Engineering steroid hormone specificity into aldo-keto reductases.
Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), 20alpha-HSDs, as well as the 5beta-reductases. To elucidate the molecular determinants of steroid hormone recognition we used rat liver 3alpha-HSD (AKR1C9) as a starting structure to engineer either 5beta-reductase or 20alpha-HSD activity. 5beta-Reductase activity was introduced by a single point mutation in which the conserved catalytic His (H117) was mutated to Glu117. The H117E mutant had a k(cat) comparable to that for homogeneous rat and human liver 5beta-reductases. pH versus k(cat) profiles show that this mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH(2)(+) character facilitates enolization of the Delta(4)-3-ketosteroid and subsequent hydride transfer to C5. Since 5beta-reductase precedes 3alpha-HSD in steroid hormone metabolism it is likely that this metabolic pathway arose by gene duplication and point mutation. 3alpha-HSD is positional and stereospecific for 3-ketosteroids and inactivates androgens. The enzyme was converted to a robust 20alpha-HSD, which is positional and stereospecific for 20-ketosteroids and inactivates progesterone, by the generation of loop-chimeras. The shift in log(10)(k(cat)/K(m)) from androgens to progestins was of the order of 10(11). This represents a rare example of how steroid hormone specificity can be changed at the enzyme level. Protein engineering with predicted outcomes demonstrates that the molecular determinants of steroid hormone recognition in AKRs will be ultimately rationalized.
Penning
T M
TM
Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, 19104-6084, Philadelphia, PA, USA.
Ma
H
H
Jez
J M
JM
D015089:20-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D013256:Steroids; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; D000429:Alcohol Oxidoreductases; D015089:20-Hydroxysteroid Dehydrogenases; D000074408:Aldo-Keto Reductases; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000449:Aldehyde Reductase; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase