Human 3β-Hydroxysteroid Dehydrogenase/ δ5→4Isomerase from Placenta: Expression in Nonsteroidogenic Cells of a Protein that Catalyzes the Dehydrogenation/Isomerization of C21 and C19 Steroids*

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Abstract
The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3.beta.-hydroxysteroid dehydrogenase/.DELTA.5.fwdarw.4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3.beta.-hydroxysteroid dehydrogenase/.DELTA.5.fwdarw.4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17.alpha.-hydroxypregnenolone to 17.alpha.-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5.alpha.-androstan-3.beta.,17.beta.-diol to 5.alpha.-dihydrotestosterone and, upon addition of NADP, reduced 5.alpha.-dihydrotestosterone to 5.alpha.-androstan-3.beta.,17.beta.-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.

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