Conversion of Human Steroid 5β-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H

Abstract

Background:The aldo-keto reductase (AKR) subfamily AKR1D comprises steroid 5␤-reductases, whereas the AKR1C subfamily comprises hydroxysteroid dehydrogenases. Results: A single E120H mutation in AKR1D1 converts human steroid 5␤-reductase into a 3␤-hydroxysteroid dehydrogenase. Conclusion: Glu 120 controls the positional specificity of hydride transfer and facilitates double bond reduction. Significance: This is an example of a perfect change-of-function that can be achieved by a single point mutation.

Rate of steroid double-bond reduction catalysed by the human steroid 5β-reductase (AKR1D1) is sensitive to steroid structure: implications for steroid metabolism and bile acid synthesis

“…Both are possible with AKR1D1. The steroid-binding pocket of AKR1D1 resembles a cylindrical cavity lined largely by hydrophobic residues, with three flexible loops forming the entrance of the binding site [21–23,39,40]. The pocket is flexible such that alternative binding modes and ligand ‘induced fit’ are known to occur with AKR1D1 and the related AKR1C enzymes [33,39,41,42].…”

“…AKR1D1 shares high sequence homology with other steroid transforming members of the AKR family. Crystal structures of AKR1D1 show that the enzyme harbours the ( α / β ) 8 -barrel core structure typical to the superfamily with the cofactor and the steroid substrate at the C-terminal end of the β -sheets [20] and the conserved catalytic tetrad (Tyr 58 , Lys 87 , Glu 120 and Asp 53 ) in the active site [21–23]. Like other members of the AKR family, the kinetics of AKR1D1 is believed to follow a sequential ordered bi-bi mechanism [24].…”

Simultaneous quantitation of nine hydroxy-androgens and their conjugates in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry

“…Recombinant rat liver 3α-hydroxysteroid dehydrogenase (AKR1C9, E.C. 1.1.1.213) and human steroid 5β-reductase mutant E120H (AKR1D1 E120H) were prepared and purified as previously described [39,40]. Charcoal dextran stripped fetal bovine serum (CD-FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA).…”

“…AKR1D1 E120H mutant has been previously shown to be a soluble recombinant source of 3β-HSD [40]. This single point mutation in AKR1D1 is sufficient to eliminate the 5b-reductase activity of the enzyme and generate an enzyme that only has 3β-HSD activity [40].…”

“…AKR1D1 E120H mutant has been previously shown to be a soluble recombinant source of 3β-HSD [40]. This single point mutation in AKR1D1 is sufficient to eliminate the 5b-reductase activity of the enzyme and generate an enzyme that only has 3β-HSD activity [40]. For the synthesis of [ 13 C 3 ]-3α-androstanediol, the reaction contained 100 mM potassium phosphate buffer (pH 6.0), 4% acetonitrile (ACN, HPLC Grade), 651 mM NADH, AKR1C9 (9.3 ng/ µL) and [ 13 C 3 ]-DHT (4 pg/ µL).…”