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====== C-25 hydroxylation, CYP306 and CYP18 ======

==== Biochemistry ====

The C-25 hydroxylation of 2,22,25-trideoxyecdysone in prothoracic glands of //Locusta migratoria// was shown to be catalyzed by a microsomal P450 enzyme (Kappler et al. 1988). CO inhibition was reversible by white light, and the activity was also inhibited by piperonyl butoxide, SKF-525A and metyrapone. The C-25 hydroxylation had a Km of 2.5 µM for 2,22,25-trideoxyecdysone.

==== CYP306 ====

An insect C-25 hydroxylase was identified as CYP306A1, a CYP2 clan P450 in //Bombyx mori// and in Drosophila. It is encoded in Drosophila by a member of the "Halloween" group of genes, the phantom (phm) gene (Niwa et al., 2004; Warren et al., 2004). In //B. mori//, microarray analysis and differential display of the PG transcriptome showed that CYP306A1 was expressed predominantly (but not solely) in the PG, and at times of high ecdysteroid production. Drosophila S2 cells transfected with the cDNA from //Bombyx mori// or Drosophila  were able to hydroxylate 2,22,25-trideoxyecdysone (ketodiol) to 2,22-dideoxyecdysone (Niwa et al., 2004; Warren et al., 2004).

==== CYP18 and CYP306 distribution and evolution ====

These two CYP2 clan P450s are closely related in sequence and result from a duplication event, forming a strongly supported monophyletic clade. They are also mostly present in close synteny, with the two genes head to head in //Drosophila// and the honey bee (Niwa et al., 2004; Claudianos et al., 2006) as well as in //Daphnia pulex// (Rewitz and Gilbert 2008). This arrangement is also found in Coleoptera, Hemiptera, Isoptera, //Calopteryx splendens//, and the collembolan //Holacanthella duospinosa// but the CYP306 and CYP18 genes are head to tail in another collembolan //Orchesella cincta// and in the amphipods //Hyalella azteca// and //Parhyale hawaiensis//. They are also head to tail in the millipede //Trogoniulus corallinus//, but tail to tail in another millipede, //Helicorthomorpha holstii//. This synteny of CYP306 and CYP18 maintained over 400 MY is remarkable, given that the function of the two genes is thought to be opposite (biosynthesis vs. inactivation - at least in Drosophila). Perhaps the risk of hormonal imbalance has favored their inheritance as a linked locus. 

CYP18 is found almost ubiquitously in arthropod genomes as a single copyy gene (see exceptions below). A CYP18 gene is even found in //[[euperipatoides|Euperipatoides rowelli]]//, (Onychophora, sister clade of Arthropoda). In stark contrast, no chelicerate has a CYP306 gene, indicating that the CYP18A1 / CYP306A1 gene duplication occurred at the base of Mandibulata (Myriapoda + Pancrustacea), see scheme below (taht does not show secondary losses in some lineages).{{ ::cyp18_306.png?400 |}}

An interesting hypothesis (Ogihara et al.,2019; Dermauw et al., 2020) is that an ancestral CYP18 had both C-25 and C-26 hydroxylase activities (or even just C-25 hydroylase activity), and that upon duplication, subfunctionalization into C-25 (CYP306) and C-26 (CYP18) hydroxylase activities emerged. This would explain the presence of 25-hydroxylated ecdysteroids in Chelicerates: scorpions, spiders, ticks and some mites (Crosby et al., 1986; Chambers et al., 1996; Feldlaufer and Hartfelter 1997; Lomas et al., 1997; Miyashita et al., 2011; Honda et al., 2017) which have a CYP18 but no CYP306. Even pycnogonids make abundant C25 and C26-hydroxylated ecdysteroids (Bückmann et al, 1986), yet presumably lack CYP306 as the other Chelicerates (or pre-Chelicerates).

To date, the substrate specificity of arthropod CYP18 has not been tested on any 25-deoxy-ecdysteroid, so that the possibility remains that some extant CYP18 of Mandibulates still have 25-hydroxylase activity. Conversely, D. melanogaster CYP306A1 was reported to have been the target of natural selection, for some unknown and apparently unquestioned reason (Orengo and Aguade, 2007). Good et al., (2014) show that it is the fastest evolving P450 in their 12 Drosophila study, and they suggest it acts on multiple substrates. 

In //Tetranychus urticae//, in the absence of both CYP18 and CYP306, ponasterone A (25-deoxy-20-hydroxyecdysone) but not 20E has been identified (Grbic et al., 2011). These two genes are also missing in //Varroa destructor//, //Metaseiulus occidentalis//, and in //Neoseiulus cucumeris//.  CYP18 is present in //Dermanyssus pteronyssinus//, //Sarcoptes scabiei//, //Psoroptes ovis//, //Aculops lycopersicii// (as probable pseudogene), //Ixodes scapularis//, the common house spider and the wolf spider //Pardosa pseudoannulata//. There are two copies in scorpions and horseshoe crabs. 

In millipede genomes (//Trigoniulus corallinus// and //Helicorthomorpha holstii//) the CYP18 and CYP306 pair of genes has generated close paralogs, with three transcripts also seen in //Chamberlinius hualienensis//. In the salmon louse (copepod) there is no CYP306 (Humble et al., 2019), so the origin of the C25 hydroxyl group of E and 20E found in this species (Sandlund et al., 2018) is unclear. Two other copepod species do carry a CYP306 gene. Similarly, CYP306 is not found in genomes of the collembolan //Folsomia candida//, but there is a CYP306 gene in other collembolans, //Sinella curviseta//, //Holacanthella duospinosa// and //Orchesella cincta//. 

CYP18A1 has been lost in //Anopheles gambiae// (Feyereisen, 2006), but this loss is restricted to the //A. gambiae// complex, as it is found in the closest species, //An. christyi// and beyond (Neafsey et al., 2015). Similarly, there is no CYP18 sequence in the genome of //Blattella germanica//, nor in the TSA of any other related species in the “Blattellinae” (//sensu// Evangelista et al., 2019), although it is readily found in Blaberidae and other Blattodea.

In Lepidoptera, CYP18 is duplicated, and the two genes have a different tissue expression pattern (Li et al., 2014). CYP18A1 is expressed in the prothoracic glands of Lepidoptera and of Drosophila (Davies et al., 2006; Guittard et al., 2011; Christesen et al., 2016; Moulos et al., 2018), but the function of this ecdysteroid-inactivating P450 in ecdysteroidogenic cells is currently unknown.

==== CYP306 and CYP18 specificity ====

CYP306A1 from several species was shown to convert 2,22,25-trideoxyecdysone ("ketodiol") to 2,22,dideoxyecdysone ("ketotriol"): //Bombyx mori//, //Drosophila melanogaster//, //Manduca sexta//, //Mamestra brassicae//, and  //Nilaparvata lugens// (Niwa et al.,2004; Warren et al.,2004; Rewitz et al., 2006; Ogihara et al.,2017; Zhou et al.,2020). 

The specificity towards other ecdysteroids is complex: Drosophila CYP306A1 can hydroxylate the 3α,5α- and 3β,5α-isomers of the "ketodiol" thus tolerating different (unnatural) A/B ring junctions (Warren et al., 2004). Niwa et al.(2004) reported hydroxylation of 3β-hydroxy-5β-cholest-7-en-6-one at the C-25 position, i.e. a substrate lacking the 14α hydroxyl group. Yet the enzymes from Drosophila and from //Bombyx mori// can discriminate between variously hydroxylated substrates. The ketodiols with one (C2),(C22) or two (C2,C22),(C2,C20) additional hydroxyl groups are not metabolized by CYP306A1 (Warren et al., 2004). 

CYP306A1 from //Helicoverpa armigera// was shown to dealkylate the model substrates 7-ethoxycoumarin and benzyloxy-methoxy resorufin (BOMR) (Shi et al. 2022). Intriguingly, CYP306A1 from //Spodoptera litura// was reported to hydroxylate the cyclic diterpenoid 7-dehydroabietanone to 19-hydroyy-7-dehydroabietanone (Liu et al. 2023). In //Bemisia tabaci//, CYP306A1 was reported to metabolize imidacloprid (Liu et al., 2023) but metabolites were not identified.

The specificity of insect CYP18 is discussed under [[ecdysteroid_metabolism|ecdysteroid catabolism]].