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Title (Primary) Four molybdenum-dependent steroid C-25 hydroxylases: Heterologous overproduction, role in steroid degradation, and application for 25-hydroxyvitamin D3 synthesis
Author Jacoby, C.; Eipper, J.; Warnke, M.; Tiedt, O.; Mergelsberg, M.; Stärk, H.-J.; Daus, B.; Martín-Moldes, Z.; Zamarro, M.T.; Díaz, E.; Boll, M.
Journal mBio
Year 2018
Department ANA
Volume 9
Issue 3
Page From e00694-18
Language englisch
Supplements https://mbio.asm.org/content/mbio/9/3/e00694-18/DC1/embed/inline-supplementary-material-1.pdf?download=true
https://mbio.asm.org/content/mbio/9/3/e00694-18/DC2/embed/inline-supplementary-material-2.pdf?download=true
https://mbio.asm.org/content/mbio/9/3/e00694-18/DC3/embed/inline-supplementary-material-3.pdf?download=true
https://mbio.asm.org/content/mbio/9/3/e00694-18/DC4/embed/inline-supplementary-material-4.docx?download=true
https://mbio.asm.org/content/mbio/9/3/e00694-18/DC5/embed/inline-supplementary-material-5.pdf?download=true
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https://mbio.asm.org/content/mbio/9/3/e00694-18/DC7/embed/inline-supplementary-material-7.docx?download=true
Keywords alkyl hydroxylases, anaerobic catabolic pathways, molybdenum enzymes, sterols, vitamin D3 biosynthesis
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.
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=21335
Jacoby, C., Eipper, J., Warnke, M., Tiedt, O., Mergelsberg, M., Stärk, H.-J., Daus, B., Martín-Moldes, Z., Zamarro, M.T., Díaz, E., Boll, M. (2018):
Four molybdenum-dependent steroid C-25 hydroxylases: Heterologous overproduction, role in steroid degradation, and application for 25-hydroxyvitamin D3 synthesis
mBio 9 (3), e00694-18