is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydro-xyandrostenedione. A cortisol-inducible operon () was previously identified in ATCC 35704 by RNA-Seq. The gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The and 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 and genes from ATCC 35704 in pETDuet for overexpression in 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, The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were of 4.96 ± 0.57 µM and of 0.87 ± 0.076 min Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.