Engineering steroid hormone specificity into aldo-keto reductases

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Aldo-keto reductases (AKRs) are crucial enzymes in steroid hormone metabolism.
• Mammalian AKRs include 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), 20alpha-HSDs, and 5beta-reductases.
• Understanding AKR substrate specificity is key to deciphering steroid hormone pathways.

Purpose of the Study:

• To elucidate the molecular basis of steroid hormone recognition by AKRs.
• To engineer specific enzymatic activities (5beta-reductase and 20alpha-HSD) into a model AKR (rat liver 3alpha-HSD, AKR1C9).

Main Methods:

• Site-directed mutagenesis was used to introduce a His117Glu mutation to confer 5beta-reductase activity.
• Loop-chimera generation was employed to engineer 20alpha-HSD activity.
• Kinetic analyses (kcat, Km) and pH-activity profiles were used to characterize enzyme function.

Main Results:

• A single point mutation (H117E) successfully introduced 5beta-reductase activity with kinetics comparable to native enzymes.
• The H117E mutation increased the acidity of Tyr55, facilitating steroid enolization and hydride transfer.
• Loop-chimeras converted 3alpha-HSD into a potent 20alpha-HSD, altering specificity from androgens to progestins by a factor of 10^11.
• Protein engineering achieved predicted functional outcomes, highlighting rational design capabilities.

Conclusions:

• Steroid hormone specificity in AKRs can be significantly altered through targeted protein engineering.
• A single mutation can confer novel enzymatic activity and alter substrate preference.
• These findings provide insights into the evolution of steroidogenic pathways and the rational design of enzymes.