Conversion of mammalian 3alpha-hydroxysteroid dehydrogenase to 20alpha-hydroxysteroid dehydrogenase using loop chimeras: changing specificity from androgens to progestins.
Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3alpha-HSD catalyzes the inactivation of androgens in the prostate by converting 5alpha-dihydrotestosterone to 3alpha-androstanediol, where excess 5alpha-dihydrotestosterone is implicated in prostate disease. By contrast, 20alpha-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20alpha-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3alpha-HSDs and 20alpha-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3alpha-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3alpha-HSD was converted to 20alpha-HSD by replacing these loops with those found in 20alpha-HSD. However, when pocket residues in 3alpha-HSD were mutated to those found in 20alpha-HSD altered specificity was not achieved. Replacement of loop A created a 17beta-HSD activity that was absent in either 3alpha- or 20alpha-HSD. Once loops A and C were replaced, the chimera had both 3alpha- and 20alpha-HSD activity. When loops A, B, and C were substituted, 3alpha-HSD was converted to a stereospecific 20alpha-HSD with a resultant shift in k(cat)/K(m) for the desired reaction of 2 x 10(11). This study represents an example where sex hormone specificity can be changed at the enzyme level.
Ma
H
H
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084, USA.
Penning
T M
TM
D015089:20-Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000728:Androgens / Q000378:metabolism*; D000818:Animals; D006801:Humans; D008958:Models, Molecular; D017354:Point Mutation; D011372:Progestins / Q000378:metabolism*; D015202:Protein Engineering; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000728:Androgens; D011372:Progestins; D011993:Recombinant Fusion Proteins; D015096:3-Hydroxysteroid Dehydrogenases; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)