Abstract
ABSTRACTSide 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.
Highlights
Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation
The subsequent hydroxylation of the side chain with water that occurs at tertiary C-25 is catalyzed by molybdenum (Mo)-dependent steroid C-25 dehydrogenase (S25DH) [10, 11], and not at primary C-26 as observed in the oxygenase-dependent pathway
The previously established five-step enrichment of the oxygen-sensitive S25DH1 (␣13␥3 complex) from wild-type Sterolibacterium denitrificans grown with cholesterol always gave low yields, low specific activities, and a partially degraded ␣1-subunit [11, 22, 24]
Summary
Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation. Crude extracts from T. aromatica expressing genes encoding S25DH1 catalyzed the hydroxylation of vitamin D3 (VD3) to the clinically relevant 25-OH-VD3 with Ͼ95% 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. Cleavage of the steran rings A and B proceeds in the so-called 2,3-secopathway involving ring-cleaving hydrolases [14, 15]; degradation of rings C and D appears to be similar in aerobic and anaerobic steroid-degrading bacteria, again using hydrolytic enzymes (Fig. 1) [16]
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