Estrogens have been classified as group 1 carcinogens by the World Health Organization and represent a significant concern given that they are found in surface waters worldwide, and long-term exposure to estrogen-contaminated water can disrupt sexual development in animals. To date, the estrogen catabolic enzymes and genes remain unknown. Using a tiered functional genomics approach, we identified three estrogen catabolic gene clusters in Sphingomonas sp. strain KC8. We identified several estrone-derived compounds, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. We characterized 17β-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase, responsible for the 17-dehydrogenation and meta-cleavage of the estrogen A ring, respectively. The characteristic pyridinestrone acid was detected in estrone-spiked samples collected from two wastewater treatment plants and two suburban rivers in Taiwan. The results significantly expand our understanding of microbial degradation of aromatic steroids at molecular level.

Steroid hormones, such as androgens, are common surface-water contaminants. However, literature on the ecophysiological relevance of steroid-degrading organisms in the environment, particularly in anoxic ecosystems, is extremely limited. We previously reported that Steroidobacter denitrificans anaerobically degrades androgens through the 2,3-seco pathway. In this study, the genome of Sdo. denitrificans was completely sequenced. Transcriptomic data revealed gene clusters that were distinctly expressed during anaerobic growth on testosterone. We isolated and characterized the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, were used as biomarkers to investigate androgen biodegradation in the largest sewage treatment plant in Taipei, Taiwan. Androgen metabolite analysis indicated that denitrifying bacteria in anoxic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assays showed androgen degradation in anoxic sewage by Thauera spp. through the action of 1-testosterone hydratase/dehydrogenase. Our integrative 'omics' approach can be used for culture-independent investigations of the microbial degradation of structurally complex compounds where isotopelabeled substrates are not easily available.

Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis, biological decomposition of lignin, and biotransformation of aromatic compounds. This cofactor is synthesized by UbiX-like prenyltransferases catalyzing the transfer of the dimethylallyl moiety of dimethylallyl-monophosphate (DMAP) to FMN. The origin of DMAP for prFMN biosynthesis and the biochemical properties of free prFMN are unknown. We show that in Escherichia coli cells, DMAP can be produced by phosphorylating prenol using ThiM or dephosphorylating DMAPP using Nudix hydrolases. We produced 14 active prenyltransferases whose properties enabled the purification and characterization of protein-free forms of prFMN. In vitro assays revealed that the UbiD-like ferulate decarboxylase (Fdc1) can be activated by free prFMN or C2'-hydroxylated prFMN under both oxidized and reduced conditions. These insights into the biosynthesis and properties of prFMN will facilitate further elucidation of the biochemical diversity of reversible UbiD (de)carboxylases.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules with essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon–halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base, and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.