title: The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization.
abstract: Human type I 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-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 3beta-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 3beta-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K(m)=4.5 microM, V(max)=53 nmol/min per mg) and the pure wild-type enzyme (K(m)=3.7 microM, V(max)=43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K(m)=25 microM, V(max)=576 nmol/min per mg) and wild-type (K(m)=28 microM, V(max)=598 nmol/min per mg) enzymes, and for NAD(+) reduction by the 3beta-HSD activities of the cytosolic (K(m)=35 microM, V(max)=51 nmol/min per mg) and wild-type (K(m)=34 microM, V(max)=46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K(m)=4.6 microM, V(max)=538 nmol/min per mg) just like the wild-type enzyme (K(m)=4.6 microM, V(max)=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 3beta-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His(261), Tyr(253)) that have been identified in the primary structure.
authors.affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Thomas
authors.forename: J I
authors.initials: JI
authors.lastname: Mason
authors.forename: G
authors.initials: G
authors.lastname: Blanco
authors.forename: M L
authors.initials: ML
authors.lastname: Veisaga
mesh_terms: D002850:Chromatography, Gel; D003460:Crystallization; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011375:Progesterone Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013251:Steroid Isomerases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D011375:Progesterone Reductase; D013251:Steroid Isomerases