Early steps of ecdysteroid biosynthesis, the "black box" and CYP307

Ecdysteroid biosynthesis is generally considered to start with a sterol (e.g. C27: cholesterol; C28:campesterol; C29: sitosterol). This sterol is converted in ecdysteroidogenic cells to its Δ-7 homolog. The nvd (neverland) gene encodes a cholesterol 7-dehydrogenase (Yoshiyama et al., 2011). The next steps are still unresolved, and have been called the “black box”. At least two enzymes are known to be involved although their precise role is unclear. One is nm-g/sro (non-molting glossy /shroud), a short chain dehydrogenase/reductase (Niwa et al., 2010). It functions along CYP307 in the “Black Box”. Until now, no clear evidence has been presented for any intermediate between 7-dehydrocholesterol and a putative “∆4-diketol” precursor which would then be reduced to the most likely product of the “black box”, the “5ß-diketol” and, depending on the species, further to the “ketodiol” (2,22,25-trideoxyecdysone)(Lafont et al., 2012). Furthermore, there is no evidence that the introduction of the 14 α-hydroxyl group is independent of the formation of the conjugated 7-ene-6-one moiety. A putative scheme for the black box was presented by Lafont et al.(2012) and is shown in adapted form below, noting that Warren et al. (2009) have proposed that 3-oxo-7-dehydrocholesterol may be an initial player in the black box:

CYP307 are enigmatic P450s of the CYP2 clan. Namiki et al., (2005) first showed that the Bombyx mori gene CYP307A2 and its Drosophila melanogaster homolog CYP307A1 (aka spook) were involved in ecdysone biosynthesis. Drosophila melanogaster has two CYP307 genes, CYP307A1 (spook) and CYP307A2. The latter was found as putative pseudogene in the initial genome release (Tijet et al., 2001), but later obtained from a difficult to sequence heterochromatic region (3R-47.1). The function of the CYP307 enzymes is still unknown 20 years after being characterized as a “key area in studies of ecdysteroid biosynthesis” (Namiki et al., 2005), but it is thought that CYP307 are involved in the “black box” steps downstream of 7-dehydrocholesterol.

CYP307 genes are known to be “unstable” with multiple instances of birth and death (Sztal et al., 2007; Rewitz and Gilbert, 2008; Sezutsu et al., 2013). Dermauw et al.(2020) expanded upon these earlier results, and confirmed the presence of two orthologous groups in Pancrustacea, the CYP307A and CYP307B genes. These cover Hexapods, Branchiopoda (Daphnia pulex) and Copepoda within “Multicrustacea” (sensu Schwentner et al., 2017). Depending on the lineage, these two groups are either present together or singly.

Rewitz and Gilbert (2008) noticed the tail to tail location of CYP307A2 and the neverland (nvd) gene in Daphnia pulex, Anopheles gambiae and Drosophila willistoni. Dermauw et al.(2020) reported that in Drosophila melanogaster the two genes are only 0.1cM apart. The synteny is also maintained in Aedes aegypti, at least four lepidopteran species, as well as Nasonia vitripennis, Cephus cinctus, Neodiprion lecontei, Athalia rosae (tail to tail), Myzus persicae, Frankliniella occidentalis, Pediculus humanus and Zootermopsis nevadensis. In Aphis gossypii there are four genes, one CYP307A (XP_027846257) located tail to tail with an nvd pseudogene (XP_027846250) and nvd (XP_027846249) and two more in tandem array (XP_027848023 and XP_027848010), as well as one CYP307B gene (XP_027854219). CYP307A2 and neverland therefore form a functional cluster.

More distant from the CYP307A and 307B sequences are clades of CYP307 in Malacostraca, Cirripedia as well as Diplura, whose relationships to the CYP307A/B clades are still unclear. There are two more clades, one includes Strigamia CYP307 and most Chelicerata, and another is specific for Acariformes. In none of these genomes can synteny of CYP307 with nvd be detected. While the TSA of the millipede Chamberlinius hualienensis revealed no CYP307, its presence in millipedes can be shown in the genomes of H. holstii and Trigoniulus corallinus. In the bark scorpion, CYP307M was recently duplicated, with the two genes differing by just four nucleotides, but with different neighboring genes, while the horseshoe crab has three CYP307 genes.

All CYP307 sequences share unusual structural features. The C-helix motif conserved GxxWxEQRR of the CYP2 clan has instead a AxCDWSxxQxxRR motif. The I helix of CYP307 lacks the conserved Thr, and has a LEDxxGGHSAvvN consensus where the CYP2 clan has LxDLFxAGx(E/D)TTS. The conserved ExxR and PERF are present and the Cys pocket motif has the consensus FxPFxxGxRxCxG. These structural features may well prove determinant in explaining the complex reaction(s) catalyzed by CYP307 enzymes. That the complex and poorly understood reactions of this famed Black Box should be catalyzed by the product of an “unstable” gene as CYP307 is somewhat paradoxical. The high degree of CYP307 “instability” contrasts with the stability and high conservation of the other P450 genes of ecdysteroid biosynthesis. It is possible that different CYP307 have different substrates but a similar product, so that comparative biochemistry may resolve the paradox, and/or that CYP307 duplications allow different timing and sites of expression as in Drosophila melanogaster and Nilaparvata lugens (Ono et al., 2006; Zhou et al., 2020).

CYP307A2 is rapidly phosphorylated in Manduca sexta prothoracic glands after prothoracicotropic hormone (PTTH) treatment (Rewitz et al., 2009), whereas other gene products (CYP306A1, CYP315A1, CYP302A1) are not. Two phosphorylation sites T165 or S168 and S438 can be identified on CYP307A2, but it is not clear whether phosphorylation is a specific response to PTTH, or whether the effect observed simply reflects the higher amounts of CYP307A2 protein in the experiment. Furthermore, the phosphorylation sites are not conserved in all CYP307 proteins.

Shi et al., 2022 reported a modest O-dealkylation activity of Helicoverpa armigera CYP307A2 towards 7-benzyloxymethoxy resorufin (BOMR).