Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.Several species of bacteria, including Nocardia restrictus and Comamonas testosteroni (formerly Pseudomonas testosteroni), are known for the ability to utilize testosterone and various other steroids as sole carbon and energy sources. The mechanism by which testosterone is degraded in N. restrictus and C. testosteroni was eagerly studied, and the main intermediate compounds in the degradation pathway of these bacteria, especially N. restrictus, were determined in 1960s (2-6, 15-18). We simultaneously identified the genes and the intermediate compounds that are accumulated by gene-disrupted mutants and revealed the testosterone degradation pathway and degradation genes in Comamonas testosteroni TA441 (9-13). In TA441, the A ring of testosterone is aromatized and metacleaved, and the resultant 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid (4,9-DSHA) is divided into two compounds through hydrolysis by TesD. The procedure after aromatization of the A ring is similar to that of the bacterial degradation of aromatic compounds such as biphenyl, and TesD shows about 40% identity with BphD, a hydrolase in biphenyl degradation. Assuming that the reaction of TesD is similar to that of BphD, the products of TesD were predicted to be 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 2-hydroxyhexa-2,4-dienoic acid, although we could not isolate and identify the products in the previous study. In this study, we isolated and identified the products of TesD in testosterone degradation with complete mass spectrometry (MS) and nuclear magnetic resonance (NMR) data. We also identified the substrate and the product of hydratase TesE, which is encoded just downstream of TesD.
MATERIALS AND METHODSPreparation of 4,9-DSHA. The tesD-disrupted mutant was incubated in 20 ml of LB medium for about 15 h. The culture was inoculated into 500 ml of one-half LB medium plus one-half C medium with 0.1% 1,4-androstadiene-3,17-dione (ADD) and incubated at 30°C for about 15 h until the medium showed an intense yellow color. C medium is a mineral medium for TA441 to grow with steroid compounds (13). The culture was centrifuged, and the supernatant was extracted twice with the same volume of ethyl acetate. The aqueous layer was then acidified with 6 M HCl and extracted twice with the same volume of ethyl acetate. The ethyl acetate...