Initial Steps in the Anoxic Metabolism of Cholesterol by the Denitrifying Sterolibacterium denitrificans

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.

Study of Anoxic and Oxic Cholesterol Metabolism by Sterolibacterium denitrificans

“…Large-scale cultivation was performed in a 200-liter fermentor. The medium used for anaerobic cultivation of S. denitrificans has been described previously (6). Cells were harvested in the exponential growth phase at an optical density at 578 nm (OD 578 ) of 1.5 (optical path length, 1 cm).…”

Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

“…toluene, are anaerobically activated by glycine radical enzymes, and the radical intermediates add to fumarate, yielding methylalkylsuccinate and benzylsuccinate, respectively (13)(14)(15). Molybdenum-containing enzymes anaerobically hydroxylate ethylbenzene (16) and cholesterol (17).…”

Substrate Uptake and Subcellular Compartmentation of Anoxic Cholesterol Catabolism in Sterolibacterium denitrificans

“…25-Hydroxycholest-4-en-3-one, 1-testosterone, androst-1-en-3,17-dione, 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (2, 3-SAOA), and 1,17-dioxo-2,3-seco-androstan-3-oic acid were produced according to previously determined methods (10,32,33). 26-Hydroxycholest-4-en-3-one (also named 27-hydroxy-4-cholesten-3-one, 99%) and cholest-4-en-3-one-26-oic acid (also named 3-oxo-4-cholestenoic acid, 99%) were obtained from Avanti Polar Lipids, and cholest-4-en-3-one-24-oic acid (also named 4-cholenic acid-3-one) and pregn-4-en-3-one-20-carboxylic acid (also named 4-pregnen-3-one-20␤-carboxylic acid) were purchased from Steraloids.…”

“…A denitrifying S. denitrificans culture (500 ml) was grown with 2 mM unlabeled cholesterol in a procedure described in a previous report (33). The residual cholesterol in the culture was quantified using the o-phthalaldehyde method (34).…”

“…Sucrose Gradient Separation of S. denitrificans Membranes-S. denitrificans membrane proteins were separated from soluble proteins as described previously (33). To separate the cytoplasmic membrane from the OM, sucrose gradient centrifugation was performed according to a previously described procedure (40) with some modifications.…”