Update of the NAD(P)H:quinone oxidoreductase (NQO) gene family

The NAD(P)H:quinone acceptor oxidoreductase (NQO) gene family belongs to the flavoprotein clan and, in the human genome, consists of two genes (NQO1 and NQO2). These two genes encode cytosolic flavoenzymes that catalyse the beneficial two-electron reduction of quinones to hydroquinones. This reaction prevents the unwanted one-electron reduction of quinones by other quinone reductases; one-electron reduction results in the formation of reactive oxygen species, generated by redox cycling of semiquinones in the presence of molecular oxygen. Both the mammalian NQO1 and NQO2 genes are upregulated as a part of the oxidative stress response and are inexplicably overexpressed in particular types of tumours. A non-synonymous mutation in the NQO1 gene, leading to absence of enzyme activity, has been associated with an increased risk of myeloid leukaemia and other types of blood dyscrasia in workers exposed to benzene. NQO2 has a melatonin-binding site, which may explain the anti-oxidant role of melatonin. An ancient NQO3 subfamily exists in eubacteria and the authors suggest that there should be additional divisions of the NQO family to include the NQO4 subfamily in fungi and NQO5 subfamily in archaebacteria. Interestingly, no NQO genes could be identified in the worm, fly, sea squirt or plants; because these taxa carry quinone reductases capable of one- and two-electron reductions, there has been either convergent evolution or redundancy to account for the appearance of these enzyme functions whenever they have been needed during evolution.


Introduction
Quinones comprise al argec lass of aromatic compounds, found widely in plants, in the general( benzoyl) formo f C 6 H 4 O 2 ,b ut also found in all organismsa sfl avonoids, electron-carrying coenzymes and metabolic end-products of oxidation. Commercially,quinones areused in making dyes, tanning hides and in photography. Quinones arep otentially dangerous, in that they can be involved in ao ne-electron reduction to formsemiquinones that will elicit oxidativestress in any cell; benzoquinones and naphthoquinones are among the most reactive in this category. Quinones were undoubtedly present in the environment and in unicellular organisms between 2a nd more than 3b illion yearsa go (at the time that partial pressureo fa mbiento xygen on thep lanetw as rising). Hence,d uring this time it is very likely there wasa strong selectiont od evelop an enzyme that would reduce quinones to hydroquinones by at wo-electron step,t hereby avoiding free radical formation, which is highly destructive to the living cell.

Background and history
The NAD(P)H:quinone acceptor oxidoreductase ( NQO) gene family is thereforeb elieved to be ancient -p robably between 2a nd more than 3b illion yearso ld. Cytosolic NQO flavoenzymes catalyse the beneficial two-electron reduction of quinones to hydroquinones. The two-electron reaction prevents the reduction of quinones by one-electronreductases, which would result in the formation of reactiveoxygenspecies (ROS) generatedb yr edoxc ycling of semiquinones in the presence of molecular oxygen. 1,2 In animals, in additiontothe likely role in detoxication of many dietaryquinones derived from plants, NQO enzymes have been shown to catalyse the reductive activation of chemotherapeutic quinones such as mitomycins and aziridinylbenzoquinones. 3,4 The hydroquinones derived from these agents appeartobechemically more reactive,either because of rapidautoxidation or rearrangement -which, in either case,w ill producer eactives pecies that can kill malignant cells. 5

Discovery of DT diaphorase
Ernster and Navazioa re credited with discovering (in the rat) the first member of the NQO family in the late 1950s. [7][8][9] This enzyme is nowo fficially named NQO1 (EC 1.6.99.2; previous names included DT diaphorase and aminoazo dye reductase,N QOR1, NMO1, NMOR1 and QR1). It was later concluded 9,10 that this enzyme is probably identicalt oa vitamin Kr eductase that had been isolated by Martius and colleagues. The mammalian NQO1 enzyme is expressed constitutively in most tissues and is strikingly upregulated during oxidativeo re lectrophilic stress; NQO1 has been shown to be am ember of the [ AHR ]g ene battery. 11 The crystal structure, 12 enzymic functions 13 and polymorphisms 14 in the NQO1 gene have all recently beens ummarised.
The human NQO1 gene on chromosome1 6q22.1 comprises six exons, spanning approximately18kilobases (kb), encodes 274 amino acids and contains, to date,9 3s ingle nucleotide polymorphisms (SNPs)within 10 kb 5 0 -ward of the first exon and 2kb3 0 -wardo ft he last exon (http://SNPper. chip.org/). Tw oa dditional variants, besides the consensus gene,w ere found in the Entrez Gene database ( Figure 1). Compared with the human consensus (reference,' wild-type') NQO1 * 1 allelec odingf or normal NQO1 enzyme and activity,t he NQO1 * 2 allele encodes an on-synonymous mutation (Pro187Ser) that has negligible NQO1 enzyme activity.T he NQO1 * 2 allelic frequency ranges between 0.22 (Caucasian) and 0.45 (Asian) in variouse thnic populations. Al arge epidemiological investigation of ab enzene-exposed population 15 has shown that NQO1* 2 homozygotes exhibit as much as as even-fold greaterr isk of bone marrowt oxicity,l eading to diseases such as aplastic anaemia and leukaemia. The NQO1* 3 allele (Arg139Try)represents an additional NQO1 polymorphism 16 that has been characterised; allelic frequencies have been reported as high as 0.05 in some ethnic groups, 17 and the NQO1* 3 allele has been linked to ah igher rate of exon 4d eletion -a na lternativelys pliced formo fN QO1 mRNA. 14 The other member 18,19 of this mammalian gene family is NQO2,w hich has recently been demonstrated to be identical to aprotein isolated from human kidney in 1962. NQO2 is 43 residues shorter than NQO1 at its C-terminus and shows 49 per cent sequence identity with NQO1. The human NQO2 gene on chromosome6 p25.2 comprises seven exons( first exon non-coding) spanning 19.8 kb,e ncodes 231 amino acids and contains, to date,2 54 SNPs within 10 kb 5 0 -wardo ft he first exon and 2kb3 0 -wardo ft he last exon (http://SNPper. chip.org/). Whereas earlier reports 20,21 had suggested an association between both NQO1 and NQO2 polymorphisms and Parkinson disease,amore recent study 22 showedn o association between alleles of either NQO1 or NQO2 and Parkinson'sd isease.
NQO1 efficiently utilises NAD(P)H as the source of reducing equivalents, but -becausethe 43-amino acid deletion in NQO2 contains aportion of the pyridine nucleotide binding site -NQO2 has been shown to prefer dihydronicotinamide ribosyl, and other similar derivatives, as acofactor. 23 Similar to NQO1, NQO2 can also bioactivate certain anti-tumour agents, such as the CB1954 nitrocompound. 13 Acrystal structure of human NQO2 has been published, 23 but not many biological functions of this enzyme have so farbeen well characterised. Curiously,NQO2 contains amelatonin-binding site, 24 which might be areasonable explanation for the anti-oxidant role ascribed to melatonin.
Both NQO1 and NQO2 activities areo bserveda th igh levels in many humans olid tumours, including lung, colon, liver and breast; this findingh as encouraged oncologists to explore the possibility of NQO1 and NQO2 as drug targets for the developmento fn ovel anti-tumour agents that can be activated by these enzymes. 13

Definition of 'Pfam' and 'clan'
Pfam is am eans of identifying similar domains tructures of all proteinst hat are summarisedi nU niProt (http://www.ebi. uniprot.org/). Both NQO1 and NQO2 were found to belong to the flavodoxin-2f amily( Pfam 02525) because they carry a domainh aving afl avodoxin-likef old. The flavodoxin-2 family is am ember of the flavoprotein clan. Based on the Pfam databased efinition: 'A clan contains twoo rm ore Pfam families that have arisen from a single evolutionaryo rigin. Evidence of their evolutionaryr elationship is usuallyd etermined by similart ertiarys tructureso r, when structures are not available,b yc ommons equence motifs.' 25 The categoryof'clans' is thus usually introduced in order to encompass twoo rm ore gene families that can be globally quite distant evolutionarily but have some segmentofthe gene products that showahigh degree of similarity.H ence in a BLASTsearch, asequencemay significantly hit more than one member of another family of the clan -aswell as all members within one family.
The flavodoxin-2 family includes bacterial and eukaryotic NAD(P)H:quinone reductases (as described above), as well as the bacterial acyl carrier protein (ACP), phosphodiesterase (EC:3.1.4.14).T he acylc arrier enzyme convertst he holo-ACP into apo-ACP by catalysing the hydrolysis of the phosphodiester linkage between Ser-36 of ACPa nd the 4 0 -phosphopantethein prosthetic group.A CP is as mall (8.8 kDa) protein that plays ac entral role in the biosynthesis of fatty acids in bacteria, plant chloroplasts and other organisms. ACPp hosphodiesterase is known to be encoded by the acpD gene in Escherichia coli and has been widely distributed in other bacteria, including Azospirillum brasilense, Bacillus halodurans, B. stearothermophilus, B. subtilis, Haemophilus influenzae, Lactococcus lactis, Mesorhizobiuml oti, Mycoplasma pneumoniae, Pseudomonas aeruginosa and Streptomyces coelicolor.
The flavodoxin_NdrI family contains ribonucleotide reductases, which areafamily of complexe nzymes that play an essential role in all organisms.T hese enzymes catalyse de novo synthesis of deoxyribonucleotides required for DNA replication and repair. 26 Finally,membersofthe FMN red family (flavin reductases) use flavins as substrates and are distinct from flavoenzymes, which have tightly bound flavins. The reduced flavincan serve to provide electrons for ferricion complexes and iron proteins. In E. coli,reactivation of ribonucleotide reductase is achieved by the formation of reduced flavins by flavinreductase.

Previous confusion about NQO genes and diaphorases
Much of the confusion that has arisen witht he nomenclature of NQO1, NQO2 and related proteins can be traced back to the widespread use of the term' diaphorase' in enzymatic studies more than 50 yearsago.Infact, this terminology is still being carried forward. 27 The term' diaphorase' has generally been used to describe a' coenzyme'( often afl avoprotein) that can transfer electrons from reduced pyridine nucleotides to electron acceptors. 9,28 In anyb iological system, therefore, there are many enzymes that can be characterised as 'diaphorases'.
The term' DT'-diaphorase waso riginally used by Ernster, to describe what is nowk nown as NQO1, because the enzyme worked with equal facility,e mploying either D PNH (NADH)o r T PNH (NADPH).E dwards et al. 29 discuss a series of 'diaphorases' defined in human tissues. Diaphorase-1 wasN ADH cytochrome b5 reductase-3 (Table 2). 30,31 Diaphorase-2 wasthe major enzyme in erythrocytes catalysing the oxidation of NADPH. Diaphorase-3 wast he principal source of oxidised coenzyme in sperm. Diaphorase-4 was characterised by Edwards et al., 29 and this enzyme was found to be identicalt ot he previously characterised DT-diaphorase. 32 Interestingly,E dwards and colleagues also showedt hat diaphorase-4 activity wasa bsenti na pproximately 4p er cent of the UK population, which is in agreement with more recent genetic studies of the prevalence of the null NQO1 * 2 allele. 14 .
All diaphorases discussed by Edwards 29 are distinct gene products but, because of their similarity based on enzymic function rather than evolutionary divergence,s earches for 'NQO1' in somed atabases will still returnd escriptions of 'cytochrome b5 reductase'. These cytochrome b5 reductase 'diaphorases' are not evolutionarily related, arev eryd ifferent functionally and are encoded by separate gene loci ( Table 2). As econd source of confusioni nvolves theu se of the generic term' quinone reductase', becauseawide variety of enzymes in prokaryotes, plants and animals can reduce quinones via both one-a nd two-electron steps.
As earch of the HUGO Gene NomenclatureC ommittee databaseu sing' NQO' provided three hits-two NQO genes plus 'NQO3A2' (which actually encodes cytochrome b5 reductase-1)-the latter appearsi nt he databaseb ecause of its previous name as ad iaphorase (see above). In fact, one of its aliasesi s' NQO3A2', which has been listed in the http:// SNPper.chip.org/ database; the authorsh avep ointed out this errort ot his organisation. As earch of the SwissProt and TrEMBL databases using' NQO' provided 168 hits (39 in SwissProt and 129 in TrEMBL), but not all of them belongto the flavodoxin-2f amily.T he term' NQO'h as thereforeb een used for other enzymes and care should be taken to avoid such confusion.  Updateoft he NQOgene family Review UPDATE ON GENE COMPLETIONSA ND ANNOTATIONS Evolutionaryd ivergence of the NQO genes Figure 2s hows the evolutionaryr elatedness of an umber of selected NQO gene products from mammals, birds,a mphibians, fish, fungia nd bacteria. Ve rtebrates carry the NQO1 and NQO2 genes while eubacteria carry the most ancient NQO3 genes. The authorss uggest naming the fungal subfamily as NQO4 and the archaebacterial subfamily as NQO5. Four plants (bottom of Figure 2) arec omplete outliers, being memberso ft he flavodoxin-1q uinone reductase family. Curiously,itwas found that frog, zebrafish and puffer fish have NQO1 but not NQO2;c onversely,c hicken and ray-finned fish have NQO2 but not NQO1.W hether the other homologous genew ill be found in any of theseg enomes in the future remains to be determined. Undoubtedly,t he NQO1 and NQO2 genes in vertebrates arose from the prokaryotic NQO3 ancestor. Fungal NQO4 genes also arose from bacterial NQO3 ,h owever it is impossible to sayw hether the eubacterial NQO3 genes or the archaebacterial NQO5 genes represent the original ancestor. One prokaryotic taxon might have captured the gene from the other prokaryotic taxon by horizontal gene transfer.
The authorsperformed aseries of BLAST searches -using NQO1, NQO2 or NQO3 sequences -and no homologous protein of significance in Ciona intestinalis, Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, Oryza sativa (rice) or Arabidopsis thaliana wasdetected. Wheat plants have been reported to showquinone reductase activity (both the one-and two-electron reductase forms), however, inducedinresponse to powdery mildew infection; 33 infection and inflammationare known to be processes that induce oxidativestress. 11 Plants appear to utilise quinonereductase by wayofevolutionarily unrelated classes of quinoneoxidoreductases, carrying out both one-and two-electron reduction reactions, 34 and also alternative NAD(P)Hdehydrogenases. 35 This findingmightthereforebean exampleofconvergent evolution: Mother Nature needed a particular enzyme in aparticular locationwithin the plant cell, did not have NQO enzymes in this phylum and therefore selected another type of quinone oxidoreductase or an alternative NAD(P)H dehydrogenase to carry out the enzymic function. Alternatively,this findingmight be an example of redundancy,inwhich multiple systemshaveevolved independently to face aparticular selective pressure.
Do plants suffer from oxidativestress? The absence of NQO genes in plants might reflect the fact that plants use carbon dioxide rather than molecular oxygen; therefore, plants may not experience oxidativestress in the wayanimals do.This is a fascinating bit of speculation that mayb ew orthyo ff urther study.T he putativep lant NQOe quivalents (QR1, QR2) contain the flavodoxin-1 domaina nd belong to the flavodoxin-1f amily.T his domaini sf ound in an umber of proteins, including flavodoxin and nitric oxide synthase (as described above).

Putativer ole of NQO in oxidative stress
BCL2 is well knownt ob ea nti-apoptotic and therefore prevents apoptosis from proceeding. 36 Proposedf unctions of BCL2 include mediation of ROSl evels and the redoxs tatus of the cell, preventiono fn ucleocytoplasmic trafficking of TRP53a nd other cell cycle regulatoryf actors, neutralisation of BAX, BAD and other proteins that heterodimerise and promote apoptosis and deterrence of mitochondrial cytochrome crelease. 37 -39 The C. elegans cell death-9 ( ced-9)gene is the homologue of the mammalian BCL2 gene, ced-9 is part of ap olycistronic locus in C. elegans that includes the cyt-1 gene encoding ap rotein similar to cytochrome b 560 . 38 Cytochrome b 560 is an inner mitochondrial membrane protein involved in system II (succinate dehydrogenase), which transfersa ne lectron from succinate to ubiquinone.I nterestingly,t his dehydrogenase has been described as am ember of the NQO genes uperfamily; 40 however, the authorsf ound that cytochrome b 560 is instead am ember of the more expansiveflavodoxin-1family(described above). They suggest that various memberso ft he flavoprotein clan, including mammalian NQO1, probably participate in aiding the cell in preventingu nwanted apoptosis. 40,41 Concluding remarks Isolation and characterisationo facytosolic enzyme in several rat tissuesi nt he 1950s ledt ot he description of a quinoner eductase in at least twoi ndependentl aboratories. Asubstantial number of publications, calling this enzyme 'DTdiaphorase', has led to confusion about the evolutionary relatedness of NAD(P)H:quinone oxidoreductases to one another.T here are two NQO genes in vertebrates,a homologous NQO3 subfamily of genes in eubacteria, a NQO4 subfamily in fungia nd a NQO5 gene subfamily in archaebacteria. Thef ascinating functions of the human NQO1 enzyme include protection against myeloid leukaemia and other types of leukaemia in workerse xposed to benzene and ap ossible anti-apoptotic role fundamental to the cell cycle.N QO2 appears to participate in the anti-oxidant properties of melatonin. Although many SNPs exist in the human NQO2 gene,little is known about any association with ap hysiological disorder or protection against environmental toxicants.