Related Experiment Videos
Engineering steroid 5 beta-reductase activity into rat liver 3 alpha-hydroxysteroid dehydrogenase
1Departments of Biochemistry and Biophysics and of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Biochemistry
|July 10, 1998
Summary
Researchers engineered 5 beta-reductase activity into 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) by altering a single amino acid. This modification demonstrates how subtle changes in enzyme structure can evolve new metabolic functions within the aldo-keto reductase superfamily.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Evolution
Background:
- Delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) are key enzymes in steroid hormone metabolism.
- Both enzymes belong to the aldo-keto reductase (AKR) superfamily and share similar catalytic tetrads, differing by a single amino acid (His117 in 3 alpha-HSD vs. Glu in 5 beta-reductase).
Purpose of the Study:
- To investigate the role of the catalytic tetrad residue at position 117 in determining the enzymatic activity of 3 alpha-HSD.
- To engineer 5 beta-reductase activity into 3 alpha-HSD by introducing a His117Glu (H117E) mutation.
Main Methods:
- Site-directed mutagenesis was used to create the H117E and H117A mutants of 3 alpha-HSD.
- Kinetic analyses (kcat, Km) were performed to characterize the enzymatic activities of the wild-type and mutant enzymes.
- pH-rate profiles were analyzed to understand the role of titratable groups in catalysis.
Main Results:
- The H117E mutant exhibited significant 5 beta-reductase activity, reducing testosterone and progesterone with kinetic parameters comparable to native 5 beta-reductase.
- The H117E mutant also retained 3 alpha-HSD activity, albeit with reduced efficiency in the subsequent reduction step.
- Tyr55 was identified as a general acid catalyst for both 5 beta-reductase and 3 alpha-HSD activities, with its acidity modulated by the residue at position 117.
Conclusions:
- The identity of amino acid 117 is crucial in dictating whether an AKR enzyme functions as a 5 beta-reductase or a 3 alpha-HSD, likely by influencing substrate orientation.
- This study provides functional evidence that modifying catalytic residues on an existing protein scaffold can lead to the evolution of new enzymatic functions within the same metabolic pathway.
- The findings highlight the plasticity of the AKR superfamily and offer insights into enzyme evolution and mechanism.