Deletion of 12 carboxyl-terminal residues from pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase affects steroid metabolism

Abstract

Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase (3alpha/beta,20beta-HSD) is 80-85% identical to human, rat, and mouse carbonyl reductases. However, pig 3alpha/beta,20beta-HSD contains an extra 12 amino acids at its COOH-terminus that these other mammalian carbonyl reductases lack. We constructed a pig 3alpha/beta,20beta-HSD mutant, G278opal, which lacks these amino acids and found that compared to wild-type 3alpha/beta,20beta-HSD, G278opal has a 10-fold lower catalytic efficiency for testosterone and progesterone. G278opal also has lower 3alpha- and 20beta-reductase and increased 3beta-reductase activity compared to wild-type 3alpha/beta,20beta-HSD. Binding of NADPH to G278opal was similar to that of wild-type 3alpha/beta,20beta-HSD. The recently determined three-dimensional structure of 3alpha/beta,20beta-HSD, without a steroid substrate, shows the 12 COOH-terminal amino acids in a random configuration. Our data indicate that the 12 COOH-terminal amino acids have a role in steroid metabolism suggesting that binding of steroid to wild-type 3alpha/beta,20beta-HSD induces a conformational change in which the 12 COOH-terminal amino acids interact with the steroid substrate.