Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation in Sterolibacterium denitrificans Chol1S.

The denitrifying betaproteobacterium Sterolibacterium denitrificans Chol1S catabolizes steroids such as cholesterol via an oxygen-independent pathway. It involves enzyme reaction sequences described for aerobic cholesterol and bile acid degradation as well as enzymes uniquely found in anaerobic steroid-degrading bacteria. Recent studies provided evidence that in S. denitrificans, the cholest-4-en-3-one intermediate is oxygen-independently oxidized to Δ4-dafachronic acid (C26-oic acid), which is subsequently activated by a substrate-specific acyl-coenzyme A (acyl-CoA) synthetase (ACS). Further degradation was suggested to proceed via unconventional β-oxidation, where aldolases, aldehyde dehydrogenases, and additional ACSs substitute for classical β-hydroxyacyl-CoA dehydrogenases and thiolases. Here, we heterologously expressed three cholesterol-induced genes that putatively code for AMP-forming ACSs and characterized two of the products as specific 3β-hydroxy-Δ5-cholenoyl-CoA (C24-oic acid)- and pregn-4-en-3-one-22-oyl-CoA (C22-oic acid)-forming ACSs, respectively. A third heterologously produced ATP-dependent ACS was inactive with C26-, C24-, or C22-oic-acids but activated 3aα-H-4α-(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) to HIP-CoA, a rather late intermediate of aerobic cholesterol degradation that still contains the CD rings of the sterane skeleton. This work provides experimental evidence that anaerobic steroid degradation proceeds via numerous alternate CoA-ester-dependent or -independent enzymatic reaction sequences as a result of aldolytic side chain and hydrolytic sterane ring C-C bond cleavages. The aldolytic side chain degradation pathway comprising highly exergonic ACSs and aldehyde dehydrogenases is considered to be essential for driving the unfavorable oxygen-independent C26 hydroxylation forward.IMPORTANCE The biological degradation of ubiquitously abundant steroids is hampered by their low solubility and the presence of two quaternary carbon atoms. The degradation of cholesterol by aerobic Actinobacteria has been studied in detail for more than 30 years and involves a number of oxygenase-dependent reactions. In contrast, much less is known about the oxygen-independent degradation of steroids in denitrifying bacteria. In the cholesterol-degrading anaerobic model organism Sterolibacterium denitrificans Chol1S, initial evidence has been obtained that steroid degradation proceeds via numerous alternate coenzyme A (CoA)-ester-dependent/independent reaction sequences. Here, we describe the heterologous expression of three highly specific and characteristic acyl-CoA synthetases, two of which play key roles in the degradation of the side chain, whereas a third one is specifically involved in the B ring degradation. The results obtained shed light into oxygen-independent steroid degradation comprising more than 40 enzymatic reactions.