TY - JOUR
T1 - The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase
AU - Devendran, Saravanan
AU - Mythen, Sean M.
AU - Ridlon, Jason M.
N1 - Publisher Copyright:
Copyright © 2018 Devendran et al.
PY - 2018
Y1 - 2018
N2 - 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.
AB - 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.
KW - 11ß-hydroxyandrostenedione
KW - Coupled enzyme assay
KW - Gut bacteria
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U2 - 10.1194/jlr.M083949
DO - 10.1194/jlr.M083949
M3 - Article
C2 - 29572237
AN - SCOPUS:85048661471
SN - 0022-2275
VL - 59
SP - 1005
EP - 1014
JO - Journal of Lipid Research
JF - Journal of Lipid Research
IS - 6
ER -