====== The mitochondrial P450 clan (Mito clan) ======

Sequences belonging to this clan are considered to be located on the inner membrane of mitochondria, as are the vertebrate members of this clan (CYP11, CYP24, CYP27). This clan, that probably should have been called **CYP11 clan** (as the other clans are named after the first family of the clan), has therefore the "mitochondrial" designation. This may be unfortunate and potentially confusing, because clan names refer to similarities in sequence, not subcellular localization, and because it is far from certain that each and all Mito clan P450s are actually mitochondrial.

The Mito clan is generally small, down to 4 sequences in the collembolan //[[Sinella|Sinella curviseta]]//, but can be larger than the CYP2 or CYP4 clan as in the cat flea //[[catflea|Ctenocephalides felis]]// where Mito clan P450s comprise 30% of the CYPome (Feyereisen, 2022).  

Strong evidence that house fly CYP12A1 is indeed a mitochondrial enzyme comes from its localization in mitochondria by immunogold histochemistry and absolute dependence on mitochondrial electron donors adrenodoxin reductase and adrenodoxin (Guzov et al., 1998). Such stringent criteria have not been applied to other members of this clan in arthropods, and assumption of mitochondrial localization is generally accepted, although //[[Drosophila|Drosophila melanogaster]]// CYP314A1, 302A1 and 315A1 tagged and expressed in S2 cells colocalize with a mitochondrial marker by confocal microscopy (Petryk et al., 2003). 

In accordance with experimental evidence, the mitochondrial CYP12A1 is predicted to be targeted to mitochondria by DeepLoc, LocTree3 and SubCons. However, this is not the case for all Mito clan P450s. Of 306 Mito clan P450s in Dermauw et al., 2020, while 97.4% were predicted to be mitochondrial by LocTree3, only 81.4% and 72.9% were predicted to be mitochondrial by DeepLoc and SubCons respectively. 

The case of CYP314A1 is of interest because it encodes an ecdysone 20-hydroxylase which is known to be found in both mitochondria and microsomes, depending on the species, the tissue and the developmental time (Lafont et al., 2012). The //[[Bombyx|Bombyx mori]]//  20-hydroxylase in eggs is microsomal and inhibited by (microsomal) NADPH cytochrome P450 reductase antibodies (Horike and Sonobe, 1999), and so is the larval cockroach enzyme (Halliday et al., 1986). It is not known whether these enzymes are encoded by CYP314A1, which is predicted to be located in the ER, not mitochondria by SubCons and DeepLoc in //[[Bombyx|Bombyx mori]]// and //[[Locusta|Locusta migratoria]]//. While LocTree3 predicted all 38 CYP314A1 to be mitochondrial, this was only the case for 12 by DeepLoc and just 9 by SubCons. 

The //Anopheles funestus// CYP314A1 shows 20 hydroxylase activity when coexpressed in //E. coli// with P450 reductase, suggesting that it is a microsomal enzyme (Ismail et al. ICE Helsinki 2022 presentation).

The “mitochondrial” in the name of this clan should therefore **NOT** be taken as definitive evidence of subcellular localization. Some P450s of this clan may be microsomal or have dual localizations. 

====== CYP302 ======

CYP302A1 is an [[ecdysteroid_metabolism|ecdysteroid C22 hydroxylase]] (dib) in Drosophila (Warren et al., 2002). No CYP302 gene can be found in the genome of the centipede //[[Strigamia|Strigamia maritima]]// (Epimorpha, order Geophilomorpha). However, CYP302 transcripts were found in other Chilopoda, //Scutigera coleoptrata// (Notostigomorpha, order Scuterigomorpha), in both //Eupolybothrus cavernicolus// and //Lithobius forficatus// (full length, Pleurostigomorpha, order Lithobiomorpha), . 

This suggests that if the loss of CYP302A1 in //[[Strigamia|Strigamia maritima]]// can be confirmed, this loss is not common to all Chilopoda. Geophilomorpha diverged from the other two lineages about 375 MYA (Fernandez et al., 2018). (there is a CYP302 transcript of //[[Strigamia|Strigamia maritima]]// (in SRR5602569) but this could not be assigned to the genome and is in fact a //[[Centruroides|Centruroides sculpturatus]]// contaminant.) 

In millipedes, the other major branch of Myriapoda, while //Helicorthomorpha holstii// and //Trigoniulus corallinus// each have one CYP302 gene, we found two CYP302 genes in //[[Chamberlinius|Chamberlinius hualienensis]]// where a further duplication has led to a third gene called CYP3197A1 which is divergent but closely related to the other two CYP302 (100/100/96 support).
Transcripts for CYP302A1 were also found in //Haploglomeris multistriata// (Pentazonia, order Glomerida ), in //Polyzonium germanicum// (Helminthomorpha, order Polyzoniida) and in //Polyxenus lagurus// (Penicillata, order Polyxenida).

Qu et al., (2015) reported the absence of CYP302 in ticks, but we found CYP302 in //[[Ixodes|Ixodes scapularis]]// and a number of other tick species. Although RefSeq calls two CYP302A1 genes in //[[Plutella|Plutella xylostella]]//, these are probably allelic variants (by one amino acid) of the same gene, as one is on a large scaffold, the other on a very small one.

Shi et al., 2022 reported a modest O-demethylation activity of //[[Harmigera|Helicoverpa armigera]]// CYP302A1 towards 7-benzyloxymethoxy resorufin (BOMR).
====== CYP315 ======

CYP315A1 is an [[ecdysteroid_metabolism|ecdysteroid C2 hydroxylase]] (sad) in Drosophila (Warren et al., 2002). The CYP315 gene is generally present in all species studied, but is not easily found in the copepods //[[Tigriopus|Tigriopus japonicus]]// and //T. californicus//. However, it is found in other copepods, //[[Paracyclopina|Paracyclopina nana]]// and //Lepeophteirus salmonis//, and phylogenetic analysis reveals that the CYP315 clade does contains two //Tigriopus japonicus// genes, CYP3022A1 and CYP3023A1, with strong support (98/97.4/73)(Dermauw et al. 2020, Suppl.Fig.1). This suggests that these two P450s are in fact the products of a CYP315 duplication and rapid divergence. Whether or not 2-deoxyecdysteroids are found in the //Tigriopus// genus is not known, and the function of these two P450s merit examination. Three CYP315 are apparent in the collembolan //[[Folsomia|Folsomia candida]]//, whereas there is just one gene in //[[Sinella|Sinella curviseta]]//. In //[[Limulus|Limulus polyphemus]]//, we found two pairs of CYP315 genes. The TSA of the millipede //[[Chamberlinius|Chamberlinius hualienensis]]// revealed no CYP315, but it was found in the genomes of //H. holstii// and //T. corallinus//. In centipedes, //[[Strigamia|Strigamia maritima]]// has two CYP315, which is intriguing given the apparent lack of CYP302.
Gilbert and Rewitz (2009) suggested that the intronless //[[Tribolium|Tribolium castaneum]]// CYP315A1 was a retrogene. However, it is located in an intron of a “tyrosine-protein kinase transmembrane receptor Ror-like” gene just as its ortholog in //[[dendroctonus_ponderosae|Dendroctonus ponderosae]]// which has seven exons and its orthologs in //Leptinotarsa decemlineata// and //Aethina tumida// which have two. We conclude that this is a case of differential intron loss in Coleoptera, rather than retrotransposition. 

Schumann et al., (2018) reported the presence of three CYP315 genes, but no other ecdysteroidogenic gene in velvet worms (Onychophora). Our analysis suggests that only two //Euperipatoides// CYP315 belong to the weakly supported monophyletic CYP315 clade (89/92.3/59), and whether they have ecdysteroid 2-hydroxylase activity is unknown.

====== CYP314 ======

CYP314A1 is an [[ecdysteroid_metabolism|ecdysteroid C20 hydroxylase]] (shd) in Drosophila (Petryk et al., 2003). Although CYP314 is found in all arthropods and forms a monophyletic clade (100/100/98), there are some exceptions. CYP314 is found in crabs (//Carcinus maenas//, //Eurypanopeus depressus//) and 20-hydroxyecdysone and ponasterone A are commonly reported from decapods. However, Sin et al., (2015) reported a lack of CYP314 in the shrimp //Neocaridina denticulata// (Decapoda, Caridae). They rightly warned of possible poor recovery of this genomic locus, as few CYP genes they reported were of full length. There is no CYP314 in the genomes of //[[Pvannamei|Penaeus vannamei]]// (Pacific white shrimp), //Penaeus monodon// or //Palaemon carinicauda//. In the spiny lobster //Sagmariasus (Jasus)(Panulirus) verreauxi// TSA, no CYP314 was recovered among 42 P450 sequences (Ventura et al., 2017). Although these authors claimed to have identified a P450 of the CYP4 clan with 20-hydroxylase function (which can in fact be shown to be a CYP3213 of the CYP2 clan), their functional expression lacks proper controls and does not support their identification. The apparent lack of CYP314 from shrimp is both remarkable and ironic, as 20-hydroxylated molting hormones are found in Panuliridae, dating back to the original isolation of crustecdysone (20-hydroxyecdysone) from //Jasus lalandei// (Hampshire and Horn 1966). Also, 20-hydroxylation of ecdysone was shown in //Panulirus argus// (James and Shiverick, 1984). The identity of the 20-hydroxylase of shrimp thus remains obscure. 

The CYP314 gene is also missing from the genome of eight ants of the Myrmicinae subfamily, notably //Atta// (3 species), //Trachymyrmex// (3 species), //Cyphomyrmex costatus// and //Acromyrmex echiniator// although it is found in all ants and 23 other species of Myrmicinae. The ants lacking CYP314 are all fungus farming ants of the Attini tribe which evolved around 50 MYA (Li et al., 2018). The lack of CYP314 is not related to the presence or absence of //Pseudonocardia// actinobacterial symbiosis. Other ecdysteroidogenic P450s are present in Attini. 

Fungus farming has also evolved in some termites, but the genome of //Macrotermes natalensis// has a CYP314 gene. 

In aphids, we found two CYP314 genes in //[[Myzus_persicae|Myzus persicae]]// and three in //Acyrthosiphon pisum// .

====== CYP428A1 ======

CYP428A1 in Lepidoptera is an unusual member of the Mito clan, but predicted to have a mitochondrial presequence and to be destined to the inner mitochondrial membrane. Its sequence is highly conserved, but very distinctive from other mito clan P450s. 

The consensus sequence around the Cys pocket motif has a three codon deletion, ASMPFG **- - -** x x CPxxG when compared with the consensus ASLPFGFGPRMCIGRR. Also, the CYP428A1 sequences have just one of the three positively charged residues known to favor binding of the adrenodoxin electron donor and have an unusual I helix, so they may use a different electron donor or may be independent of an external source of reducing equivalents (e.g. if acting on an endo-hydroperoxide). 

CYP428A1 is not found in Trichoptera or in non-Ditrysian Lepidoptera, although genomic data in those groups are scant.