The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S]

Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogena...
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Gespeichert in:

Autor*in:	Saravanan Devendran [verfasserIn] Sean M. Mythen [verfasserIn] Jason M. Ridlon [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2018

Schlagwörter:	gut bacteria coupled enzyme assay 11β-hydroxyandrostenedione

Übergeordnetes Werk:	In: Journal of Lipid Research - Elsevier, 2021, 59(2018), 6, Seite 1005-1014
Übergeordnetes Werk:	volume:59 ; year:2018 ; number:6 ; pages:1005-1014

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1194/jlr.M083949

Katalog-ID:	DOAJ057237700

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allfields	10.1194/jlr.M083949 doi (DE-627)DOAJ057237700 (DE-599)DOAJ553ae85530034e28a1ae662436911736 DE-627 ger DE-627 rakwb eng QD415-436 Saravanan Devendran verfasserin aut The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S] 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids. gut bacteria coupled enzyme assay 11β-hydroxyandrostenedione Biochemistry Sean M. Mythen verfasserin aut Jason M. Ridlon verfasserin aut In Journal of Lipid Research Elsevier, 2021 59(2018), 6, Seite 1005-1014 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:59 year:2018 number:6 pages:1005-1014 https://doi.org/10.1194/jlr.M083949 kostenfrei https://doaj.org/article/553ae85530034e28a1ae662436911736 kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520330996 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 59 2018 6 1005-1014
spelling	10.1194/jlr.M083949 doi (DE-627)DOAJ057237700 (DE-599)DOAJ553ae85530034e28a1ae662436911736 DE-627 ger DE-627 rakwb eng QD415-436 Saravanan Devendran verfasserin aut The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S] 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids. gut bacteria coupled enzyme assay 11β-hydroxyandrostenedione Biochemistry Sean M. Mythen verfasserin aut Jason M. Ridlon verfasserin aut In Journal of Lipid Research Elsevier, 2021 59(2018), 6, Seite 1005-1014 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:59 year:2018 number:6 pages:1005-1014 https://doi.org/10.1194/jlr.M083949 kostenfrei https://doaj.org/article/553ae85530034e28a1ae662436911736 kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520330996 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 59 2018 6 1005-1014
allfields_unstemmed	10.1194/jlr.M083949 doi (DE-627)DOAJ057237700 (DE-599)DOAJ553ae85530034e28a1ae662436911736 DE-627 ger DE-627 rakwb eng QD415-436 Saravanan Devendran verfasserin aut The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S] 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids. gut bacteria coupled enzyme assay 11β-hydroxyandrostenedione Biochemistry Sean M. Mythen verfasserin aut Jason M. Ridlon verfasserin aut In Journal of Lipid Research Elsevier, 2021 59(2018), 6, Seite 1005-1014 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:59 year:2018 number:6 pages:1005-1014 https://doi.org/10.1194/jlr.M083949 kostenfrei https://doaj.org/article/553ae85530034e28a1ae662436911736 kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520330996 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 59 2018 6 1005-1014
allfieldsGer	10.1194/jlr.M083949 doi (DE-627)DOAJ057237700 (DE-599)DOAJ553ae85530034e28a1ae662436911736 DE-627 ger DE-627 rakwb eng QD415-436 Saravanan Devendran verfasserin aut The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S] 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids. gut bacteria coupled enzyme assay 11β-hydroxyandrostenedione Biochemistry Sean M. Mythen verfasserin aut Jason M. Ridlon verfasserin aut In Journal of Lipid Research Elsevier, 2021 59(2018), 6, Seite 1005-1014 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:59 year:2018 number:6 pages:1005-1014 https://doi.org/10.1194/jlr.M083949 kostenfrei https://doaj.org/article/553ae85530034e28a1ae662436911736 kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520330996 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 59 2018 6 1005-1014
allfieldsSound	10.1194/jlr.M083949 doi (DE-627)DOAJ057237700 (DE-599)DOAJ553ae85530034e28a1ae662436911736 DE-627 ger DE-627 rakwb eng QD415-436 Saravanan Devendran verfasserin aut The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S] 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids. gut bacteria coupled enzyme assay 11β-hydroxyandrostenedione Biochemistry Sean M. Mythen verfasserin aut Jason M. Ridlon verfasserin aut In Journal of Lipid Research Elsevier, 2021 59(2018), 6, Seite 1005-1014 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:59 year:2018 number:6 pages:1005-1014 https://doi.org/10.1194/jlr.M083949 kostenfrei https://doaj.org/article/553ae85530034e28a1ae662436911736 kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520330996 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 59 2018 6 1005-1014
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source	In Journal of Lipid Research 59(2018), 6, Seite 1005-1014 volume:59 year:2018 number:6 pages:1005-1014
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callnumber-first	Q - Science
author	Saravanan Devendran
spellingShingle	Saravanan Devendran misc QD415-436 misc gut bacteria misc coupled enzyme assay misc 11β-hydroxyandrostenedione misc Biochemistry The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S]
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topic_title	QD415-436 The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S] gut bacteria coupled enzyme assay 11β-hydroxyandrostenedione
topic	misc QD415-436 misc gut bacteria misc coupled enzyme assay misc 11β-hydroxyandrostenedione misc Biochemistry
topic_unstemmed	misc QD415-436 misc gut bacteria misc coupled enzyme assay misc 11β-hydroxyandrostenedione misc Biochemistry
topic_browse	misc QD415-436 misc gut bacteria misc coupled enzyme assay misc 11β-hydroxyandrostenedione misc Biochemistry
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title	The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S]
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title_full	The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S]
author_sort	Saravanan Devendran
journal	Journal of Lipid Research
journalStr	Journal of Lipid Research
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author_browse	Saravanan Devendran Sean M. Mythen Jason M. Ridlon
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doi_str_mv	10.1194/jlr.M083949
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title_sort	desa and desb genes from clostridium scindens atcc 35704 encode steroid-17,20-desmolase[s]
callnumber	QD415-436
title_auth	The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S]
abstract	Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.
abstractGer	Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.
abstract_unstemmed	Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.
collection_details	GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700
container_issue	6
title_short	The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S]
url	https://doi.org/10.1194/jlr.M083949 https://doaj.org/article/553ae85530034e28a1ae662436911736 http://www.sciencedirect.com/science/article/pii/S0022227520330996 https://doaj.org/toc/0022-2275
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doi_str	10.1194/jlr.M083949
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up_date	2024-07-04T00:52:24.031Z
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The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase[S]

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