| Hydroxysteroid dehydrogenases (HSDs) are NAD(P)(H) dependent oxidoreductases that play pivotal roles in the biosynthesis and inactivation of all steroid hormones. They belong to the short-chain dehydrogenase/reductase (SDR) and aldo-keto reductase (AKR) gene superfamilies. Mammalian 3alpha-HSDs are AKRs that catalyze the interconversion of male hormones in androgen target tissues and regulate the concentration of neuroactive steroids in the brain, they are thus implicated in the development of prostate cancer and premenstrual syndrome. By contrast, mammalian 20alpha-HSDs are AKRs that regulate progesterone levels and are important for maintaining normal pregnancy. All HSDs in the AKR superfamily have high amino acid sequence identity and are predicted to adopt the same (alpha/beta)8 structure, yet they display strict positional and stereo-specificities. Using rat liver 3alphaalpha-HSD (AKR1C9, EC 1.1.1.213) as template, we mutated nicotinamide binding pocket residues and characterized their biochemical and biophysical properties. This study revealed that there are two different modes of cofactor binding in 3alpha-HSD and that these modes were not solely dependent on tail residues that accommodated the 2'-AMP portion of NADP(H) but also rely on interactions of the head residues that bind the nicotinamide ring. To define the determinants of steroid hormone recognition, we cloned and expressed rat ovarian 20alpha-HSD (AKR1C8, EC 1.1.1.149), then purified and characterized its kinetic and inhibitory properties in detail. This overexpression system provides an abundant source of 20alpha-HSD for crystallization and protein engineering studies. We then mutated the steroid binding residues of 3alpha-HSD to the corresponding residues in 20alpha-HSD. Kinetic studies indicated that these point mutants were unable to introduce 20alpha-HSD activity into 3alpha-HSD. By replacing the A, B and C loops in 3alpha-HSD which define the steroid pocket, with the corresponding loops of 20alpha-HSD, 3alpha-HSD was changed to 20alpha-HSD. Replacement of loop A created a bifunctional 3alpha/17beta-HSD. Replacement of loops A and C created a bifunctional 3alpha/20alpha-HSD. When loops A, B and C were replaced, 3alpha-HSD was converted to a stereospecific 20alpha-HSD with a resultant shift in kcat/ Km for the desired reaction of 2 x 1011. This study represents the first example where sex hormone specificity has been changed at the enzyme level. |
