using phylogenetic trees of the Nox family have been performed, it should be noted that this approach is sometimes misleading, e.g due to ‘long branch attraction’, when distantly related genes derived from a wide variety of species are used [54,205–207] Further increases in genomic data for various eukaryotes will help to clarify the origins of Nox subfamilies The function of EF-hands is variable between Nox subfamilies, which may be in agreement with their sequence diversity [98] The EF-hands of the animal enzyme Nox5, in the Ca2+-bound state, appear to interact with the Nox superdomain, leading to superoxide production [66] In the resting state of Duox, another EF-hand-containing Nox in animals, the EF-hands may exert an inhibitory effect by normally FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS H Sumimoto associating with the Nox superdomain; for Duox activation, the association is probably disrupted by binding of Ca2+ to the EF-hands [65] In plants, the EF-hands of Rboh localize within the Rac-binding region at the N-terminus, and binding of Ca2+ to the EF-hands prevents Rac from binding to the N-terminal region, resulting in inactivation of this oxidase [76] Structural studies of EF-hands complexed with a Nox superdomain or Rac will help to extend our understanding of the molecular mechanisms that govern oxidase regulation Acknowledgements This work was supported in part by CREST of JST (Japan Science and Technology Agency) and by Grants-in-Aid for Scientific Research and Targeted Proteins Research Program (TPRP) from the Ministry of Education, 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supplementary material is available online: Fig S1 Amino acid sequences of p22phox, p47phox, p67phox and p40phox in the amphioxus B floridae are found in the database of the DOE’s Joint Genome Institute (JGI) http://genome.jgi/psf.org/euk_home.html This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3277 .. .Structure, regulation and evolution of Nox H Sumimoto Introduction Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious byproducts of aerobic metabolism... enzymes, and not EF-hand-containing oxidases such as Nox5 and Duox, although these two families are found in a variety of species of protostomes and deuterostomes (Fig 3) Thus, Nox5 and Duox... chain of this residue forming hydrogen bonds with oxygens of the 3-phosphate; NH1 and NH2 of Arg105 form hydrogen bonds with the 4-OH and 5-OH of the inositol moiety; and the aromatic ring of Tyr59