Ring A Cleaving Beta-Diketone Hydrolase Is a Key Enzyme of Steroid Degradation in Anaerobic Bacteria

Abstract

Bacterial degradation of ubiquitous and persistent steroids such as steroid hormones is important for their removal from the environment. Initial studies of steroid degradation in anaerobic bacteria suggested that ring-cleaving hydrolases are involved in oxygen-independent sterane skeleton degradation. However, the enzymes involved in ring A cleavage of the common intermediate androsta-1,4-diene-3,17-dione have remained unknown. Here, we enriched a ring A hydrolase from cholesterol/nitrate grown Sterolibacterium denitrificans and from Escherichia coli after heterologous expression of its gene. This enzyme specifically cleaves the cyclic 1,3-diketone of the central degradation intermediate, androsta-1,3,17-trione to 1,17-dioxo-2,3-seco-androstan-3-oate (DSAO), a hallmark reaction of anaerobic steroid degradation. The highly conserved ring A hydrolase was identified in all known and many previously unknown steroid-degrading proteobacteria. Using enriched enzymes, we enzymatically produced DSAO from the chemically synthesised androsta-1-en-3,17-dione precursor, allowing the identification of subsequent metabolites involved in ring A degradation. The results obtained suggest the involvement of an additional hydrolase, an aldolase, and a β-oxidation-like cascade for complete ring A degradation to form the three-ring 5,10-seco-1,2,3,4-tetranorandrosta-5,17-dione. The results identified a key enzyme of anaerobic steroid degradation that may serve as a functional marker for monitoring steroid contaminant degradation at anoxic environmental sites.

Keywords: beta‐diketon hydrolase; ring hydrolase; sterane skeleton; steroid degradation.

FIGURE 1

Lower panel: O₂‐independent 2,3‐seco sterane skeleton degradation pathway in Stl. denitrificans. ADD, androsta‐1,4‐diene‐3,17.dione; 3‐HSA, 3‐hydroxy‐9,10‐seco‐androsta‐1,3,5(10)‐trien‐9,17‐dione; 3,4‐DHSA, 3,4‐dihydroxy‐9,10‐seco‐androsta‐1,3,5(10)‐triene‐9,17‐dione; 4,9‐DHSA, 4,9‐di‐seco‐3‐hydroxy‐4,5,17‐trioxoandrosta‐1(10),2‐diene‐4‐oate; HDD, 2‐hydroxyhexa‐2,4‐dieonate; HIP, 9,17‐dioxo‐1,2,3,4,10,19‐hexaorandrostan‐5‐oate; 1‐AD, androsta‐1‐en‐3,17‐dione; 1,3,17‐ATO, androsta‐1,3,17‐trione; DSAO, 1,17‐dioxo2,3‐seco‐androstan‐3‐oate; KshAB, 3‐ketosteroid 9α‐hydroxylase; HsaAB, flavin‐dependent monooxygenase; HsaC, meta‐cleavage enzyme; HsaD, carbon–carbon hydrolase; 1‐TDH, 1‐testosterone dehydrogenase. “R” represents either a hydroxyl or a keto group.

FIGURE 2

Activity assay and enrichment of steroid ring A hydrolase (SRAH). (A) Time‐dependent conversion of androsta‐1‐en‐3,17‐dione (1,3,17‐ATO) to 1,17‐dioxo‐2,3‐seco‐androstan‐3‐carboxylate (DSAO). UPLC chromatograms at 0 and 30 min using 1 mg mL⁻¹ SRAH enriched from crude extracts of Stl. denitrificans. (B) SDS‐PAGE analysis of active pools obtained during the enrichment of SRAH from crude extracts of Stl. denitrificans. Extract: 20 μg crude extracts, (NH₄)₂SO₄: 15 μg protein after (NH₄)₂SO₄ precipitation, Butyl: 10 μg protein after Butyl‐Sepharose chromatography, CaptoQ: 7.5 μg protein after Capto Q ion exchange chromatography, Superdex 200: 5 μg protein after size‐exclusion chromatography using a Superdex 200 Increase 10/300 GL. (C) SDS‐PAGE analysis of active pools during the enrichment of SRAH after heterologous expression of the encoding gene by StrepTactin affinity chromatography: 20 μg SRAH producing E. coli BL21 soluble proteins, flow through: unbound protein fraction, StrepTacin XT: 3 μg SRAH after StrepTactin XT affinity chromatography. (D) Blue Native PAGE analysis of enriched SRAH after heterologous production in E. coli .

FIGURE 3

Phylogenetic analysis of SRAH‐like proteins (A) and gene clusters of known and potential androgen‐degrading bacteria (B). (A) Phylogenetic tree illustrating the relationships among selected SRAH‐related enzymes retrieved from the refseq_protein database (NCBI). Protein alignment was performed using ClustelW, and the phylogenetic tree was constructed through the Maximum Likelihood method, employing the Poisson correction model with 1000 Bootstraps values. Known androgen‐degrading bacteria are denoted in red, and branches leading to putative androgen‐degrading bacteria are also highlighted in red. (B) Representation of gene clusters associated with the encoding genes of SRAH and the three structural subunits of 1‐TDH from various proteobacterial genomes. The figure displays the amino acid sequence identities of putative SRAHs and catalytic α‐subunits of 1‐TDH to those of Stl. denitrificans. Hundred percentage refers to the corresponding enzymes from Stl. denitrificans.

FIGURE 4

(A) Time‐dependent conversion of DSA‐CoA to 5‐OH‐SDA‐CoA in desalted soluble proteins of Stl. denitrificans and (B) proposed ring A degradation pathway. (A) Semi‐quantitative representation of the conversion of detected CoA‐ester intermediates at 0, 2.5, 5, 15, 30, 60 and 120 min of incubation with Stl. denitrificans desalted soluble extracts. Error bars indicate the standard deviation of three independent replicates. (B) Schematic representation of the proposed ring A degradation pathway in denitrifying steroid degraders, involving CoA‐ester intermediates. Substrates that were not confirmed in this or previous studies are depicted in grey.