Characterization of rat and mouse NAD+-dependent 3alpha/17beta/20alpha-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis.
In this study, we characterized rat and mouse aldo-keto reductases (AKR1C16 and AKR1C13, respectively) with 92% sequence identity. The recombinant enzymes oxidized non-steroidal alcohols using NAD+ as the preferred coenzyme, and showed low 3alpha/17beta/20alpha-hydroxysteroid dehydrogenase (HSD) activities. The substrate specificity differs from that of rat NAD+-dependent 3alpha-HSD (AKR1C17) that shares 95% sequence identity with AKR1C16. To elucidate the residues determining the substrate specificity of the enzymes, we performed site-directed mutagenesis of Tyr24, Asp128 and Phe129 of AKR1C16 with the corresponding residues (Ser, Tyr and Leu, respectively) of AKR1C17. The double mutation (Asp128/Tyr-Phe129/Leu) had few effects on the substrate specificity, while the Tyr24/Ser mutant showed only 3alpha-HSD activity, and the triple mutation of the three residues produced an enzyme that had almost the same properties as AKR1C17. The importance of the residue 24 for substrate recognition was verified by the mutagenesis of Ser24/Tyr of AKR1C17 which resulted in a decrease in 3alpha-HSD activity and appearance of 17beta- and 20alpha-HSD activities. AKR1C16 is also 92% identical with rat NAD+-dependent 17beta-HSD (AKR1C24), which possesses Tyr24. The replacement of Asp128, Phe129 and Ser137 of AKR1C16 with the corresponding residues (Glu, Ser and Phe, respectively) of AKR1C24 increased the catalytic efficiency for 17beta- and 20alpha-hydroxysteroids.
Endo
Satoshi
S
Laboratory of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan. sendo@gifu-pu.ac.jp
Sanai
Masaharu
M
Horie
Kenji
K
Matsunaga
Toshiyuki
T
Ishikura
Shuhei
S
Tajima
Kazuo
K
El-Kabbani
Ossama
O
Hara
Akira
A
D000438:Alcohols / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004960:Estradiol Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D006913:Hydroxysteroid Dehydrogenases / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D014018:Tissue Distribution; D014443:Tyrosine / Q000737:chemistry
D000438:Alcohols; D011994:Recombinant Proteins; D014443:Tyrosine; D006913:Hydroxysteroid Dehydrogenases; C524623:AKR1C13 protein, mouse; C524624:AKR1C16 protein, mouse; D004960:Estradiol Dehydrogenases