Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D ₃ 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.

Microbial Functional Responses to Cholesterol Catabolism in Denitrifying Sludge

“…denitrificans harbors eight alpha subunits (molybdopterin-containing subunits) of C25DH, and the substrate specificity of each subunit (S25dA1–8) has been studied through proteome analysis ( 14 ). We identified homologues of S25dA1–A7, but the expression level of S25dA1 homologues in our cholesterol-treated sludge was highest; this is congruent with previous studies that indicated that S25dA1 is responsible for the side chain hydroxylation of cholest-4-en-3-one due to its high substrate specificity ( 14 , 36 ). Interestingly, our maximum likelihood tree clearly separated subunits with side chain specificity (S25dA1–4) and subunits without specificity (S25dA5–A7) into two lineages and grouped homologues of each subunit into distinct clades (except S25dA2).…”

Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms

“…Based on genomics and metabolomics studies, S. denitrificans appeared to utilize a range of steroid C25 dehydrogenases for oxygen-independent C25-hydroxylations of steroids with different isoprenoid side chains, followed by an unusual conversion to C26-oic acids (Warnke et al 2017 ). This hypothesis was very recently confirmed by successful heterologous overexpression of paralogous S25DH-like genes in Thauera aromatica K172 and characterization of substrate specificity for four different enzymes (Jacoby et al 2018 ). After complete degradation of the aliphatic side chain, androst-4-en-3,17-dione (AD) and/or androst-1,4-dien-3,17-dione (ADD) enter the central degradation pathway via 2,3- seco -AB-rings opening (Chiang and Wael 2011 ; Wang et al 2013 ; Wang et al 2014 ).…”

“…This hypothesis was very recently confirmed by successful production of recombinant S25DH isoenzymes (S25DH 1–4 ) in T. aromatica K172 and Azoarcus sp. CIB and characterization of substrate specificity for individual enzymes (see below and Jacoby et al 2018 ). The attempts to obtain overexpress S25DH enzymes in E. coli systems yielded either non or trace activity only (Jacoby et al 2018 ; Rugor et al 2017b ).…”

“…These results clearly showed that S. denitrificans utilizes different S25DH-like enzymes for conversion of different substrates with S25DH 2 hydroxylating 7-dehydrocholesterol and S25DH 4 hydroxylating campest-4-en-3-one and β-sitost-4-en-3-one. The role of other S25DH-like enzyme is not yet established (Jacoby et al 2018 ).…”

“…This approach allows hydroxylation of VD3 with a final concentration of 1.4 g/L (3.5 mM) of calcifediol within 3 days. The potential application of S25DH for biocatalytic synthesis of calcifediol seems to be especially attractive since recent establishment of the overexpression system as recombinant S25DH1 preparation is 6.5-fold more active than wild-type extract and enables conversion of 0.38 g/L of VD3 in 3 h (Jacoby et al 2018 ). The proposed system was also applied for hydroxylation of various 3-ketosteroids such as: cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one with a final concentration in the range of 2.2 g/L, or sterols such as: cholesterol, 7-dehydrocholesterol, with a final concentration in the range of 0.8 g/L (Rugor et al 2017a ).…”