Structure of X5P-1 [(2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid] (a) and structure of X5P-2 {9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid [IUPAC name (3aS,4S,7aS)-7a-methyl-1,5-dioxooctahydroindene-4-propanoic acid]} (b).

Degradation pathway of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid in steroid degradation by Comamonas testosteroni TA441 with structures of DNPH derivatives identified in this study (A, B, C, P1, and P2). Using P1 and P2, presented as authentic, 2-hydroxy-4-hex-2-enolactone was identified as the product of TesE from the reaction solution without treatment with DNPH. Isolation of 2-hydroxy-4-hex-2-enolactone indicates that the direct product of the TesE reaction is 4-hydroxy-2-oxohexanoic acid, which was considered to be converted to 2-hydroxy-4-hex-2-enolactone under the acidic conditions of the isolation procedure. Reactions of TesG and TesF and compounds in brackets are speculation. CoA, coenzyme A.

Three-dimensional HPLC analysis of the culture of TesD-disrupted mutants incubated with ADD (a), 4,9-DSHA (b), and purified 4,9-DSHA treated with TesD (c). The vertical axis indicates wavelength (nanometers), the horizontal axis indicates retention time (minutes), and the UV absorbance of each compound is represented in contour. For the analysis, a Waters 2690 HPLC was used with an Inertsil ODS-3 column (2.1 by 250 mm; GL Science), and elution was carried out using a linear gradient from 20% solution A (CH₃CN-CH₃OH-TFA [95:5:0.05]) and 80% solution B (H₂O-CH₃OH-TFA [95:5:0.05]) to 65% solution A and 35% solution B over 10 min and then maintained for 3 min and changed to 20% solution A for 5 min. Abbreviations are as follows: Ts, testosterone; 4-AD, 4-androstene-3,17-dione; 17OH-ADD, 17-hydroxy-1,4-androstadiene-3-one; 4,9-DSHA-N, 4-aza-9,17-dioxo-9,10-secoandrosta-1,3,5(10)-triene-3-carboxylic acid.

Three-dimensional HPLC analysis of purified 4,9-DSHA (a); purified 4, 9-DSHA treated with TesD (b); purified 2-hydroxyhexa-2,4-dienoic acid (c); purified 2-hydroxyhexa-2,4-dienoic acid right after addition of TesE (d), 30 min after addition of TesE (e), and 60 min after addition of TesE (f); and purified 2-hydroxyhexa-2,4-dienoic acid solution right after addition of TesE followed by treatment with DNPH (g), 30 min after addition of TesE followed by treatment with DNPH (h), 60 min after addition of TesE followed by treatment with DNPH (i), and 120 min after addition of TesE followed by treatment with DNPH (j). HPLC conditions and abbreviations are the same as those described in the legend of Fig. 1 for a to f and the same as those described in the legend of Fig. 1 except for elution (65% solution A and 35% solution B, constant) for g to j.