Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3β-hydroxysteroid dehydrogenase/Isomerase

Abstract 3 β -Hydroxysteroid dehydrogenase/steroid Δ 5→4 -isomerase (3 β -HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants – H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3 β -HSD/isomerase (monomeric M r =42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.0 l). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3 β -HSD activity, whereas the K m and V max values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The V max (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean V max (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3 β -HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar V max values for substrate oxidation by the 3 β -HSD activity. The 3 β -HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD + with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD + was dramatically decreased. Based on these kinetic measurements, His 261 appears to be a critical amino acid residue for the 3 β -HSD activity, and Tyr 253 or Tyr 254 participates in the isomerase activity of human type I (placental) enzyme.