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

Saravanan Devendran, Sean M. Mythen, Jason M. Ridlon

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Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11?-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 20?-hydroxysteroid dehydrogenase (20?-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 DesABdependent formation of 11ß-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17ß-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 11?-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.- Devendran, S., S. M. Mythen, and J. M. Ridlon. The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase.

Original languageEnglish (US)
Pages (from-to)1005-1014
Number of pages10
JournalJournal of Lipid Research
Issue number6
StatePublished - 2018


  • 11ß-hydroxyandrostenedione
  • Coupled enzyme assay
  • Gut bacteria

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology


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