Steroids as Environmental Compounds Recalcitrant to Degradation: Genetic Mechanisms of Bacterial Biodegradation Pathways

Abstract

Steroids are perhydro-1,2-cyclopentanophenanthrene derivatives that are almost exclusively synthesised by eukaryotic organisms. Since the start of the Anthropocene, the presence of these molecules, as well as related synthetic compounds (ethinylestradiol, dexamethasone, and others), has increased in different habitats due to farm and municipal effluents and discharge from the pharmaceutical industry. In addition, the highly hydrophobic nature of these molecules, as well as the absence of functional groups, makes them highly resistant to biodegradation. However, some environmental bacteria are able to modify or mineralise these compounds. Although steroid-metabolising bacteria have been isolated since the beginning of the 20th century, the genetics and catabolic pathways used have only been characterised in model organisms in the last few decades. Here, the metabolic alternatives used by different bacteria to metabolise steroids (e.g., cholesterol, bile acids, testosterone, and other steroid hormones), as well as the organisation and conservation of the genes involved, are reviewed.

Keywords: 2,3-seco pathway; 4,5-seco pathway; 9,10-seco pathway; bile acids; biodegradation; steroid hormones; sterols.

Figure 1

Chemical structure of cholesterol and some of its mammalian derivatives, and selected synthetic steroids.

Figure 2

Genetic organisation of the mce4 cluster involved in sterol uptake in Actinobacteria.

Figure 3

Initial reactions catalysed by cholesterol oxidase and different hydroxysteroid dehydrogenases in steroid degradation through the 9,10-seco pathway.

Figure 4

Genetic organisation of the genes encoding the 9,10-seco pathway involved in cholesterol (Mycobacterium tuberculosis H37Rv, Rhodococcus jostii RHA1), or in cholic acid and testosterone (R. jostii RHA1, Comamonas thiooxidans CNB-1 (formerly, Comamonas testosteroni CNB-2), Pseudomonas stutzeri Chol1 (formerly Pseudomonas sp. Chol1), and Pseudomonas putida DOC21 catabolism. Genes coding enzymes involved in cholesterol or bile acids side chain degradation are shown in green; genes participating in ring A/B degradation are in orange; in purple are shown genes coding for ring C/D degradation; blue color indicates genes coding transport systems.

Figure 5

Catabolism of the cholesterol side chain and C₂₄-branched chain of β-sitosterol in Actinobacteria.

Figure 6

Cholic acid metabolism in Pseudomonas putida DOC21 and P. stutzeri Chol1 through the 9,10-seco pathway. Preliminary evidence suggests that oxidation of the A-ring occurs simultaneously with C₁₇ side chain degradation. Hydroxyl groups at C₇ and C₁₂ are maintained during degradation of the molecule, the affecting 3aα-H-4α(3′-propanoate)7a-β-methylhexahydro-1,5- indanedione-hydroxylated derivatives metabolism. 3′,7-diOH-HIP, 3aα-H-4α(3′(R)-hydroxy-3′propanoate)-7-hydroxy-7aβ-methylhexahydro-1,5-indanedione; 3′,7-diOH-HIP-CoA, 3aα-H-4α(3′(R)-hydroxy-3′propanoyl-CoA)-7-hydroxy-7aβ-methylhexahydro- 1,5-indanedione; 3′,5,7-triOH-HIP-CoA, 3aα-H-(3′(R)-hydroxy-3′propanoyl-CoA)-5,7-dihydroxy-7aβ-methylhexahydro-1-indanone; 4OH-COCHEA-CoA, 2-(2-carboxyethyl)-4-hydroxy-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA; 7-OH-HIEC-CoA, (7aS)-7a-methyl-7-hydroxy-1,5-dioxo-2,3,5,6,7,7a-hexahydro-1H-indene-4-carboxyl-CoA; 5,7-diOH-HIC-CoA, 3aα-H-4α(3′-carboxyl-CoA)-5,7-dihydroxy-7aβ-methylhexahydro-1-indenone.

Figure 7

Proposed 9,10-seco pathway from androst-4-en-3,17-dione to central metabolites, pyruvate, acetyl-CoA, propionyl-CoA, and succinyl-CoA. AD, androst-4-en-3,17-dione; ADD, androst-1,4-dien-3,17-dione; 9OH-AD, 9-hydroxy-androst-4-en-3,17-dione; 9OH-ADD, 9-hydroxy-androst-1,4-dien-3,17-dione; 3-HAS, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione; 3,4-DHSA, 3,4-dihydroxy-9,10- secoandrosta-1,3,5(10)-triene-9,17-dione; 4,9-DSHA, 4,5,9,10-diseco-3-hydroxy-5-9-17- trioxoandrosta-1(10),2-diene-4-oic acid; HHD, 2-hydroxy-2,4-hexadienoic acid; HIP, 3aα-H-4α(3′-propanoate)7a-β-methylhexahydro-1,5-indanedione; 5OH-HIP-CoA, 3aα-H-4α(3′-propanoyl-CoA)-5-hydroxy-7a-β-methylhexahydro-1-indanone; 5OH-HIC-CoA, 3aα-H-4α(3′-carboxyl-CoA)-5-hydroxy-7a-β-methylhexahydro-1-indanone; HIEC-CoA, (7aS)-7a-methyl-1,5-dioxo-2,3,5,6,7,7a-hexahydro-1H-indene-4-carboxyl-CoA; COCHEA-CoA, 2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA; MOODA-CoA, 4-methyl-5-oxo- octanedioyl-CoA. Actinobacterial enzymes from cholesterol metabolism are indicated in red, actinobacterial enzymes involved in cholic acid catabolism are indicated in orange, and those from catabolism of testosterone and cholic acid from Comamonas spp. are written in blue.

Figure 8

Metabolism of 17β-estradiol in Sphingomonas sp. KC8. (a) Reactions of the 4,5-seco pathway. HIP, 3aα-H-4α(3′-propanoate)7a-β-methylhexahydro-1,5-indanedione. (b) Genetic organisation of the three clusters codifying the enzymes required for 17β-estradiol assimilation (Genome Accession NZ_CP016306). Annotation from the genome: KC8_RS16375, putative dioxygenase; KC8_RS16385, Rieske (2Fe 2S) protein; KC8_RS05200, MaoC dehydratase; KC8_RS05205, hypothetical protein; KC8_RS05210, acyl-CoA dehydrogenase; KC8_RS05215, ferredoxin oxidoreductase; KC8_RS05220, VOC family protein; KC8_RS05230, TetR transcriptional regulator; KC8_RS05235, cytochrome P450; KC8_RS05240, hypothetical protein; KC8_RS05245, lipid-transfer protein; KC8_RS05250, MaoC dehydratase; KC8_RS05255 and KC8_RS05260, enoyl-CoA hydratases; KC8_RS05265, acetyl-CoA acetyltransferase; KC8_RS05270, 3-hydroxy-3-mthylglutaryl-CoA synthase; KC8_RS05275, Short-chain oxidoreductase; KC8_RS05280, acyl-CoA dehydrogenase; KC8_RS00980, CoA acyltransferase; KC8_RS00985, steroid Δ-isomerase; KC8_RS00990, MaoC dehydratase; KC8_RS00995, short-chain dehydrogenase/reductase; KC8_RS01000, acyl-CoA dehydrogenase; KC8_RS01005, short-chain oxidoreductase; KC8_RS01010, phenylacetic acid degradation protein PaaY; KC8_RS01015, acetyl-CoA acetyltransferase; KC8_RS01020, acyl-CoA dehydrogenase; KC8_RS01025, acyl-CoA dehydrogenase; KC8_RS01030, enoyl-CoA hydratase; KC8_RS01035, monooxygenase; KC8_RS01040, lipid-transfer protein (Ltp); KC8_RS01045, thiolase; KC8_RS01050, CoA transferase, β-subunit; KC8_RS01055, CoA transferase, α-subunit; KC8_RS01060, meta-dioxygenase. Genes encoding key enzymes for A ring degradation are depicted in red; genes coding β-oxidation related enzymes putatively catalyzing degradation of A/B ringare indicated in green; genes for C/D-ring degradation are depicted in purple.

Figure 9

Metabolic alternatives to the 4,5-seco pathway proposed for estrogen mineralisation/biotransformation in different bacterial species.

Figure 10

Anaerobic catabolism of cholesterol, in Sterolibacterium denitrificans, and testosterone, in Steroidobacter denitrificans, by the 2,3-seco pathway. Putative points of convergence between both metabolic mechanisms are also suggested.

Figure 11

Genetic organisation of the genes encoding 2,3-seco pathway functions for cholesterol degradation in Sterolibacterium denitrificans. Annotation in the genome (accession LT837803): SEDNCHOL_11188, aldehyde dehydrogenase; SEDNCHOL_11189, acyl-CoA synthetase 1 (ACS1); SEDNCHOL_11190, short-chain dehydrogenase; SEDNCHOL_11191, SEDNCHOL_11192, and SEDNCHOL_11193 steroid C25 dehydrogenase, γ-, β- and α- subunit, respectively; SEDNCHOL_11194 and SEDNCHOL_11195, proteins with unknown functions; SEDNCHOL_11196, AcmB; SEDNCHOL_11197, putative transcriptional regulatory protein; SEDNCHOL_11198, putative aldolase; SEDNCHOL_11199, enoyl-CoA hydratase; SEDNCHOL_11200 and SEDNCHOL_11201, acyl-CoA dehydrogenases; SEDNCHOL_11202, oxidoreductase; SEDNCHOL_10299, acyl-CoA synthetase 2 (ACS2); SEDNCHOL_10300 and SEDNCHOL_10301, acyl-CoA dehydrogenases; SEDNCHOL_10302, protein of unknown function; SEDNCHOL_10303, putative metallo-β-lactamase; SEDNCHOL_10304, phytoene dehydrogenase-like protein; SEDNCHOL_10305, putative C₂₂ acyl-CoA synthetase; SEDNCHOL_10306 and SEDNCHOL_10307, acyl-CoA dehydrogenases; SEDNCHOL_10308, aldolase; SEDNCHOL_10309 and SEDNCHOL_10310, enoyl-CoA hydratases; SEDNCHOL_10168, CoA transferase; SEDNCHOL_10169, plasmid stabilization system; SEDNCHOL_10170 and SEDNCHOL_10171, proteins of unknown function; SEDNCHOL_10172, SEDNCHOL_10173, and SEDNCHOL_10174, A, B, and C subunits of AtcABC, respectively; SEDNCHOL_10175 and SEDNCHOL_10176, proteins of unknown function; SEDNCHOL_10177; putative electron-transfer flavoprotein, β-subunit; SEDNCHOL_10178, protein of unknown function; SEDNCHOL_10317, SEDNCHOL_10318, and SEDNCHOL_10321, IpdABC-like proteins; SEDNCHOL_10319, probably subunit of benzoylsuccinyl-CoA thiolase; SEDNCHOL_10320, Propanoyl-CoA C-acyltransferase; SEDNCHOL_10322, enoyl-CoA hydratase; SEDNCHOL_10323 and SEDNCHOL_10324, acyl-CoA dehydrogenases; SEDNCHOL_10325, thiolase; SEDNCHOL_10326, MarR family transcriptional regulator; SEDNCHOL_10327, outer membrane protein; SEDNCHOL_10328, putative Filamentous hemagglutinin family N-terminal domain containing protein; SEDNCHOL_10329, protein of unknown function; SEDNCHOL_10330, N-acetyltransferase; SEDNCHOL_10331, RseB; SEDNCHOL_10332, enoyl-CoA hydratase; SEDNCHOL_10333 and SEDNCHOL_10334, proteins of unknown function; SEDNCHOL_10335, CoA transferase; SEDNCHOL_10336, putative IpdC; SEDNCHOL_10337 and SEDNCHOL_10338, CoA transferases; SEDNCHOL_10339, acetyl-CoA acetyltransferase; SEDNCHOL_10340, protein of unknown function; SEDNCHOL_10341, enoyl-CoA hydratase; SEDNCHOL_10763, putative 2-phospho-L-lactate guanylyltransferase; SEDNCHOL_10764, 2-phospho-L-lactate transferase; SEDNCHOL_10765, Coenzyme F420:L-glutamate ligase; SEDNCHOL_10766, acyl-CoA synthetase 3 (ACS3); SEDNCHOL_10767, steroid Δ–isomerase; SEDNCHOL_10768, enoyl-CoA hydratase; SEDNCHOL_10769, short chain dehydrogenase; SEDNCHOL_10770 and SEDNCHOL_10771, acyl-CoA dehydrogenases; SEDNCHOL_10772, short chain alcohol dehydrogenase. Genes encoding enzymes involved in cholesterol side chain degradation are shown in green; genes participating in A ring degradation are shown in orange; and in purple is shown genes coding for ring C/D degradation.