title: C-26 vs. C-27 hydroxylation of insect steroid hormones: regioselectivity of a microsomal cytochrome P450 from a hormone-resistant cell line.
abstract: Hydroxylation of steroids at one of the side chain terminal methyl groups, commonly linked to C-26, represents an important regulatory step established in many phyla. Discrimination between the two sites, C-26 and C-27, requires knowing the stereochemistry of the products. 26-Hydroxylation of the insect steroid hormone 20-hydroxyecdysone by a microsomal cytochrome P450 was previously found to be responsible for hormonal resistance in a Chironomus cell line mainly producing the (25S)-epimer of 20,26-dihydroxyecdysone. Here, we studied the 25-desoxy analog of 20-hydroxyecdysone, ponasterone A, to elucidate the stereochemistry of the expected 26-hydroxy product, inokosterone, which occurs as C-25 epimers in nature. We identified the predominant metabolite as the C-25 R epimer of inokosterone on comparison by RP-HPLC with the (25R)- and (25S)-epimers the stereochemistry of which was confirmed by X-ray crystallography. (25R)-inokosterone was further oxidized to the 26-aldehyde identified by mass spectroscopy, borohydride reduction and metabolic transformation to 26-carboxylic acid. The (25S)-epimers of inokosterone and its aldehyde were minor products. With 20-hydroxyecdysone as substrate, we newly identified the (25R)-epimer of 20,26-dihydroxyecdysone as a minor product. In conclusion, the present stereochemical studies revealed high regioselectivity of the Chironomus enzyme to hydroxylate both steroids at the same methyl group, denoted C-27.
authors.affiliation: Institute of General Zoology and Endocrinology, University of Ulm, Germany. hartmut.kayser@uni-ulm.de
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kayser
authors.forename: Philipp
authors.initials: P
authors.lastname: Eilinger
authors.forename: Philippe
authors.initials: P
authors.lastname: Piechon
authors.forename: Trixie
authors.initials: T
authors.lastname: Wagner
mesh_terms: D000818:Animals; D002460:Cell Line; D002683:Chironomidae / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D026461:Ecdysteroids / Q000737:chemistry* / Q000378:metabolism; D006900:Hydroxylation; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology*
chemical_list: D026461:Ecdysteroids; D019476:Insect Proteins; D003577:Cytochrome P-450 Enzyme System