Abstract
The anoxic metabolism of the ubiquitous triterpene cholesterol is challenging because of its complex chemical structure, low solubility in water, low number of active functional groups, and the presence of four alicyclic rings and two quaternary carbon atoms. Consequently, the aerobic metabolism depends on oxygenase catalyzed reactions requiring molecular oxygen as co-substrate. Sterolibacterium denitrificans is shown to metabolize cholesterol anoxically via the oxidation of ring A, followed by an oxygen-independent hydroxylation of the terminal C-25 of the side chain. The anaerobic hydroxylation of a tertiary carbon using water as oxygen donor is unprecedented and may be catalyzed by a novel molybdenum containing enzyme.
Highlights
The complete aerobic mineralization of cholesterol by various genera of bacteria, such as Arthrobacter, Corynebacterium, Mycobacterium, Nocardia, Pseudomonas, and Rhodococcus has been studied in some detail [2, 3] (Fig. 2A)
Anoxic metabolism of cholesterol is mechanistically interesting since this triterpene is intrinsically difficult to attack due to its complex chemical structure, low solubility in water, low number of active functional groups, and the presence of four alicyclic rings and two quaternary carbon atoms (Fig. 1)
The large scale assay for the production of intermediates S4 and S5 was performed as follows: 300 ml of soluble proteins extracted from S. denitrificans cells were precipitated at 25% ammonium sulfate saturation at 4 °C
Summary
Materials and Bacterial Strain [4C-14C]Cholesterol was obtained from American Radiolabeled Chemicals Inc./Biotrend Chemikalien GmbH 18O2 (99 atom %) and 18O-labeled water (97 atom %) were obtained from Campro Scientific GmbH (Berlin, Germany). The chemicals used were of analytical grade and were purchased from Sigma-Aldrich (Deisenhofen, Germany), Merck (Darmstadt, Germany), or Roth (Karlsruhe, Germany). S. denitrificans Chol-1ST (ϭ DSMZ13999) [21] was obtained from the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (Braunschweig, Germany)
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