EXPRESSION AND KINETIC-PROPERTIES OF A RECOMBINANT 3-ALPHA-HYDROXYSTEROID DIHYDRODIOL DEHYDROGENASE ISOENZYME OF HUMAN LIVER

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydr ogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme forms to the genes, a cDNA, which has been reported t o code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrod iol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the re combinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone an d 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha -hydroxysteroid dehydrogenase and chlordecone reductase previously pur ified, which suggests that these human liver enzymes are identical, Fu rthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-inda nol oxidation by the recombinant isoenzyme were consistent with a sequ ential ordered mechanism in which NADP(+) binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other hum an liver dihydrodiol dehydrogenases, and was a competitive inhibitor ( K-i =20 nM) that bound to the enzyme-NADP(+) complex.