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

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 (S25...

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Autores principales: Jacoby, Christian, Eipper, Jens, Warnke, Markus, Tiedt, Oliver, Mergelsberg, Mario, Stärk, Hans-Joachim, Daus, Birgit, Martín-Moldes, Zaira, Zamarro, María Teresa, Díaz, Eduardo, Boll, Matthias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016249/
https://www.ncbi.nlm.nih.gov/pubmed/29921665
http://dx.doi.org/10.1128/mBio.00694-18
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author Jacoby, Christian
Eipper, Jens
Warnke, Markus
Tiedt, Oliver
Mergelsberg, Mario
Stärk, Hans-Joachim
Daus, Birgit
Martín-Moldes, Zaira
Zamarro, María Teresa
Díaz, Eduardo
Boll, Matthias
author_facet Jacoby, Christian
Eipper, Jens
Warnke, Markus
Tiedt, Oliver
Mergelsberg, Mario
Stärk, Hans-Joachim
Daus, Birgit
Martín-Moldes, Zaira
Zamarro, María Teresa
Díaz, Eduardo
Boll, Matthias
author_sort Jacoby, Christian
collection PubMed
description 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, S25DH(1) and three isoenzymes (S25DH(2), S25DH(3), and S25DH(4)) 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 S25DH(1) catalyzed the hydroxylation of vitamin D(3) (VD(3)) to the clinically relevant 25-OH-VD(3) with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH(1) was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD(3) synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family.
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spelling pubmed-60162492018-06-26 Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D(3) Synthesis Jacoby, Christian Eipper, Jens Warnke, Markus Tiedt, Oliver Mergelsberg, Mario Stärk, Hans-Joachim Daus, Birgit Martín-Moldes, Zaira Zamarro, María Teresa Díaz, Eduardo Boll, Matthias mBio Research Article 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, S25DH(1) and three isoenzymes (S25DH(2), S25DH(3), and S25DH(4)) 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 S25DH(1) catalyzed the hydroxylation of vitamin D(3) (VD(3)) to the clinically relevant 25-OH-VD(3) with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH(1) was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD(3) synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family. American Society for Microbiology 2018-06-19 /pmc/articles/PMC6016249/ /pubmed/29921665 http://dx.doi.org/10.1128/mBio.00694-18 Text en Copyright © 2018 Jacoby et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Jacoby, Christian
Eipper, Jens
Warnke, Markus
Tiedt, Oliver
Mergelsberg, Mario
Stärk, Hans-Joachim
Daus, Birgit
Martín-Moldes, Zaira
Zamarro, María Teresa
Díaz, Eduardo
Boll, Matthias
Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D(3) Synthesis
title Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D(3) Synthesis
title_full Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D(3) Synthesis
title_fullStr Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D(3) Synthesis
title_full_unstemmed Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D(3) Synthesis
title_short Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D(3) Synthesis
title_sort four molybdenum-dependent steroid c-25 hydroxylases: heterologous overproduction, role in steroid degradation, and application for 25-hydroxyvitamin d(3) synthesis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016249/
https://www.ncbi.nlm.nih.gov/pubmed/29921665
http://dx.doi.org/10.1128/mBio.00694-18
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