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====== C-22 hydroxylation and CYP302 ======

====Biochemistry====

C-22 hydroxylation occurs with retention of configuration in the ovaries of //Schistocerca gregaria// (Greenwood and Rees, 1982): The 22-pro-//R// and 22-pro-//S// hydrogen atoms of cholesterol are stereospecifically eliminated and retained respectively during ecdysteroid formation. This indicates that C-22 hydroxylation in ecdysone biosynthesis is direct as expected from a P450-catalyzed reaction. The C-22 hydroxylation of 2,22-dideoxyecdysone in prothoracic glands of //Locusta migratoria// was shown to be catalyzed by a mitochondrial P450 enzyme (Kappler et al. 1988). It had a Km of 1.5 µM for 2,22-dideoxyecdysone, and was inhibited competitively by 2-deoxyecdysone and by 2,22,25-trideoxyecdysone.

====CYP302====

CYP302A1, a mitochondrial CYP clan P450 was identified as a C-22 hydroxylase, encoded in Drosophila by the "Halloween" gene called disembodied (dib) (Chavez et al., 2000, Warren et al., 2002). CYP302A1 is expressed in embryos, as well as in the larval ring gland and follicle cells of adult ovaries. Transient expression in Drosophila S2 cells coupled with the use of radiolabeled 2,22-dideoxyecdysone was used to identify the reaction (Warren et al., 2002). The CYP302A1 gene was rapidly induced by prothoracicotropic hormone (PTTH) in vitro (Niwa et al., 2005). 


====CYP302 distribution and evolution====

CYP302A1 is generally a single copy gene in all arthropods, with some exceptions as noted below:

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 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, there are 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.
Transcripts for CYP302A1 are 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 CYP302A1 is found 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.

====CYP302A1 specificity====

Information on the specificity of CYP302A1 is scant. The conversion of 2,22-dideoxyecdysone to 2-deoxyecdysone was shown to occur with heterologously expressed CYP302A1 from //Drosophila melanogaster// (Warren et al., 2002), //Bombyx mori// (Niwa et al., 2005), //Manduca sexta// (Rewitz et al., 2006), //Anopheles gambiae// (Pondeville et al., 2008) and //Mamestra brassicae// (Ogihara et al., 2017). Interestingly, the //Nilaparvata lugens// CYP302A1 was unable to convert 2,22-dideoxyecdysone to 2-deoxyecdysone. RNAi has no developmental effect in nymphs, although RNAi in adult females severely affected egg production and embryonic development (Zhou et al., 2020).  

The Drosophila CYP302A1 did not metabolize 22-deoxyecdysone (Warren et al., 2002). 

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