The conversion of ecdysone to 20-hydroxyecdysone does not occur in the prothoracic glands of insects but occurs in many peripheral tissues, such as the fat body, midgut, and Malpighian tubules. Similarly, in Crustacea the reaction occcurs in peripheral tissues rather then in the ecdysone producing Y organs. The P450 nature of the enzyme catalyzing the 20-hydroxylation of ecdysone has been well established (Smith, 1985) following the initial reports of 1977 (Bollenbacher et al., 1977, Feyereisen, 1977, Johnson and Rees, 1977). An NADPH and O₂-dependent enzyme system, inhibited by typical pharmacological P450 inhibitors, was studied in several insect species. The evidence for P450 involvement was strengthened by the light-sensitive carbon monoxide inhibition observed in some of the most thorough studies (Feyereisen and Durst, 1978, Smith et al., 1979, Greenwood and Rees, 1984). Several studies have shown that the 20-hydroxylation reaction is competitively inhibited by the product, 20E.

The agreement on the P450 nature of the reaction was accompanied by a lack of consensus on the subcellular localization of E20MO. Some studies showed a microsomal activity, other studies showed a mitochondrial activity, and yet other studies indicated the presence of both microsomal and mitochondrial activities in the same tissue (Smith, 1985; Lafont et al. 2005). For instance, it has been reported to be mosty microsomal in imaginal discs of Pieris brassicae (Blais and Lafont, 1986) and in Gryllus bimaculatus midgut (Liebrich and Hoffmann, 1991). In the midgut of Diploptera punctata (Halliday et al., 1986) and in embryos of Bombyx mori (Horike and Sonobe, 1999), the activity is essentially microsomal, and can be inhibited by antibodies to insect P450 reductases. This is clear evidence that the enzyme derives its reducing equivalents from the usual microsomal redox partner in those cases. In Spodoptera littoralis fat body, E20MO activity is predominantly mitochondrial, with a small amount of microsomal activity (Hoggard and Rees, 1988).In whole body homogenates of third-instar Drosophila, and in the midgut of larval Spodoptera frugiperda the E20MO activity is distributed 1:3 between mitochondrial and microsomal fractions (Smith, 1985, Yu, 1995), and it is also distributed in both fractions in larvae of the house fly, and of the flesh fly Neobellieria bullata (Darvas et al., 1993). Weirich et al. (1996) compared the apparent Km, and specific activities of the mitochondrial and microsomal E20MO activities of Manduca sexta larval midgut. They concluded that at physiological ecdysone titers, despite a higher specific activity, the mitochondrial E20MO would contribute less than one-eigth the activity of the microsomal form.

The existence of several genes encoding P450s with E20MO activity could account for the dual localization and complex regulation of the enzyme's activity. On the other hand, post-translational modifications of a single gene product could also lead to the microsomal and mitochondrial forms, and this seems possible given the unusual N-terminal sequences of CYP314A1 proteins (see below). There is currently no evidence of alternative splicing of CYP314A1. Interestingly, both microsomal and mitochondrial activities of the Spodoptera littoralis fat body E20MO were reported to be reversibly activated by phosphorylation (Hoggard and Rees, 1988, Hoggard et al., 1989). Cases of post-translational modifications of P450 enzymes by reversible phosphorylation are not very common, and this observation would suggest that the S. littoralis fat body E20MOs are products of the same gene.

Petryk et al. (2003) identified Drosophila CYP314A1 as the product of the shade (shd) gene. Expression of CYP314A1 in Drosophila S2 cells enabled the NADPH-dependent hydroxylation of ecdysone to 20-hydroxyecdysone by cell homogenates. Embryonic lethality of shd mutants indicates that CYP314A1 encodes the only significant E20MO activity in Drosophila at that stage. A CYP314A1 protein modified at the C-terminus by the addition of three copies of the hemaglutinin epitope was targeted to mitochondria of S2 cells (Petryk et al., 2003).

The sequences of the predicted CYP314A1 proteins are unusual. They are clearly members of the mitochondrial CYP clan and have several intron positions in common with other mitochondrial P450 genes (Ranson et al., 2002a; Rewitz et al., 2007). However, they have usually just one or two of the three positively charged residues residues thought to confer high affinity to adrenodoxin. Their exact N-terminal sequence is also variable and somewhat unclear in the absence of proteomic data to confirm the annotation prediction of the N-terminus.

Regulation of E20MO activity, developmental changes, induction and inhibition, are discussed by Lafont et al. (2005) and Rewitz et al. (2006).

CYP314A1 is generally found in all arthropods as a single gene, but there are some notable exceptions.

In aphids, CYP314A1 is duplicated. There are two CYP314 genes in Myzus persicae and three in Acyrthosiphon pisum.

CYP314A1 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 CYP314A1 in the genomes of 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 CYP314A1 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 CYP314A1 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 CYP314A1 are all fungus farming ants of the Attini tribe which evolved around 50 MYA (Li et al., 2018). The lack of CYP314A1 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 CYP314A1 gene.

Beyond ecdysone, no other substrate has been reported for CYP314A1 as of now. C-20 hydroxylation of ecdysone was shown for the CYP314A1 enzyme from Manduca sexta (Rewitz et al., 2006), Bombyx mori (Maeda et al., 2008), Leptinotarsa decemlineata (Kong et al., 2014), Mamestra brassicae (Ogihara et al. 2017), and Nilaparvata lugens (Zhou et al. 2020).

Interestingly, 20-hydroxylation of 26-hydroxyecdysone, ecdysonoic acid, and 3-epiecdysone has been observed in Pieris brassicae (Lafont et al., 1980).