Channeling c1 metabolism toward s-adenosylmethionine-dependent conversion of estrogens to androgens in estrogen-degrading bacteria

Abstract

Bacterial degradation of endocrine disrupting and carcinogenic estrogens is essential for their elimination from the environment. Recent studies of the denitrifying, estrogen-degrading Denitratisoma strain DHT3 revealed the conversion of estrogens to androgens by a putative cobalamin-dependent methyl-transferase encoded by the emtABCD genes. The methyl donor and its continu-ous regeneration to initiate estradiol catabolism have remained unknown. Here, large-scale cultivation of the denitrifying bacterium Denitratisoma oestradiolicum with estrogen provided the biomass required for quantitative biochemical analy-ses. Soluble fractions of extracts from estradiol-grown cells catalyzed the S-adenosyl-L-methionine (SAM)-and Ti(III)-citrate-dependent conversion of 17β-estradiol/es-trone to the respective androgens at 0.15 nmol min-1 mg-1. Kinetic studies of 17β-estradiol methylation and reverse 1-dehydrotestosterone demethylation reactions indicated that the exergonic methyl transfer from SAM to the putative cobalamin drives the endergonic methyl transfer from the methylcobalamin in-termediate to the phenolic ring A. Based on a high-quality circular genome from D. oestradiolicum, proteogenomic analyses identified a 17β-estradiol-induced gene cluster comprising emtABCD genes together with genes involved in SAM regeneration viaL-serine andL-methionine. Consistent with this finding,L-methi-onine/ATP orL-serine/ATP/tetrahydrofolate/L-homocysteine substituted for SAM as methyl donors, further confirmed by the incorporation of the13 C-methyl-group from13 C-L-methonine into methyl(III)cobalamine and the estrone methyl-ation product androsta-1,4-diene-3-one. This work demonstrates that during bacterial estrogen catabolism, the C1 pool is channeled toward the initiating methyl transfer to ring A. The effective cellular SAM regeneration system may serve as a model for whole-cell SAM-dependent methylation reactions of biotechnological interest. IMPORTANCE Estrogens comprise a group of related hormones occurring in pre-dominantly female vertebrates, with endocrine disrupting and carcinogenic poten-tial. Microbial biodegradation of estrogens is essential for their elimination from surface waters and wastewater. Aerobic bacteria employ oxygenases for the initial cleavage of the aromatic ring A. In contrast, anaerobic degradation of estrogens is initiated by methyl transfer-dependent conversion into androgens involving a putative cobalamin-dependent methyltransferase system. The methyl donor for this un-precedented reaction and its stoichiometric regeneration have remained unknown. With the biomass obtained from large-scale fermentation of an estrogen-degrading denitrifying bacterium, we identified S-adenosyl-methionine (SAM) as the methyl donor for the cobalamin-mediated methyl transfer to estrogens. To continuously sup-ply C1 units to initiate estrogen degradation, genes for SAM regeneration from estradiol-derived catabolites are highly upregulated. Data presented here shed light into biochemical processes involved in the globally important microbial degradation of estrogens.