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

Abstract

Human type I 3 -hydroxysteroid dehydrogenase/ isomerase (3 -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 membranespanning 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 3 -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 3 -HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K m =4·5 µM, V max =53 nmol/min per mg) and the pure wild-type enzyme (K m =3·7 µM, V max =43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K m =25 µM, V max =576 nmol/min per mg) and wild-type (K m =28 µM, V max =598 nmol/min per mg) enzymes, and for NAD + reduction by the 3 -HSD activities of the cytosolic (K m =35 µM, V max =51 nmol/min per mg) and wild-type (K m =34 µM, 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 µM, V max =538 nmol/min per mg) just like the wild-type enzyme (K m =4·6 µM, V max =536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergentsolubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3 -HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His 261 , Tyr 253 ) that have been identified in the primary structure.

Rational proteomics V: Structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3β-hydroxysteroid dehydrogenase/isomerase type 1

“…2) is consistent with the relatively short distance of 6.7Ǻ between residues 282 and 311 in the model. This deletion mutant of 3β-HSD_1 had substrate and cofactor kinetics that were almost identical to those of the wild-type enzyme [39].…”

Flavonoid inhibition of overexpressed human 3β-hydroxysteroid dehydrogenase type II

“…Both types I [31,32] and II human isozymes [33] have successfully been expressed in a baculovirus system. Although the structure of native enzyme has not yet been elucidated, the type I isozyme has been crystallized [34] using soluble enzyme, which is modified by removal of the membrane-associated regions [35]. In the present study, 3␤-HSD type II was overexpressed using a baculovirus expression system and was not distributed in the soluble fraction, but was broadly detected in all membrane-associate fractions of Sf9 cells.…”

Human 3β-hydroxysteroid dehydrogenase types 1 and 2: Gene sequence variation and functional genomics

“…None of the HSD3B1 nonsynonymous cSNPs observed during our gene resequencing studies were located in any of these regions. In addition, two putative membrane-binding domains have been identified in these enzymes that are located between residues 72 and 89 in the N-terminus of the proteins and between resides 283 and 310 in the C-terminal region [11,37]. Two of the nonsynonymous cSNPs in HSD3B1 (Ile79Val and Phe286Leu), and one in HSD3B2 (Asp74Asn), were located in these putative membrane-binding domain regions.…”

“…Since the HSD3B1 Val79 and Leu286 and the HSD3B2 Asn74 alterations in amino acid sequence were located within putative membrane-binding domains [11,37], confocal microscopy was performed to determine the possible effects of these polymorphisms on the subcellular localization of these isoforms. Specifically, with calnexin as an endoplasmic reticulum marker, immunofluorescent studies were performed using COS-1 cells transiently transfected with constructs encoding HSD3B1 WT, Val79, Leu286 and a combination of the codon 79 and 286 variants, as well as HSD3B2 WT and Asn74 constructs.…”

“…HSD3B-catalyzed oxidation and isomerization are essential steps in the formation of all classes of active steroid hormones [7]. In humans, HSD3B1 is expressed in the placenta and peripheral tissues, while HSD3B2 is expressed predominantly in the adrenal gland, ovary and testis [8][9][10][11]. Deficiency of HSD3B2 is associated with a rare form of congenital adrenal hyperplasia [12].…”