Study of Anoxic and Oxic Cholesterol Metabolism by Sterolibacterium denitrificans

Abstract

The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1ST . The second enzyme of the proposed pathway, cholest-4-en-3-one-⌬ 1 -dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14 C-labeled products from [ 14 C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans

“…Recently, the initial reactions involved in the anaerobic metabolism of cholesterol and testosterone were reported (19)(20)(21), albeit the ring cleavage details of the anaerobic pathways are yet to be unraveled. In this study, we adopted a 13 C metabolomic approach to investigate the anaerobic degradation of testosterone using S. denitrifi cans as a model organism.…”

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

“…In a previous investigation, AcmA was identified as an NAD ϩ -dependent enzyme belonging to the short-chain dehydrogenase/reductase family (31). Here, we showed that AcmA is a periplasmic protein.…”

“…The existence of free NAD(P) ϩ in the periplasm seems unlikely for various reasons (53), although there is no direct experimental evidence regarding this matter. The primary structure of AcmA is highly similar to that of 3␤-hydroxysteroid dehydrogenase isolated from various bacteria, including Comamonas testosteroni (31). The 3␤-hydroxysteroid dehydrogenase of C. testosteroni (formerly named Pseudomonas testosteroni (54)) was also reported to be a periplasmic protein (55).…”

“…In this study, we applied a 13 C metabolomic approach to demonstrate that the denitrifying S. denitrificans adopts a highly similar strategy to degrade cholesterol. The highly similar protein patterns of the anaerobically and aerobically grown cells (31) suggest that S. denitrificans may use the same catabolic enzymes to degrade cholesterol regardless of oxygen availability. It is worth mentioning that the denitrifying S. denitrificans cells produce mainly 17-hydroxyl catabolic intermediates (Fig.…”

“…Sterolibacterium denitrificans DSMZ 13999 can degrade cholesterol under both oxic and anoxic conditions (30,31). Recently, we examined the cholesterol catabolic pathway in aerobically grown S. denitrificans (10).…”