Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3β-hydroxysteroid dehydrogenase/Isomerase1Supported by NIH Grant HD20055 (JLT).1

3 beta-Hydroxysteroid dehydrogenase/steroid Delta(5 --> 4)-isomerase (3 beta-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 beta-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 1). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3 beta-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 beta-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 beta-HSD activity. The 3 beta-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 wildtype 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 beta-HSD activity, and Tyr(253) Or Tyr(254) participates in the isomerase activity of human type I (placental) enzyme. (C) 1998 Elsevier Science Ltd. All rights reserved.