Characterization of Recombinant Mouse Type 5 17β-Hydroxysteroid Dehydrogenase from Mus musculus and Epitestosterone...

17β-Hydroxysteroid dehydrogenase type 5 from Mus musculus (17β-HSD5_Mm) performs preferential NADPH-dependent conversion of androstenedione (AD) into testosterone (T). Unlike the well-characterized 17β-HSD types 1, 2, 3, 4, and 7, which belong to the short-chain dehydrogenase/reductase family, 17β-HSD5_Mm is a less-studied protein belonging to the aldo-keto reductase (AKR) family. For the first time, physicochemical and kinetic characteristics of the purified recombinant 17β-HSD5_Mm enzyme preparation were determined. Using site-directed mutagenesis, an enzyme was constructed in which tyrosine at position 24 was replaced by alanine. When analyzing phenotype of the mutant form of 6×His-17β-HSD5_Mm (Y24A), kinetic constants of the reaction of 17β-reduction of AD and androstadienedione (ADD) were calculated, and it was also revealed that, along with formation of T, the mutant catalyzed formation of epitestosterone (E) from AD. Critical T/E ratio serves as a reference parameter in anti-doping control within professional sports. These findings contribute to the understanding of functional characteristics of the AKR1-family aldo-keto reductases, provide evidence of alteration in stereospecificity of the 17β-HSD5_Mm (Y24A) mutant enzyme, and demonstrate feasibility of enzymatically producing E using the mutant enzyme.