1. Volume 24, Issue 6, Pages 712-724.e7 (June 2017)
Biochemical Mechanisms and Catabolic Enzymes Involved in Bacterial Estrogen Degradation Pathways 
Yi-Lung Chen, Chang-Ping Yu, Tzong-Huei Lee, King-Siang Goh, Kung-Hui Chu, Po-Hsiang Wang, Wael Ismail, Chao-Jen Shih, Yin-Ru Chiang 
Cell Chemical Biology 
Volume 24, Issue 6, Pages e7 (June 2017)
DOI: /j.chembiol
Copyright © 2017 Elsevier Ltd Terms and Conditions

2. Figure 1 UPLC-ESI-HRMS Analysis
UPLC-ESI-HRMS analysis indicated substrate consumption (A) and sequential production of 4-hydroxyestrone (B) and pyridinestrone acid (C) by E1-grown strain KC8 cells. The steroid substrate was composed of unlabeled E1 and [3,4C-13C]E1 (mixed in a 2:1 molar ratio). The bacterial culture was sampled every 30 min. The samples were extracted using ethyl acetate, and the estrogen metabolites were analyzed through UPLC-ESI-HRMS. The predicted elemental composition of individual metabolite ions was calculated using MassLynx Mass Spectrometry software (Waters).
Cell Chemical Biology  , e7DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

3. Figure 2
Identification of the Gene Clusters for the Aerobic Degradation of Natural Estrogens in Sphingomonas sp. Strain KC8
(A) The 4,5-seco pathway, an aerobic estrogen catabolic pathway, established in Sphingomonas sp. strain KC8. Asterisk indicates a cosubstrate; dagger indicates a nitrogen donor. See Figures S2 and S5 for experimental support.
(B) Putative gene clusters for estrogen degradation found in three estrogen-degrading aerobes. In gene clusters I and II, percent identities (blastp) of the corresponding gene products are given compared with the products of strain KC8 genes. In gene cluster III, ORFs homologous between different bacterial strains are connected with dotted lines, and numbers (%) indicate the identity of the deduced amino acid sequences with those of the C. testosteroni genes involved in the degradation of steroidal C/D rings.
(C) Global gene expression profiles (RNA sequencing) of strain KC8 grown with E2 or testosterone. Each spot represents a gene. The linear regression line is based on the data points of the selected housekeeping genes (see Table S1 for gene information).
Cell Chemical Biology  , e7DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

4. Figure 3
The Yeast-Based Estrogen Assay Suggested that Pyridinestrone Acid Exhibits Negligible Estrogenic Activity
(A) Yeast estrogen bioassay results for individual intermediates of the 4,5-seco pathway. A570nm of the solvent; 1% (v/v) DMSO was set to zero. Results are representative of three individual experiments.
(B) Time course of substrate consumption, intermediates production, and estrogenic activities in the cell suspension of strain KC8 incubated with E1. Data are shown as means ± SE of three experimental measurements.
Cell Chemical Biology  , e7DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

5. Figure 4
Functional Characterization and Phylogenetic Analysis of 4-Hydroxyestrone 4,5-Dioxygenase
(A and B) ESI-MS (A) and UV/Vis absorption (B) spectra of the HPLC-purified ring-cleaved product.
(C) Phylogenetic tree of selected extradiol dioxygenases. Table S2 provides detailed information on these extradiol dioxygenases. The evolutionary history was inferred using the maximum-likelihood method in MEGA 7 (Kumar et al., 2016). Numbers are bootstrap values in percentage of 1,000 replicates. Asterisk indicates that the enzyme can also function on 2,3-dihydroxybiphenyl; dagger marks the uncharacterized enzymes encoded by the putative extradiol dioxygenase genes (KC8_16655 and corresponding genes; Figure 2B) of three estrogen-degrading aerobes.
Cell Chemical Biology  , e7DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

6. Figure 5
UPLC-ESI-MS/MS Detection of 13C-Labeled Pyridinestrone Acid in 0.33 μM [3,4C-13C]E1-Treated Activated Sludge Collected from Two Wastewater Treatment Plants, DHSTP and LKCSTP
(A) Extracted ion chromatograms (m/z = for 13C-labeled pyridinestrone acid) of the activated sludge samples.
(B) MS/MS spectra of the 13C-labeled pyridinestrone acid. The predicted elemental composition of individual ions was calculated using MassLynx Mass Spectrometry software (Waters).
Cell Chemical Biology  , e7DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions

7. Figure 6
Temporal Changes in E1 Consumption and 13C-Labeled Pyridinestrone Acid Production in 0.33 μM [3,4C-13C]E1-Treated Water Collected from the Tamsui River, N1–N5 and Gaoping River, S1–S3
The water samples were extracted with ethyl acetate, and the extracts were analyzed by UPLC-ESI-MS/MS. Asterisk indicates that maximal amounts of 13C-labeled E1 (0.31 μM) and pyridinestrone acid (0.02 μM) detected in the water samples were set to 100%.
See also Table S3 (physical and chemical parameters of the sampling sites) and Figures S6 and S7 (MS/MS spectra).
Cell Chemical Biology  , e7DOI: ( /j.chembiol )
Copyright © 2017 Elsevier Ltd Terms and Conditions