Figure 1.
The aerobic catabolic pathways of cholesterol by bacteria.
The ring identification (A–D) and carbon numbering systems (1–27) of steroids are shown in cholesterol. (A) The classical 9,10-seco-pathway demonstrated in G. cholesterolivorans DSMZ 45229. (B) The alternative 2,3-seco-pathway proposed in this study using S. denitrificans DSMZ 13999 as the model organism. 25-hydroxycholest-4-en-3-one was the last detected intermediate reported in the previous studies [32], [33]. First ring cleavage intermediates appearing in the catabolic pathways are highlighted in boxes. In this study, α,α′-D and tert-butyl alcohol served as the inhibitors for the 9,10-seco-pathway and 2,3-seco-pathway, respectively.
Table 1.
UPLC-HRMS and UV absorption behavior of the intermediates involved in aerobic cholesterol catabolism byGordonia cholesterolivorans DSMZ 45229.
Figure 2.
Aerobic cholesterol catabolic pathway byS denitrificans was inhibited by tert-butyl alcohol but not by α,α′-D.
(A) UPLC-HRMS analysis of ethyl-acetate extracts of cholesterol-grown bacterial cells with or without α,α′-D. (AI) G. cholesterolivorans DSMZ 45229 grown with cholesterol (2 mM), (AII) G. cholesterolivorans grown with cholesterol and α,α′-D (5 mM), (AIII) S. denitrificans DSMZ 13999 grown with cholesterol, and (AIV) S. denitrificans grown with cholesterol and α,α′-D. (B) UPLC-HRMS analysis of ethyl-acetate extracts of cholesterol-grown S. denitrificans cells with different concentrations of tert-butyl alcohol. (BI) The aerobic growth without tert-butyl alcohol, (BII) in the presence of 2.5% (v/v) tert-butyl alcohol, and (BIII) in the presence of 5% tert-butyl alcohol. Abbreviations: TIC, total ion current; 26-OH, 26-hydroxycholest-4-en-3-one; 25-OH, 25-hydroxycholest-4-en-3-one; PCA, pregn-4-en-3-one-20-carboxylic acid; AD, androst-4-en-3,17-dione; ADD, androsta-1,4-diene-3,17-dione; *, unidentified nitrogen compounds.
Figure 3.
Aerobic cholesterol catabolism byS. denitrificans DSMZ 13999.
(A) Time course of cholesterol consumption and intermediate production in a S. denitrificans fed-batch culture. [4C-13C]cholesterol (1 mM) was fed to a starved S. denitrificans culture (OD600nm = 0.9) after 2 mM cholesterol was exhausted. 0.1 mM of estrone was added as the internal control. The culture was incubation at 28°C for 30 hours with shaking (180 rpm). The steroids sampled at different time intervals were extracted with ethyl acetate three times, and the dominant intermediates were then separated and quantified using a reverse-phase HPLC system. Data are averages of three determinations (standard deviations <0.1). (B) High-resolution mass spectra of the dominant 13C-labeled intermediates detected in ethyl-acetate extracts of S. denitrificans cells grown on [4C-13C]cholesterol (1 mM). *The predicted elemental composition of individual intermediates was calculated using MassLynx™ Mass Spectrometry Software (Waters). See Figure S1 for other detected 13C-labeled intermediates derived from [4C-13C]cholesterol.
Figure 4.
The structure elucidation and the investigation of the ring cleavage mechanism of compound 1 (1,17-dioxo-2,3-seco-androstan-3-oic acid, DSAO).
(A) The interpretations of COSY and key HMBC spectra of compound 1. (B) The chemical structure of compound 1. *The oxygen atoms were labeled with 18O in the H218O-incorporation assay. (C) ESI-mass spectra (positive ion mode) of DSAO. (CI) DSAO purified from the anaerobic control assay. (CII) DSAO purified from the 18O2-treated assay. (CIII) DSAO purified from the 18O-labeled H2O-treated assay. For detailed NMR spectral data of compound 1, see Table S2.
Figure 5.
APCI-mass spectra (positive mode) of UPLC-separated 25-hydroxycholest-4-en-3-one (25-OH) and 26-hydroxycholest-4-en-3-one (26-OH).
(A) 25-OH produced in the anoxic control assay. (B) 25-OH produced in the 18O2-treated assay. (C) 25-OH produced in the H218O-treated assay. (D) 26-OH produced in the anoxic control assay. (E) 26-OH produced in the 18O2-treated assay. (F) 26-OH produced in the H218O-treated assay.