UPDATE ONRGeEvNieEwCOMPLETIONS AND ANNOTATIONS Vasiliou, Ross and Nebert Evolutionary divergence of the NQO genes Figure 2 shows the evolutionary relatedness of a number of selected NQO gene products from mammals, birds, amphi- bians, ﬁsh, fungi and bacteria. Vertebrates carry the NQO1 and NQO2 genes while eubacteria carry the most ancient NQO3 genes. The authors suggest naming the fungal sub- family as NQO4 and the archaebacterial subfamily as NQO5. Four plants (bottom of Figure 2) are complete outliers, being members of the ﬂavodoxin-1 quinone reductase family. Curiously, it was found that frog, zebraﬁsh and puffer ﬁsh have NQO1 but not NQO2; conversely, chicken and ray-ﬁnned ﬁsh have NQO2 but not NQO1. Whether the other hom- ologous gene will be found in any of these genomes in the future remains to be determined. Undoubtedly, the NQO1 and NQO2 genes in vertebrates arose from the prokaryotic NQO3 ancestor. Fungal NQO4 genes also arose from bacterial NQO3, however it is impossible to say whether the eubac- terial NQO3 genes or the archaebacterial NQO5 genes rep- resent the original ancestor. One prokaryotic taxon might have captured the gene from the other prokaryotic taxon by horizontal gene transfer. The authors performed a series 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 was detected. Wheat plants have been reported to show quinone reductase activity (both the one- and two-electron reductase forms), however, induced in response to powdery mildew infection;33 infection and inﬂammation are known to be processes that induce oxidative stress.11 Plants appear to utilise quinone reductase by way of evolutionarily unrelated classes of quinone oxidoreductases, carrying out both one- and two-electron reduction reactions,34 and also alternative NAD(P)H dehydrogenases.35 This ﬁnding might therefore be an example of convergent evolution: Mother Nature needed a particular enzyme in a particular location within the plant cell, did not have NQO enzymes in this phylum and therefore selected another type of quinone oxidoreductase or an alterna- tive NAD(P)H dehydrogenase to carry out the enzymic func- tion. Alternatively, this ﬁnding might be an example of redundancy, in which multiple systems have evolved indepen- dently to face a particular selective pressure. Do plants suffer from oxidative stress? 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 oxidative stress in the way animals do. This is a fascinating bit of speculation that may be worthy of further study. The putative plant NQO equivalents (QR1, QR2) contain the ﬂavodoxin-1 domain and belong to the ﬂavo- doxin-1 family. This domain is found in a number of proteins, including ﬂavodoxin and nitric oxide synthase (as described above). Putative role of NQO in oxidative stress BCL2 is well known to be anti-apoptotic and therefore prevents apoptosis from proceeding.36 Proposed functions of BCL2 include mediation of ROS levels and the redox status of the cell, prevention of nucleocytoplasmic trafﬁcking of TRP53 and other cell cycle regulatory factors, neutralisation of BAX, BAD and other proteins that heterodimerise and promote apoptosis and deterrence of mitochondrial cyto- chrome c release.37 – 39 The C. elegans cell death-9 (ced-9) gene is the homologue of the mammalian BCL2 gene, ced-9 is part of a polycistronic locus in C. elegans that includes the cyt-1 gene encoding a protein similar to cytochrome b560.38 Cytochrome b560 is an inner mitochondrial membrane protein involved in system II (succinate dehydrogenase), which transfers an electron from succinate to ubiquinone. Interest- ingly, this dehydrogenase has been described as a member of the NQO gene superfamily;40 however, the authors found that cytochrome b560 is instead a member of the more expansive ﬂavodoxin-1 family (described above). They suggest that various members of the ﬂavoprotein clan, including mammalian NQO1, probably participate in aiding the cell in preventing unwanted apoptosis.40,41 Concluding remarks Isolation and characterisation of a cytosolic enzyme in several rat tissues in the 1950s led to the description of a quinone reductase in at least two independent laboratories. A substantial number of publications, calling this enzyme ‘DT- diaphorase’, has led to confusion about the evolutionary relatedness of NAD(P)H:quinone oxidoreductases to one another. There are two NQO genes in vertebrates, a homologous NQO3 subfamily of genes in eubacteria, a NQO4 subfamily in fungi and a NQO5 gene subfamily in archaebacteria. The fascinating functions of the human NQO1 enzyme include protection against myeloid leukaemia and other types of leukaemia in workers exposed to benzene and a possible anti-apoptotic role fundamental to the cell cycle. NQO2 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 a physiological disorder or protection against environmental toxicants. Acknowledgments The authors thank their colleagues and many e-mails for valuable discussions and a critical reading of this manuscript. They appreciate the help of Jadwiga K. Kepa and Marian Miller with the graphics. The bioinformatics searches and writing of this paper were funded, in part, by NIH grants R01 EY11490 (V.V.), R01 ES09554 (D.R.) and P30 ES06096 (D.W.N.). 334 q HENRY STEWART PUBLICATIONS 1473-9542. HUMAN GENOMICS. VOL 2. NO 5. 329–335 MARCH 2006