The engineered, cytosolic form of human type I 3β-hydroxysteroid dehydrogenase/isomerase: Purfication, characterization and crystallization

Abstract

Human type I 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membrane-spanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3β-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD+ and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38.8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42.0 kDa). Michaelis-Menten constants measured for 3β-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (Km=4.5 μM, Vmax = 53 nmol/min per mg) and the pure wild-type enzyme (Km = 3.7 μM, Vmax = 43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (Km = 25 μM, Vmax = 576 nmol/min per mg) and wild-type (Km = 28 μM, Vmax = 598 nmol/min per mg) enzymes, and for NAD+ reduction by the 3β-HSD activities of the cytosolic (Km = 35 μM, Vmax = 51 nmol/min per mg) and wild-type (Km = 34 μM, Vmax = 46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (Km = 4.6 μM, Vmax = 538 nmol/min per mg) just like the wild-type enzyme (Km = 4.6 μM, Vmax = 536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3β-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His261, Tyr253) that have been identified in the primary structure.