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.

Keywords: 9α-Hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid; Bacterial steroid degradation; Cholic acid; Comamonas testosteroni.