A patchwork pathway for oxygenase-independent degradation of side chain containing steroids
Markus Warnke
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorChristian Jacoby
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorTobias Jung
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorMichael Agne
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorMario Mergelsberg
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorRobert Starke
Department of Molecular Systems Biology, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Search for more papers by this authorNico Jehmlich
Department of Molecular Systems Biology, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Search for more papers by this authorMartin von Bergen
Department of Molecular Systems Biology, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany
Search for more papers by this authorHans-Hermann Richnow
Department of Isotope Biogeochemistry, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Search for more papers by this authorThomas Brüls
CEA, DRF, IG, Genoscope, Evry, France
CNRS-UMR8030, Université d'Evry Val d'Essonne and Université Paris-Saclay, Evry, France
Search for more papers by this authorCorresponding Author
Matthias Boll
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
For correspondence. E-mail [email protected]; Tel. (+49) 761 2032649; Fax (+49) 761 22 2032626.Search for more papers by this authorMarkus Warnke
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorChristian Jacoby
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorTobias Jung
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorMichael Agne
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorMario Mergelsberg
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Search for more papers by this authorRobert Starke
Department of Molecular Systems Biology, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Search for more papers by this authorNico Jehmlich
Department of Molecular Systems Biology, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Search for more papers by this authorMartin von Bergen
Department of Molecular Systems Biology, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany
Search for more papers by this authorHans-Hermann Richnow
Department of Isotope Biogeochemistry, Helmholtz Centre of Environmental Sciences, Leipzig, Germany
Search for more papers by this authorThomas Brüls
CEA, DRF, IG, Genoscope, Evry, France
CNRS-UMR8030, Université d'Evry Val d'Essonne and Université Paris-Saclay, Evry, France
Search for more papers by this authorCorresponding Author
Matthias Boll
Institute of Biology II, Microbiology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
For correspondence. E-mail [email protected]; Tel. (+49) 761 2032649; Fax (+49) 761 22 2032626.Search for more papers by this authorSummary
The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen-independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo-, phyto- and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin-containing steroid dehydrogenases are involved in C25-hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26-oic acids. Side chain degradation to androsta-1,4-diene-3,17-dione (ADD) via aldolytic C–C bond cleavages involves acyl-CoA synthetases/dehydrogenases specific for the respective 26-, 24- and 22-oic acids/-oyl-CoAs and promiscuous MaoC-like enoyl-CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA-ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase-dependent reactions.
Supporting Information
Additional Supporting Information may be found in the online version of this article at the publisher's web-site.
| Filename | Description |
|---|---|
| emi13933-sup-0001-suppinfo001.pdf1.7 MB |
Table S1. Metabolites detected in cholesterol-grown cells by UPLC–ESI-HRMS. For all compounds identified (bold red), the calculated and observed masses and the assignment to the 1,4-diene-3-one (ADD) or 4-en-3-one (AD) ring A structure, according to the characteristic fragmentation ion spectra pattern of ring A (see Supporting Information Fig. S1) is given. For CoA esters, extraction was by acidic acetonitrile, for all others by ethyl acetate. UPLC separation of all compounds listed with the given retention times was by a single ammonium acetate buffer based program. The detection of cholesterol, cholest-4-en-3-one and cholesta-1,4-diene-3-one is not shown, as UPLC-based detection of these hydrophic initial intermediates requires a different separation method. The presence of CoA esters was confirmed by the detection of characteristic fragments obtained from the CoA moiety (see main text). n.a. = not available, n.d. = not detected. Table S2. COG classification of Stl. denitrificans protein coding sequences (84% of the CDSs are classified in at least one COG group). Table S3. Bacterial genomes most closely related to Stl. denitrificans in terms of bidirectional best hit (BBH) and syntenic relationships. Table S4. Distribution of Stl. denitrificans CDSs into beta-, gammaproteobacterial or actinobacterial classes based on synteny analysis (see main text). 11877 = Pseudomonas syringae pv. syringae FF5 NZ_ACXZ, 3891 = Thauera terpenica 58Eu WGS TTPv1_TTP, 3966 = Rhodococcus ruber IEGM 231 WGS RHRU231v1_ RHRU231. |
| emi13933-sup-0002-suppinfo002.xlsx6.7 MB |
Table S5. Original data of differential proteome analyses: Proteome.xlsx. Here, the abundance of proteins (in categories ‘general’, ‘clustered’ and ‘differentially regulated’) is listed and compared during growth with cholesterol (A), sitosterol (B), stigmasterol (C) and ergosterol (D) vs testosterone (E), propionate (F), palmitate (G) and cholesterol aerobic (H). |
| emi13933-sup-0003-suppinfo003.xlsx244.1 KB | Table S6. List of differentially regulated genes: Proteome_Regulation.xlsx. Here, the log2 ratios and p-values are shown for protein abundancies during growth with cholesterol (A), sitosterol (B), stigmasterol (C) and ergosterol (D) vs testosterone (E), propionate (F), palmitate (G) and cholesterol aerobic (H). |
| emi13933-sup-0004-suppinfo004.xlsx13.8 KB | Table S7. List of differentially regulated genes encoding S25 DH (ABC) and a chaperone (D) during growth with different carbon sources: Proteom_Regulation_S25DH.xlsx. |
| emi13933-sup-0005-suppinfo005.pdf1.7 MB | Table S8. Identities of cholesterol-induced gene products from Stl. denitrificans to those of aerobic model organisms that degrade side-chain containing steroids. Model organisms used: Mycobacterium tuberculosis H37Rv, Rhodococcus jostii RHA1 (both actinobacteria), Comamonas testosteroni ATCC 11996 (betaproteobacterium), Pseudomonas sp. Chol1 and Steroidobacter (Sdo.) denitrificans (both gammaproteobacteria). For genes from Stl. denitrificans Chol1S that are involved in anaerobic cholesterol degradation the annotation/function is shown together with the respective identification number/locus tag. For simpler presentation, the term ‘SDENv1_’ was omitted from identification numbers. Gene identities to known aerobic steroid degraders below 20% or e-values higher that e−10 were not considered. Best hits are highlighted in bold letters. Testos = testosterone; Prop = propionate. Table S9. Doubling time of Stl. denitrificans cells grown with different carbon sources. For calculations the mean of five biological replicates of the different growth cultures were considered. Fig. S1. LC–ESI-MS/MS-based discrimination between 4-en-3-one (AD) and 1,4-diene-3-one (ADD) forms of steroid standards. Characteristic mass peaks are shown of the AD-forms (A) androst-4-en-3,17-dione, (D) cholest-4-en-3-one and the ADD forms (B) androsta-1,4-diene-3,17-dione and (C) cholesta-1,4-diene-3-one. Fig. S2. Nucleotide composition statistics of the Stl. denitrificans genome (from top to bottom: GC%. tetra-nucleotide bias and coding density). Fig. S3. Read-based coverage of the Stl. denitrificans genome (obtained by random subsampling of reads to an average of ∼ 2.5 genome coverage). Fig. S4. Transformation of obtained protein group areas receiving normalized data during proteome analyses of cells grown with different growth substrates as indicated. Fig. S5. Principle component analysis of Stl. denitrificans incubated with different substrates. (A/H) cholesterol (anaerobic/aerobic), (B) β-sitosterol, (C) stigmasterol, (D) ergosterol, (E) testosterone, (F) propionate and (G) palmitate. Fig. S6. Heterologous expression of SDENv1_11189 and its identification as cholest-4-en-3-one-26-oyl-CoA synthetase. (A) SDS-PAGE of the gene product purified by Strep-Tactin affinity chromatography; (B) UPLC-based assay demonstrating the CoA-, ATP- and time-dependent conversion of cholest-4-en-3-one-26-oic acid (1) to cholest-4-en-3-one-26-oyl-CoA (2). Identification of products was by co-elution and mass spectrometric analysis using authentic standards as reference compounds. Fig. S7. Growth of Stl. denitrificans with testosterone (E), palmitate (F), propionate (G) and cholesterol (H) aerobically. The apparent biphasic growth curves in (F) are due to the consumption and stepwise addition of nitrate (5 mM) to avoid toxic nitrite accumulation. |
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