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

Abstract

The denitrifying betaproteobacterium 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, the cholest-4-en-3-one intermediate is oxygen-independently oxidized to Δ-dafachronic acid (C-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-Δ-cholenoyl-CoA (C-oic acid)- and pregn-4-en-3-one-22-oyl-CoA (C-oic acid)-forming ACSs, respectively. A third heterologously produced ATP-dependent ACS was inactive with C-, C-, or C-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 C hydroxylation forward. 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 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 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.

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D 3 Synthesis

“…Further degradation to androsta-1,4-diene-3,17-dione (ADD) proceeds via oxidation and activation to a C-26-oyl-coenzyme A (CoA) component, followed by modified β-oxidation like reaction sequences ( Fig. 1 ) ( 8 , 13 ). Finally, cleavage of the steran rings A and B proceeds in the so-called 2,3- seco -pathway involving ring-cleaving hydrolases ( 14 , 15 ); degradation of rings C and D appears to be similar in aerobic and anaerobic steroid-degrading bacteria, again using hydrolytic enzymes ( Fig.…”

The 7α‐hydroxysteroid dehydratase Hsh2 is essential for anaerobic degradation of the steroid skeleton of 7α‐hydroxyl bile salts in the novel denitrifying bacterium Azoarcus sp. strain Aa7

“…strain Aa7, that is able to grow with bile salts as sole carbon source under anoxic conditions with nitrate as electron acceptor. There are only a few studies on the bacterial degradation of steroid compounds under denitrifying conditions, which mainly focused on the metabolism of cholesterol and testosterone (Chiang et al ., ; Fahrbach et al ., ; Wang et al ., ; ; Yang et al ., ; Warnke et al ., ; ). To the best of our knowledge, this is the first study showing bacterial degradation of the steroid skeleton of bile salts under denitrifying conditions.…”

Microbial Functional Responses to Cholesterol Catabolism in Denitrifying Sludge

“…The transformation of 25-hydroxycholest-4-en-3-one to 26-hydroxycholest-4-en-3-one appeared to be the most highly rate-limiting step in the overall pathway, accumulating 25-hydroxycholest-4-en-3-one in Stl. denitrificans cultures ( 18 ). We did not detect this particular 25-hydroxysteroid intermediate in SCN samples; instead, homologues of the C25DH molybdopterin-containing subunits (S25dA1–A7) were identified, suggesting the occurrence of anaerobic 25-hydroxylation in the SCN samples.…”

“…Subsequently, the activated side chain is degraded through three cycles of β-oxidation reactions to produce the androgen metabolite androst-4-en-3,17-dione (AD) ( 12 ). Recently, the coenzyme A (CoA)-ester metabolites and genes involved in this anaerobic side chain degradation have been identified ( 14 ), and the substrate specificity of acyl-CoA synthetase required for side chain activation prior to each β-oxidation has also been functionally characterized ( 18 ). Most metabolites used in the cholesterol degradation possess corresponding 1,4-diene structures ( 12 , 14 ), which are transformed by a 3-ketosteroid Δ 1 -dehydrogenase AcmB ( 15 ).…”