Comparative Analysis of Bile-Salt Degradation in Sphingobium sp. Strain Chol11 and Pseudomonas stutzeri Strain Chol1 Reveals Functional Diversity of Proteobacterial Steroid Degradation Enzymes and Suggests a Novel Pathway for Side Chain Degradation.

The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ4,6-variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations of strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase (ACAD) Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ22-ACAD Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts, while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δscd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshABChol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ4,6-steroids, while KshABChol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228, led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of proteobacterial enzymes for bile-salt degradation and suggest a novel side chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with nonnative substrates.

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 ß-oxidation, where aldolases, aldehyde dehydrogenases, and additional ACSs substitute for classical ß-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 3ß-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)-7aß-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.

Degradation of Bile Acids by Soil and Water Bacteria.

Bile acids are surface-active steroid compounds with a C5 carboxylic side chain at the steroid nucleus. They are produced by vertebrates, mainly functioning as emulsifiers for lipophilic nutrients, as signaling compounds, and as an antimicrobial barrier in the duodenum. Upon excretion into soil and water, bile acids serve as carbon- and energy-rich growth substrates for diverse heterotrophic bacteria. Metabolic pathways for the degradation of bile acids are predominantly studied in individual strains of the genera Pseudomonas, Comamonas, Sphingobium, Azoarcus, and Rhodococcus. Bile acid degradation is initiated by oxidative reactions of the steroid skeleton at ring A and degradation of the carboxylic side chain before the steroid nucleus is broken down into central metabolic intermediates for biomass and energy production. This review summarizes the current biochemical and genetic knowledge on aerobic and anaerobic degradation of bile acids by soil and water bacteria. In addition, ecological and applied aspects are addressed, including resistance mechanisms against the toxic effects of bile acids.

Investigations on the Degradation of the Bile Salt Cholate via the 9,10-Seco-Pathway Reveals the Formation of a Novel Recalcitrant Steroid Compound by a Side Reaction in Sphingobium sp. Strain Chol11.

Bile salts such as cholate are steroid compounds from the digestive tracts of vertebrates, which enter the environment upon excretion, e.g., in manure. Environmental bacteria degrade bile salts aerobically via two pathway variants involving intermediates with Δ1,4- or Δ4,6-3-keto-structures of the steroid skeleton. Recent studies indicated that degradation of bile salts via Δ4,6-3-keto intermediates in Sphingobium sp. strain Chol11 proceeds via 9,10-seco cleavage of the steroid skeleton. For further elucidation, the presumptive product of this cleavage, 3,12ß-dihydroxy-9,10-seco-androsta-1,3,5(10),6-tetraene-9,17-dione (DHSATD), was provided to strain Chol11 in a co-culture approach with Pseudomonas stutzeri Chol1 and as purified substrate. Strain Chol11 converted DHSATD to the so far unknown compound 4-methyl-3-deoxy-1,9,12-trihydroxyestra-1,3,5(10)7-tetraene-6,17-dione (MDTETD), presumably in a side reaction involving an unusual ring closure. MDTETD was neither degraded by strains Chol1 and Chol11 nor in enrichment cultures. Functional transcriptome profiling of zebrafish embryos after exposure to MDTETD identified a significant overrepresentation of genes linked to hormone responses. In both pathway variants, steroid degradation intermediates transiently accumulate in supernatants of laboratory cultures. Soil slurry experiments indicated that bacteria using both pathway variants were active and also released their respective intermediates into the environment. This instance could enable the formation of recalcitrant steroid metabolites by interspecies cross-feeding in agricultural soils.