@entity1 type I 3beta- @entity14128 dehydrogenase/isomerase ( @entity11187 /isomerase) is an integral membrane protein of @entity1 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 @entity11187 was purified using affinity chromatography with Cibacron Blue 1000. The @entity2125 and @entity1219 used to elute the enzyme were removed by size-exclusion centrifugation. @entity692 chromatography yielded a 26-fold purification of the enzyme. @entity214 -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 @entity11187 substrate ( @entity2494 ) 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 ( @entity49953 ) 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 @entity2125 reduction by the @entity11187 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 @entity2125 (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 @entity11187 /isomerase will clarify the mechanistic roles of potentially critical @entity1522 (His(261), Tyr(253)) that have been identified in the primary structure.

The engineered, cytosolic form of @entity1 type I 3beta- XXXX dehydrogenase/isomerase: purification, characterization and crystallization.

@entity1 :: ('9606', 'Species') :: ['human', 'Human'] @entity2125 :: ('MESH:D009243', 'Chemical') :: ['NAD(+)', 'NADH'] @entity1522 :: ('CHEBI:33709', 'Chemical') :: ['amino acids'] @entity1219 :: ('CHEBI:26710', 'Chemical') :: ['NaCl'] @entity2494 :: ('MESH:D003687', 'Chemical') :: ['dehydroepiandrosterone'] @entity14128 :: ('CHEBI:35350', 'Chemical') :: ['hydroxysteroid'] @entity692 :: ('MESH:D017886', 'Chemical') :: ['Hydroxylapatite'] @entity49953 :: ('MESH:C003528', 'Chemical') :: ['5-androstene-3,17-dione'] @entity11187 :: ('3283', 'Gene') :: ['3beta-HSD/isomerase', '3beta-HSD'] @entity214 :: ('CHEBI:8984', 'Chemical') :: ['SDS']
@entity14128 :: (CHEBI:35350,Chemical) :: ['hydroxysteroid']
