Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens

Abstract

We have purified a steroid-inducible 20a-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%.Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17ai,21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 Clostridium scindens is the only bacterium isolated known to synthesize 20a-hydroxysteroid dehydrogenase (20a-HSDH) and steroid-17-20-desmolase activities (3, 51). Preliminary studies with cell extracts indicated that both neutral steroid-transforming activities were coinducible in C. scindens cultured in the presence of specific C21 steroids. In addition, it was found that both conversions required a pyridine nucleotide coenzyme, bivalent metal cations, and the same adrenocorticosteroid substrates for maximal activity (22). 20a-HSDH (EC 1.1.1.149) is widely distributed in nature. The enzyme has been found previously in bird testes, fungi (8, 11), and a great variety of mammalian tissues. In vertebrate species, 20a-HSDH is thought to be a key enzyme involved in tissue-specific regulation of steroid hormone metabolism (10, 39). Various forms of 20a-HSDH are found in the major steroid-producing tissues of mammals (adrenals, ovaries, testes, and placenta) and in liver, kidney, muscle, lymphatic organs, fibroblasts, and hematopoietic cells (4,28,29,45,47,49). Multiple forms of 20a-HSDH have been distinguished in several tissues, largely on the basis of steroid substrate specificity, pyridine nucleotide requirement, and intracellular location (2, 12, 37). A soluble enzyme from human placenta (36), rat ovary (35,50), and the testes of two species (33, 40) has been purified to homogeneity and well characterized. This report describes the purification and partial characterization of a novel form of 20a-HSDH from C. scindens.( corticosterone, 11,21-dihydroxy-4-pregnene-3,20-dione; deoxycorticosterone, 21-hydroxy-4-pregnene-3,20-dione; progesterone, 4-pregnene-3,20-dione; 17a-hydroxyprogesterone, 17a-hydroxy-4-pregnene-3,20-dione; pregnenolone, 3p-hydroxy-5-pregnen-20-one; 20a-dihydrocortisol, 11P,17,20a,21-tetrahydroxy-4-pregnene-3-one; MES, MOPS,propane-sulfonic acid); CHES, (2[N-cyclohexylamino]-ethanesulfonic acid); PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Chemicals. The following chemicals were purchased: unlabeled steroids, Steraloids Inc., Wilton, N.H., and Sigma Chemical Co., St. Louis, Mo.; [1,2-3H]cortisol (40.6 Ci/ mmol), Amersham Corp; high-pressure liquid chromatography (HPLC) solvents, Burdick and Jackson; brain-heart infusion medium, Difco Laboratories; protease inhibitors, Boehringer Mannheim; pyridine nucleotide...

Hydroxysteroid dehydrogenases (HSDs) in bacteria – A bioinformatic perspective

“…Furthermore, a 20␣-HSD enzyme [55] does also exist only as a fragment of 11 amino acids (Q7M1A0). A pattern search, as explained above, identified a protein (B0ND76) in a similar bacterium, Clostridium scindens.…”

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens

“…Clostridium strain 19 was later named Clostridium scindens (type strain American Type Culture Collection (ATCC) 35704) which means “to cut” (6). Previously, our lab purified and characterized an NAD(P) + -dependent 20α-hydroxysteroid dehydrogenase (HSDH) from cell extracts of C. scindens ATCC 35704 in addition to a partially purified steroid-17,20-desmolase (SDase); however, the genes encoding these enzymes have yet to be identified (7, 8). …”

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

“…Cortisol-inducible steroid-17,20-desmolase was partially purified, and 20α-HSDH was purified to apparent electrophoretic homogeneity (SDS-PAGE) by traditional chromatographic separation (7,8). The application of genome-wide transcriptomics (RNA-Seq) identified a polycistronic cortisol-inducible operon (desABCD) which included a gene encoding 20α-HSDH (desC) (9).…”