Deletion of 12 carboxyl-terminal residues from pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase affects steroid metabolism.
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.
Nakajin
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142, Japan. nakajin@hoshi.ac.jp
Itoda
M
M
Ohno
S
S
Takase
N
N
Toyoshima
S
S
Baker
M E
ME
D015089:20-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D002245:Carbon Dioxide / Q000737:chemistry; D002384:Catalysis; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D007700:Kinetics; D009154:Mutation; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D013552:Swine; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism*
D002245:Carbon Dioxide; C041069:carboxyl radical; D013739:Testosterone; D011374:Progesterone; D000429:Alcohol Oxidoreductases; D015089:20-Hydroxysteroid Dehydrogenases; C115140:3 alpha-beta, 20 beta-hydroxysteroid dehydrogenase