Conversion of mammalian 3alpha-hydroxysteroid dehydrogenase to 20alpha-hydroxysteroid dehydrogenase using loop chimeras: changing specificity from androgens to progestins

Abstract

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.

Figure 1

(a) HSDs regulate the occupancy of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. (b) The (α/β)₈ barrel of 3α-HSD is turned on its side to show the positions of the five loops that comprise the mature steroid binding site. Testosterone is bound with its A-ring in the active site with its β-face toward the A-face of NADP⁺. Colored fragments indicate the loops swapped in the chimeras. Loop A is blue, loop B is yellow, and loop C is red.

Figure 2

Schematic of loop chimera construction. The arrows indicate the locations of oligonucleotide primers that were used for PCRs, and the small letters indicate the oligonucleotide sequences referenced in the text. The numbers indicate the nucleotide positions of the mutated loops. The positions of the swapped loops in the ternary complex also are shown in Fig. 1b.

Figure 3

Comparison of the substrate binding pockets of 3α- and 20α-HSDs. (a) The 3α-HSD testosterone binding pocket viewed from the top of the (α/β)₈ barrel. (b) The 20α-HSD testosterone binding pocket viewed from the top of the (α/β)₈ barrel to show the clashes with steroid ligand. (c) Side view to show the residues that directly interact with the edge of steroid hormones. (d) Side view to show the residues that directly interact with the α and β faces of steroid hormones. Testosterone and the binding pocket residues in 3α-HSD are labeled in green, and progesterone and the binding pocket residues in 20α-HSD are labeled in red.

Figure 4

Converting 3α-HSD into 20α-HSD by using chimeric constructs. The log k_cat/K_m values represented in Table 3 are plotted to demonstrate the progression from 3α- to 20α-HSD activity. (A) The log k_cat/K_m for 3α-HSD activity. (B) The log k_cat/K_m for 17β-HSD and 20α-HSD activity.

Figure 5

20α-OHP is identified as the product of [4-¹⁴C] progesterone reduction catalyzed by the double- and triple-loop chimeras. Chimeras Lp-AC, Lp-ABC, and homogenous recombinant 20α-HSD all convert progesterone into 20α-OHP, but chimeras Lp-AB and Lp-BC failed to display this activity.