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 methyltransferase encoded by the emtABCD genes. The methyl donor and its continuous 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 analyses. Soluble fractions of extracts from estradiol-grown cells catalyzed the S-adenosyl-l-methionine (SAM)- and Ti(III)-citrate-dependent conversion of 17β-estradiol/estrone 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 intermediate 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 via l-serine and l-methionine. Consistent with this finding, l-methionine/ATP or l-serine/ATP/tetrahydrofolate/l-homocysteine substituted for SAM as methyl donors, further confirmed by the incorporation of the 13C-methyl-group from 13C-l-methonine into methyl(III)cobalamine and the estrone methylation 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 predominantly female vertebrates, with endocrine disrupting and carcinogenic potential. 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 unprecedented 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 supply 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.

Methyltransferases: Functions and Applications

“…In this kind of reactions, B 12 ‐cofactors (AdoCbl) and methylcobalamin (MeCbl) catalyse many biological processes, which are essential in the metabolism of microorganisms, animals and humans. These processes use methyl tetrahydrofolate, methanol, and methanethiol as methyl donors [41,104] and are not further discussed here.…”

“…[38a] Given the number of MT genes with as yet unknown structures, it is possible there are further structural classes of SAM-dependent MTs waiting to be found, either concentrated within clades or spread across all domains of life. [41]…”

A fully reversible 25-hydroxy steroid kinase involved in oxygen-independent cholesterol side-chain oxidation

“…DHT3 can degrade side chain containing steroids thereby using the same pathway for steroid side chain degradation as established for S. denitrificans. In other denitrifying bacteria that are restricted to the degradation of steroids without side chains such as estradiol (Denitratisoma oestradiolicus) (25,26) (29). Our data indicate that 25phospho-CDO hydrolysis will be similar if not even slightly more negative, around 20 kJ mol -1 more negative than standard sugar-phosphate esters (-13 kJ mol -1 ) (29).…”

Biocatalytic One-Carbon Transfer – A Review

“…This observation was confirmed by Boll and coworkers. 136 The mechanism is somewhat reminiscent of the SAM-dependent enzymes NovO and CouO (Scheme 13) and SibL (Scheme 14c), except that rearomatization cannot take place for the quaternary carbon. For a comparison of cobalamin-and SAM-dependent enzymes, the reader is referred to a review by Drennan and co-workers.…”