Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D3 Synthesis

Abstract

Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation. Aerobic, steroid-degrading bacteria employ oxygenases for isoprenoid side chain and tetracyclic steran ring cleavage. In contrast, a Mo-containing steroid C-25 dehydrogenase (S25DH) of the dimethyl sulfoxide (DMSO) reductase family catalyzes the oxygen-independent hydroxylation of tertiary C-25 in the anaerobic, cholesterol-degrading bacterium Sterolibacterium denitrificans Its genome contains eight paralogous genes encoding active site α-subunits of putative S25DH-like proteins. The difficult enrichment of labile, oxygen-sensitive S25DH from the wild-type bacteria and the inability of its active heterologous production have largely hampered the study of S25DH-like gene products. Here we established a heterologous expression platform for the three structural genes of S25DH subunits together with an essential chaperone in the denitrifying betaproteobacterium Thauera aromatica K172. Using this system, S25DH₁ and three isoenzymes (S25DH₂, S25DH₃, and S25DH₄) were overproduced in a soluble, active form allowing a straightforward purification of nontagged αβγ complexes. All S25DHs contained molybdenum, four [4Fe-4S] clusters, one [3Fe-4S] cluster, and heme B and catalyzed the specific, water-dependent C-25 hydroxylations of various 4-en-3-one forms of phytosterols and zoosterols. Crude extracts from T. aromatica expressing genes encoding S25DH₁ catalyzed the hydroxylation of vitamin D₃ (VD₃) to the clinically relevant 25-OH-VD₃ with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH₁ was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD₃ synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family.IMPORTANCE Steroids are ubiquitous bioactive compounds, some of which are considered an emerging class of micropollutants. Their degradation by microorganisms is the major process of steroid elimination from the environment. While oxygenase-dependent steroid degradation in aerobes has been studied for more than 40 years, initial insights into the anoxic steroid degradation have only recently been obtained. Molybdenum-dependent steroid C₂₅ dehydrogenases (S25DHs) have been proposed to catalyze oxygen-independent side chain hydroxylations of globally abundant zoo-, phyto-, and mycosterols; however, so far, their lability has allowed only the initial characterization of a single S25DH. Here we report on a heterologous gene expression platform that allowed for easy isolation and characterization of four highly active S25DH isoenzymes. The results obtained demonstrate the key role of S25DHs during anoxic degradation of various steroids. Moreover, the platform is valuable for the efficient enzymatic hydroxylation of vitamin D₃ to its clinically relevant C-25-OH form.

Keywords: alkyl hydroxylases; anaerobic catabolic pathways; molybdenum enzymes; sterols; vitamin D3 biosynthesis.

FIG 1

(A) Proposed anoxic degradation pathway of cholesterol in Sterolibacterium denitrificans. The activation of the side chain is mediated by S25DH, which hydroxylates the tertiary C-25 carbon with water. ADD, androsta-1,4-diene-3-one; 2,3-DSAO, 1,17-dioxo-2,3-seco-androstan-3-oic acid. (B) Conversion of vitamin D₃ (VD₃) to 25-OH-VD₃ by S25DH using ferricyanide as the electron acceptor.

FIG 2

Genes encoding S25DH subunits and chaperone. (A) Arrangement of genes encoding putative α_1-4 (MoCo-containing), β₃,₄ (4Fe-4S cluster-containing), and γ_3,4 (heme b-containing) subunits, as well as the chaperone (SdhD) of S25DHs. (B) Constructs prepared for the heterologous production of S25DH₁, S25DH₂, S25DH₃, and S25DH₄.

FIG 3

Aerobic conversion of 1 mM VD₃ to 25-OH-VD₃ by crude extracts of Thauera aromatica K172 producing S25DH₁ (12 mg ml⁻¹). Symbols: ● VD₃; ▲, 25-OH-VD₃.

FIG 4

Enrichment of recombinant S25DHs. SDS-PAGE analysis of S25DH activity-containing fractions during the enrichment of recombinant S25DH₁, S25DH₂, S25DH₃, and S25DH₄ from T. aromatica cell extracts. Lane 1; 20 µg supernatant after centrifugation at 150,000 × g; lane 2, 10 µg protein after DEAE-Sepharose chromatography; lane 3, 5 µg protein obtained after Reactive Red chromatography. The positions of the corresponding α-, β- and γ-subunits of S25DHs are indicated by arrows to the right of the S25DH₄ gel. The positions of molecular size markers (in kilodaltons) are indicated to the left of the gels.

FIG 5

Enrichment of β-sitost-4-en-3-one converting S25DH₄ from S. denitrificans grown with β-sitosterol and nitrate. Separation of protein fractions forming 25-OH-β-sitost-4-en-3-one by 12.5% SDS-PAGE after ultracentrifugation (16 µg of cell extract of supernatant) (UZ), chromatography on DEAE-Sepharose (24 µg protein) (DEAE), Q-Sepharose (24 µg protein) (Q), Reactive Red-agarose (20 µg) (RR), and Cibacron blue-agarose (10 µg) (CB). The arrows point to the protein bands that were identified as α₄β₄γ₄ of S25DH₄; the proteins migrating at 50 and 55 kDa were identified as degradation products of the α₄-subunit [α₄ (d₁) and α₄ (d₂)]. Lane M contains molecular size markers (in kilodaltons).