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

ABSTRACTVarious bacteria, mainly actinobacteria and proteobacteria, are capable of aerobic estrogen degradation. In a previous study, we used the obligate aerobic alphaproteobacteriumSphingomonassp. strain KC8 as a model microorganism to identify the initial metabolites involved in the oxygenolytic cleavage of the estrogen A ring: 4-hydroxyestrone, ameta-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.IMPORTANCEEstrogens, 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.