Expansion and evolution of insect GMC oxidoreductases
© Iida et al; licensee BioMed Central Ltd. 2007
Received: 27 July 2006
Accepted: 11 May 2007
Published: 11 May 2007
The GMC oxidoreductases comprise a large family of diverse FAD enzymes that share a homologous backbone. The relationship and origin of the GMC oxidoreductase genes, however, was unknown. Recent sequencing of entire genomes has allowed for the evolutionary analysis of the GMC oxidoreductase family.
Although genes that encode enzyme families are rarely linked in higher eukaryotes, we discovered that the majority of the GMC oxidoreductase genes in the fruit fly (D. melanogaster), mosquito (A. gambiae), honeybee (A. mellifera), and flour beetle (T. castaneum) are located in a highly conserved cluster contained within a large intron of the flotillin-2 (Flo-2) gene. In contrast, the genomes of vertebrates and the nematode C. elegans contain few GMC genes and lack a GMC cluster, suggesting that the GMC cluster and the function of its resident genes are unique to insects or arthropods. We found that the development patterns of expression of the GMC cluster genes are highly complex. Among the GMC oxidoreductases located outside of the GMC gene cluster, the identities of two related enzymes, glucose dehydrogenase (GLD) and glucose oxidase (GOX), are known, and they play major roles in development and immunity. We have discovered that several additional GLD and GOX homologues exist in insects but are remotely similar to fungal GOX.
We speculate that the GMC oxidoreductase cluster has been conserved to coordinately regulate these genes for a common developmental or physiological function related to ecdysteroid metabolism. Furthermore, we propose that the GMC gene cluster may be the birthplace of the insect GMC oxidoreductase genes. Through tandem duplication and divergence within the cluster, new GMC genes evolved. Some of the GMC genes have been retained in the cluster for hundreds of millions of years while others might have transposed to other regions of the genome. Consistent with this hypothesis, our analysis indicates that insect GOX and GLD arose from a different ancestral GMC gene than that of fungal GOX.
Underlying biosynthesis and metabolism in all organisms is a large array of enzymes that catalyze a vast number of chemical reactions. Among these, oxidation-reduction reactions are the most prevalent and fundamental. Oxido-reductases typically entail electron transfer between the primary substrate and a co-factor such as NAD(P), FAD, or a cytochrome. Although similar structural domains are found in these enzymes, their primary amino acid sequences are generally not similar and therefore it is difficult to discern if they share a common evolutionary ancestor. An exceptional group in this regard is the family of GMC-FAD oxidoreductases  that shares an evolutionary conserved ca. 30 amino acid sequence comprising a beta-alpha-beta motif of the ADP-binding subdomain of FAD. Moreover, the GMC oxidoreductases contain five other blocks of conserved sequences dispersed throughout their primary sequence , supporting the hypothesis that they are evolutionarily homologous throughout.
Since the discovery of the GMC oxidoreductase family, several new enzymes have been added to this family [3, 4]. Some of the unusual additions are hydroxynitrile lyase, which does not appear to catalyze an oxidation-reduction reaction , and celliobiose dehydrogenase, which contains an additional heme domain, not present in the archetypal GMC oxidoreductases . In this study, we have searched the newly sequenced genomes of prokaryotic and eukaryotic organisms and have discovered a large number of previously unidentified GMC oxidoreductase genes in insects. Surprisingly, most of these newly identified genes are clustered in a conserved order and orientation, and are located in a large intron of the flotillin-2 gene in four distantly related insect species: Drosophila melanogaster, Anopheles gambiae, Apis mellifera, and Tribolium castaneum. We speculate that this insect GMC gene cluster may function in ecdysteroid metabolism. In addition, we report that the two glucose-metabolizing GMC enzymes in insects, GOX and GLD, are evolutionarily distinct from GOX in fungi and likely arose from a different ancestral GMC gene.
Results and discussion
The identification of a GMC oxidoreductase gene cluster in Drosophila
Prior to the sequence determination of the D. melanogaster genome, glucose dehydrogenase (Gld) located on the 3rd chromosome (3R, 84D1-2) was the only known GMC oxidoreductase family member in Drosophila. Upon completion of the genome sequencing , we surveyed the entire genome for genes that belong to the GMC family based on the amino acid sequence characteristics. In addition to Gld, two GMC homologues, NinaG  and CG6142, are located on the 3rd chromosome (3R, 97A1 and 86E7, respectively) and 12 other GMC homologues are located on the X-chromosome (12F5-13A1). These genes had been tentatively annotated as putative homologues of either choline dehydrogenase or Gld by the Berkeley Drosophila Genome Project .
However, the functions of the 12 GMC genes located on the X-chromosome are unknown except CG9504, which was recently identified by H. Takeuchi and coworkers as ecdysone oxidase (EO) . Although these twelve genes share sequence similarity with choline dehydrogenase and GLD, their sequence similarity to these two enzymes is not significantly greater than to any other GMC oxidoreductases. This indicates that they are unlikely to encode either choline dehydrogenase or GLD, arguing against the initial annotation of these genes by the Berkeley Drosophila Genome Project. Moreover, choline dehydrogenase has not been reported in insects and we have not been able detect this enzyme in Drosophila by biochemical assays (D. R. Cavener, unpublished data).
Evolutionary conservation of the GMC cluster in insect genomes
The predicted number of GMC genes in the insect genomes
In order to identify orthologs of the Drosophila GMC genes in the other three insect genomes, all amino acid sequences of the newly discovered GMC genes were aligned together with several outgroup GMC enzymes, including choline dehydrogenase from E. coli, C. elegans, and humans, two NinaG genes from Drosophila and beetles, and fungal glucose oxidase. A considerable proportion of the alignment contained gaps or highly diverged residues due to the long divergence time among these distantly related species. However, the average sequence distance across all pairwise comparisons (0.63) was below the saturated distance (~0.94) estimated from the average amino acid frequencies across all sequences analyzed (see Additional File 1 for the sequence alignment).
These subfamilies were also identified when a different phylogenetic algorithm (maximum parsimony method) or a different sequence substitution model (Jones-Taylor-Thornton amino acid substitution model ) was used for phylogenetic reconstruction (Additional File 3). The phylogenetic tree reconstructed by the maximum parsimony method showed that each of the 13 subfamilies remained clustered as a monophyletic clade except for GMCβ and κ subfamilies although the bootstrap scores were not high. It has been known that the maximum parsimony method becomes unreliable when the extent of homoplasy (backward and parallel substitutions) is high, a problem often found when sequences are diverged considerably . To take into account the problem of multiple substitutions, we also built a neighbor-joining tree by assuming a more complex substitution model (the Jones-Taylor-Thornton model). The results showed that each subfamily was clustered as a monophyletic group with a good bootstrap support (>88 for all subfamilies except for the GMC κ, which had the bootstrap score, 70).
The classification of these subfamilies is further supported by the striking evolutionary conservation in their order and orientation within the GMC gene cluster among the four distantly related species (Figure 2). The most readily identifiable are GMCα found at the 5' end of the cluster and four tandemly-arrayed families, GMCδ, ε, ζ, and θ, in the middle of the cluster. These genes have a single copy in the same orientation, except GMCθ, which has two copies in some species. GMCγ has also retained a well-conserved position between GMCα and GMCβ genes. Although A. gambiae has two GMCγ genes in opposing directions, the other three species contain a single copy in the same orientation. The other subfamilies also show conserved positions among different species despite that they contain the varied number of gene copies (0–3) in different species. Two copies of D. melanogaster GMCβ genes are located in the relative position conserved among the other species (between GMCγ and GMCδ) though EO-β1 is located between GMCα and GMCγ genes. The GMCι subfamily is missing from A. mellifera, but in the other species, it is always located at the 3' end of the cluster. The GMCκ subfamily is present only in A. gambiae and T. castaneum, and in both species, the GMCκ genes are located between the GMCι genes.
Evolution of the GMC cluster
The overall conservation of the GMC cluster among insect species is striking, as microsynteny is typically not conserved among these highly diverged species. For example, only 30% of A. gambiae genes that are homologous to D. melanogaster genes in the Adh region retain microsynteny, where each syntenic region includes only two or three genes . The conservation of the cluster region is highly specific to the cluster, and does not extend to the flanking regions. We examined the location of some A. gambiae genes that are apparently homologous to D. melanogaster genes to see if any of the genes surrounding the D. melanogaster cluster maintained microsynteny in Anopheles. Examined genes included Rut, CG14411, CG14411, and CG14407 in the 3' direction of the cluster and CG9009, Eag, Hiw, CG5530, CG5560, and CG15027 in the 5' direction of the cluster, which covered about 300 kb in the area surrounding the cluster. We found that the only gene that maintained microsynteny with the cluster was Flo-2, which contains the cluster within one of its introns. In fact, in all four species, the cluster locates within the homologous intron of Flo-2, and the first 16 amino acids of FLO-2 are encoded by an exon located at the 3' of the cluster (Figure 1 and Additional File 4). Flo-2 is transcriptionally oriented in the opposite direction to almost all of the GMC genes within the cluster (Figure 2). Sequences of FLO-2 homologues in these three insect species are highly conserved with D. melanogaster FLO-2 (77–88% pairwise amino acid identity).
The fact that four core genes (GMCδ, ε, ζ, and θ) in the middle of the GMC cluster have remained in tandem and in the same orientation over hundreds of millions of years strongly suggests that this cluster, partly or entirely, has been maintained by natural selection. None of the four core GMC cluster genes is a close homologue to any of the other GMC oxidoreductases for which enzyme substrate specificity has been determined, and therefore their catalytic activities remain to be determined.
The presence of ecdysone oxidase (EO-β1) gene in the cluster suggests that the cluster may encode a series of enzymes that are involved in ecdysone metabolism. Ecdysone oxidase catalyzes the oxidation of ecdysone to dehydroecdysone within pathways involved in degradation of edysone and/or generation of unique ecdysteroids . A diversity of ecdysteroids is produced in insects, and their tissue- and developmental stage-specific modification and degradation is important in the orchestration of insect development [20–22]. We speculate that the GMC cluster comprises a network of coordinately regulated suite of genes that act to modify developmental and physiological processes in tissue and spatially distinct patterns. By maintaining these genes in a cluster, combinatorial regulatory elements can efficiently coordinate their regulation.
Why then is GMC cluster located in a large intron of the Flo-2 gene? The parsimonious hypothesis is that the ancestral GMC gene or cluster was accidentally transposed into the Flo-2 gene and has never had the opportunity to leave without destroying itself or the Flo-2 gene. A more compelling possibility is that the transcriptional regulation of the GMC complex and Flo-2 are intimately tied together. Conservation of other gene clusters, including the vertebrate globin gene cluster and the insect homeobox gene clusters, appears to be due to a requirement of these genes to be coordinately regulated by local cis-acting mechanisms. Flo-2 encodes the lipid raft protein flotillin-2. As lipid rafts contain cholesterol and their derivatives including steroids  and steroid binding proteins have been detected in lipid rafts , Flo-2 and the GMC genes, including ecdysone oxidase, may be coordinately regulated in support of a common developmental or physiological function. We speculate that cis-acting control elements may exist in the GMC cluster and act to coordinately regulate the expression of the GMC genes, and perhaps the Flo-2 gene as well.
Duplication of genes and exons
Homologous genes typically arise from tandem duplication events that result in two or more homologues tandemly arrayed. The GMC cluster has retained much of its history of gene duplication events that gave rise to the cluster. At a subfamily level, this is most apparent for the GMCα, γ, δ, ε, and ζ subfamilies. As these genes are tandemly arrayed (Figure 2) and phylogenetically form a distinct group of subfamilies (bootstrap value, 100; Figure 3), we postulate that these genes arose from the same ancestral gene prior to the divergence of the major insect subfamilies.
Duplication events are also seen within a subfamily. Four of the subfamilies in the GMC cluster, GMCα, δ, ε, and ζ, have remained as single-copy genes in the genome, whereas GMCβ, γ, θ, ι, and κ, are multi-copy genes. Particularly, each pair of GMCγ genes (A. gambiae), GMCθ genes (D. melanogaster and A. gambiae), GMCκ genes (T. castaneum), and GMCι genes (D. melanogaster) is supported by a very high bootstrap value (99–100) and is arrayed tandemly. This indicates that duplication events of the GMC genes had occurred after the emergence of the four insect species from a common ancestor.
Diversification of GLD and GOX in insects
In addition to GMC genes in the cluster, we discovered several other GMC genes that reside outside the cluster in D. melanogaster, T. castaneum, A. gambiae, and A. mellifera (Table 1). While the identity of these genes is largely unknown, our phylogenetic analysis suggests that several of them belong to a gene subfamily containing glucose dehydrogenase (GLD) and glucose oxidase (GOX) ("insect GLD/GOX/GLXr," supported by a bootstrap value, 100; Figure 3 and Table 1). These two enzymes catalyze the conversion of β-D-glucose to δ-gluconolactone but differ in the electron acceptor [1, 27].
Apparent orthologues of the previously identified GLD in D. melanogaster were found in all four insect species (bootstrap value, 100; Figure 3). The Gld genes of D. melanogaster and A. gambiae share a very similar exon/intron structure while honeybee Gld structure is more divergent. Similar patterns in developmental expression are also observed between honeybees and Drosophila (D. L. Cox-Foster, unpublished). Because GLD is an essential gene in Drosophila for exoskeleton metabolism [28, 29], we speculate that all other arthropods with exoskeletons contain GLD.
However, GOX-1 and GLXr-2 also share some expression patterns; for example, both are highly expressed in hemocytes  and are induced in similar patterns by immune challenge . These data indicate that these genes may share some common regulatory elements for those expression patterns, further suggesting that genetic linkage between these two genes has been under an evolutionary constraint.
Pairwise amino acid sequence identity of GLD, GOX, and GLXr proteins
10. Human CHD
11. Ecol CHD
12. Anig GOX
13. Pama GOX
14. Aory GOX
Evolution of GMC genes
Some GMC genes in the cluster, namely GMCθ,λ, andβ subfamilies, have homologues in one or more species that exist outside of the cluster, which suggests that the GMC cluster may have been the birthplace for all insect GMC genes including GLD and GOX. This hypothesis is supported by several facts as follows. (1) GLXr-1 is located in the GMC cluster of A. mellifera whereas all other GOX, GLD, and GLXr genes are located outside the GMC cluster. Importantly, GLD is present on the same chromosome as the GMC cluster in three of the four species. The Gld genes of A. gambiae, T. castaneum, and A. mellifera are located 30 Mb, 9 Mb and 1 Mb apart from the GMC cluster, respectively. (2) A cluster of three tandemly duplicated GMCβ genes in A. mellifera are present outside of the GMC cluster (GMCβ7–9) but are still on the same chromosome, and these have the closest relationship with GMCβ6 in the cluster (bootstrap value, 94). (3) One of the T. castaneum GMCθ genes, which apparently arose from a duplication event, is located ~250 kb away from the cluster on the same chromosome (GMCθ6). (4) The closest homolog of the cluster-localized A. mellifera GMCλ1 is T. castaneum GMCλ1 located outside the cluster on a different linkage group from that of the cluster (bootstrap value, 100). Together these data are consistent with the hypothesis that the GMC genes have undergone tandem duplication in the GMC cluster and then one or more copies have relocated outside of the cluster, frequently on the same chromosome, before some have been further dispersed to other chromosomes.
Relocation of genes outside the cluster would likely occur by transposition for two reasons. First, the most common event of transposition is "local hopping" to a nearby region on the same chromosome , and secondly, transposition would allow excising one or more genes without disrupting the Flo-2 gene. Other larger scale chromosome rearrangements (e.g., inversion and translocations) would likely disrupt the Flo-2 gene in which the GMC cluster resides. In summary, we propose that the location of the highly conserved core genes (GMCδ, ε, ζ, and θ) is constrained due to shared regulatory elements within or flanking the cluster, whereas the other GMC genes are less constrained and have spawned a number of new GMC genes that have relocated to other regions of the genome.
Insects contain a cluster of GMC oxidoreductase genes that is highly conserved in gene composition, gene order, transcriptional orientation, and presence in a large intron of the Flo-2 gene. In addition, a smaller number of GMC oxidoreductase genes exists outside of this cluster but may have originated from the cluster and evolved independently. Although fungal GOX and insect GLD are closely related functionally, their relatively low sequence similarity suggests that they arose independently from an ancient GMC gene. In contrast several glucose oxidase and glucose dehydrogenase genes within insects have a high degree of sequence similarity consistent with the hypothesis that these two genes have more recently arose from a common GLD/GOX common ancestor since the divergence of insects.
The presence of all GMC-related sequences in A. gambiae, A. mellifera, and T. castaneum genomes was detected by TBLASTN using D. melanogaster GLD sequence against the database at the National Center for Biotechnology Information (NCBI) . The preliminary sequence data for T. castaneum genome was provided to the NCBI from Baylor College of Medicine Human Genome Sequencing Center . One of the outgroup sequences used in our phylogenetic study, Aspergillus oryzae putative GOX, was obtained from the DOGAN (Database Of the Genomes Analyzed at NITE [National Institute of Technology and Evaluation in Japan]) . All other outgroup sequences were obtained from the NCBI.
When there was a predicted protein sequence in the public database that corresponded to the BLAST hit, we evaluated the sequence based on the alignments with GMC genes in other insect species. When the sequence was reasonably aligned with the putative homolog, we used the predicted sequence. However, when the sequence had a major insertion/deletion and/or did not have major conserved regions, we manually annotated a sequence. The details of sequence information can be found in Additional Files 7, 8, and 9.
Multiple alignments of protein sequences were carried out using CLUSTALW [36, 37]. We reconstructed a phylogenetic tree using a neighbor-joining algorithm  implemented in MEGA3 . The pairwise distance matrix was estimated based on the Poisson correction model  with exclusions of gaps for each pair of sequence comparison (pairwise gap deletion). A bootstrap re-sampling analysis with 500 replicates was also performed to evaluate the inferred tree topology .
Primers used for this study were as follows: AmGLXr-2, 5'-CGGCCCGGAGAATCATCAG-3', 5'-ATCCGCATTTACATTTCTTTGGTCTC-3' (which amplifies two products of alternatively spliced isoforms, 548 bp for Isoform I and 455 bp for Isoform II); AmGOX-1, 5'-CTGGACTGGAAGTATTACACTACGAAC-3', 5'-ACGATTGGTGATTGTGAAGGTTCT-3'; AmEF1α (elongation factor 1 alpha), 5'-ATGGGCAAGGGCTCGTTCAAGTA-3', 5'-CTTTCCGTCAGCGTTACCATCTTTGC-3'; DmGMCα1 (CG9503), 5'-TGGTGGTTATCTGACAGTTGGTGAGG-3', 5'-ATGGCTTTCGTTCGGGATAATGC-3'; DmEO-β1 (CG9504), 5'-ATGCCATTGTTTCTGCTCTTCGGTT-3', 5'-AACCAGTAGTCATCGGAATCGGC-3'; DmGMCβ3 (CG9512), 5'-AAAATGTTGGGCGGCACGAATGG-3', 5'-TCCTGAGTGCCCAAGATGTCCATTT-3'; DmGMCγ1 (CG12398), 5'-ATCCCGATGGTGATTTCAATGGT-3', 5'-CAGAATCACCTCTCGTTTGGCTC-3'; DmGMCδ1 (CG9514), 5'-GACGGGTTTCGGTTTCTATCAGTTCA-3', 5'-AATCTCTTCATAGCCTGCGTTTCACC-3'; DmGMCε1 (CG9517), 5'-CATTGGGCATCGTTGGGTAATCCG-3', 5'-TTGGAAACGATTGCGGGTGACAGT-3'; DmGMCθ2 (CG9521), 5'-TTCAAGGATGTGCTGCCGTATTTCAA-3', 5'-ACAGCATCAGTAGTTGGGGCGTATTG-3'; DmGMCι1 (CG9522), 5'-CGGAGGAGTGGAGAACATAGTGC-3', 5'-CAATCCCCGACAGCATCAGCAACT-3'; DmGMCζ1 (CG9518), 5'-TTCAATCCCACAGCCGTCACCTTTC-3', 5'-GTCTATTTGCCTGCCGCTTTACTTTGT-3'.
Genomic library screening and 5' RACE
The Apis mellifera genomic library (RZPD, Germany) was screened twice: the first time with the Gox-1 probe produced by the above primers, and the second time with the Glxr-2 probe. The latter probe contained a mixture of two probes produced by the above primers (for Glxr-2) and the following set: 5'-GACGGGGCTCTCGCAACTG-3', 5'-GGCGCACCTCCAGTAGTCGT-3'. Sequences for Glxr-2 primers were obtained from two fragments of the EST contig 59 found in the adult bee brain cDNA library : BB160017A20G03 and BB170027B20D05. The 5' ends of Gox-1 and Glxr-2 genes were isolated by 5' RACE System (Invitrogen) and sequenced.
Total RNA was isolated using TRI Reagent (SIGMA) followed by DNase treatment (Ambion). To assess the gene expression patterns, we performed RT-PCR (Promega) of total RNA (1 μg) from different developmental stages of A. mellifera or D. melanogaster using appropriate gene-specific primer sets. For Drosophila GMC cluster genes, band signal intensity was compared visually and categorized to either undetectable ("-") or relative expression ("+" to "++++"). As control, reactions for eIF2α were run to confirm the uniform quantity and quality of samples. For A. mellifera Gox-1 and Glxr-2, RT-PCR products were subjected to Southern blot analysis and probed with each gene's fragment generated by the primers used for genomic library screening. Signal intensity was detected and analyzed by STORM scanner and ImageQuant (Molecular Dynamics) and was normalized to the level of Ef1α signals.
We thank Dr. Gene Robinson (University of Illinois, Urbana-Champaign) for providing EST fragments for the Glxr-2 gene of A. mellifera, and Dr. Peichuan Zhang (University of California, San Francisco) for technical assistance. This study was partially supported by grants NIH DK62049 and NIH AR49816 (D.R.C.).
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