Characterization of a novel type of human microsomal 3 alpha-hydroxysteroid dehydrogenase - Unique tissue distribution and catalytic properties

We report characterization of a novel member of the short chain dehydrogena se/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons, The recombinant protein produced using the bacul ovirus system is localized in the microsomal fraction of Sf9 cells and is a n integral membrane protein with cytosolic orientation of its catalytic dom ain. The enzyme exhibits an oxidoreductase activity toward hydroxysteroids with NAD(+) and NADH as the preferred cofactors, The enzyme is most efficie nt as a 3 alpha -hydroxysteroid dehydrogenase, converting 3 alpha -tetrahyd roprogesterone (allopregnanolone) to dihydroprogesterone and 3 alpha -andro stanediol to dihydrotes- tosterone with similar catalytic efficiency (V-max values of 13-14 nmol/min/mg microsomal protein and K-m values of 5-7 muM). Despite similar to 44-47% sequence identity with retinol/ 3 alpha -hydroxy sterol dehydrogenases, the enzyme is not active toward retinols, The corres ponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain, heart, colon, testis, placenta, lu ng, and lymph node, Thus, the new short chain dehydrogenase represents a no vel type of microsomal NAD(+)-dependent 3 alpha -hydroxysteroid dehydrogena se with unique catalytic properties and tissue distribution.