title: Identification of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid and β-oxidation products of the C-17 side chain in cholic acid degradation by Comamonas testosteroni TA441.
abstract: Comamonas testosteroni degrades testosterone into 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 2-hydroxyhexa-2,4-dienoic acid via aromatization of the A-ring. The former compound is suggested to be degraded further by β-oxidation, but the details of the process remain unclear. In this study, we identified 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid as an intermediate compound in the β-oxidation of this compound. ORF32, located in one of the two main steroid degradation gene clusters, was shown to be indispensable for the conversion of this compound. A homology search indicated that ORF32 encodes a hydratase for the CoA-ester, suggesting that ORF32 encodes a hydratase that adds a water molecule to a double bond at C-6 of the CoA-ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. From the culture of an ORF32-disrupted mutant incubated with cholic acid for a short period (around two days, when a considerable number of intermediate compounds were detected by HPLC), 7α,12α-dihydroxy-3-oxochola-1,4-dien-24-oic acid, 7α,12α-dihydroxy-3-oxochol-4-en-24-oic acid, 12α-hydroxy-3-oxochola-4,6-dien-24-oic acid, 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid, 12α-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid, 7α,12α-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid, 12α-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid, 7α-hydroxy-3-oxopregna-4,17(20)-diene-20-carboxylic acid, and 3-oxopregna-4,6,17(20)-triene-20-carboxylic acid were isolated as intermediate compounds of C-17 side-chain degradation. The presence of these compounds implies that the process of degradation of the C-17 side chain in C. testosteroni will be similar to the process in Pseudomonas. The final two compounds, which have a double bond at the C-17(20) position, are here identified for the first time, to the best of our knowledge, as intermediate compounds in bacterial steroid degradation; their composition suggests that the remaining three carbons at the C-17 position would be removed oxidatively as a propionic acid derivative.
authors.affiliation: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Electronic address: masae@postman.riken.go.jp.
authors.forename: Masae
authors.initials: M
authors.lastname: Horinouchi
authors.affiliation: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Koshino
authors.affiliation: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Michal
authors.initials: M
authors.lastname: Malon
authors.affiliation: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hirota
authors.affiliation: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Kudo
mesh_terms: D019826:Cholic Acid / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D020604:Comamonas testosteroni / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics; D015394:Molecular Structure; D005810:Multigene Family; D009635:Norandrostanes / Q000032:analysis* / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics*; D010084:Oxidation-Reduction
chemical_list: D009635:Norandrostanes; D006913:Hydroxysteroid Dehydrogenases; D019826:Cholic Acid
keywords: 9α-Hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid; Bacterial steroid degradation; Cholic acid; Comamonas testosteroni