Metabolites Involved in Aerobic Degradation of the A and B Rings of Estrogen

Abstract

Various bacteria, mainly actinobacteria and proteobacteria, are capable of aerobic estrogen degradation. In a previous study, we used the obligate aerobic alphaproteobacterium Sphingomonas sp. strain KC8 as a model microorganism to identify the initial metabolites involved in the oxygenolytic cleavage of the estrogen A ring: 4-hydroxyestrone, a meta-cleavage product, and a dead-end product pyridinestrone acid. In this study, we identified the downstream metabolites of this aerobic degradation pathway using ultraperformance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS). 4-Norestrogen-5(10)-en-3-oyl-coenzyme A and its closely related deconjugated (non-coenzyme A [non-CoA]) structure, 4-norestrogenic acid, were detected in the estrone-grown strain KC8 cultures. The structure of 4-norestrogenic acid was elucidated using nuclear magnetic resonance (NMR) spectroscopy. The extracellular distribution and the accumulation of 4-norestrogenic acid in the bacterial cultures indicate that the estrogen-degrading bacteria cannot degrade this deconjugated product. We also observed temporal accumulation and subsequent consumption of a common steroid metabolite, 3aα-H-4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), in the bacterial cultures. The metabolite profile and genomic analyses shed light on the biochemical mechanisms involved in the degradation of the A and B rings of natural estrogens. In this proposed aerobic pathway, C-4 of the meta-cleavage product is removed by a 2-oxoacid oxidoreductase through oxidative decarboxylation to produce the 4-norestrogen-5(10)-en-3-oyl-CoA. Subsequently, the B ring is cleaved by hydrolysis. The resulting A/B-ring-cleaved product is transformed into a common steroid metabolite HIP through β-oxidation reactions. Accordingly, the A and B rings of different steroids are degraded through at least three peripheral pathways, which converge at HIP, and HIP is then degraded through a common central pathway. IMPORTANCE Estrogens, often detected in surface waters worldwide, have been classified as endocrine disrupting chemicals and carcinogens. Bacterial degradation is crucial for removing natural estrogens from natural and engineered ecosystems; however, current knowledge regarding the biochemical mechanisms and catabolic enzymes involved in estrogen biodegradation is very limited. Our estrogen metabolite profile and genomic analyses on estrone-degrading bacteria enabled us to characterize the aerobic estrogen degradation pathway. The results greatly expand our understanding of microbial steroid degradation. In addition, the characteristic metabolites, dead-end products, and degradation genes can be used as biomarkers to investigate the fate and biodegradation potential of estrogens in the environment.

Microbial degradation of steroid sex hormones: implications for environmental and ecological studies

“…Subsequently, the removal of C‐1 and C‐10 through aldolytic cleavage results in the production of HIP. Except for 4‐norestrogen‐5(10)‐en‐3‐oyl‐CoA, no other CoA esters proposed in this aerobic pathway have been detected; however, at least five deconjugated (non‐CoA) metabolites corresponding to these hypothetical CoA esters have been detected in the bacterial cultures (Wu et al , ). The dead‐end‐products pyridinestrone acid and 4‐norestrogenic acid are less biodegradable and tend to accumulate in bacterial cultures (Wu et al , ) or environmental samples (Chen et al , , ); thus, these compounds may serve as biomarkers for investigating environmental aerobic oestrogen biodegradation.…”

Suitability of Immobilized Systems for Microbiological Degradation of Endocrine Disrupting Compounds

“…The oecC cluster, which is responsible for the induction of active 4,5-dioxygenase, probably plays an important role in the catabolism of estrogens [ 25 ]. Wu et al [ 50 ] noted that a meta cleavage product is converted by 2-oxoacid oxidoreductase to 4-norestrogen-(10)-en-3-oyl-CoA. Further transformations include sequences of ester intermediate reactions leading to hydrolytic cleavage of the B ring.…”

“…Hydrolysis by 2-hydroxycyclohexanecarboxyl-CoA dehydrogenase is facilitated by the deficiency of electrons on the carbonyl carbon of the B ring. The end product of the pathway is 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (HIP) [ 50 ]. Similarly, 17β-estradiol degradation is carried out by the Acinetobacter strain DSSKY-A-001.…”

“…The obtained product is further oxidized to ( Z )-8-(7α-methyl-1n-5-dioxo-octahydro-1 H -inden-4-yl)-2o-6-dioxy-4-butenoic acid with the participation of the B ring cleaving oxygenase. In addition, hydroxylation at position 4 of the 17β-estradiol ring and conversion of 4-hydroxy-17β-estradiol to 4-hydroxyestrone were also observed [ 10 , 50 ].…”

“… Estrogens biodegradation by bacteria; the 4,5-seco pathway reactions are shown in blue; HIP–9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid [ 10 , 12 , 21 , 25 , 50 ]. …”