Human placental Δ5-3β hydroxysteroid dehydrogenase activity (Δ5-33 HSDH): Intracellular distribution, kinetic properties, retroinhibition and influence of membrane delipidation

Ferré, Françoise; Breuiller, M.; Cédard, L.; Duchesne, Marie-Josèphe; Saintot, M.; Descomps, Bernard; Paulet, AndréCrastes de

doi:10.1016/0039-128x(75)90050-1

Cited by 77 publications

(10 citation statements)

References 16 publications

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“…In enzyme preparations that utilized BME [6][7][8], the K m values of substrate steroids were 10-fold lower than those reported by laboratories which avoided the use of BME [2,16,19], which reduces disulfide bonds. The dilemma has finally been resolved by the current study on the effects of BME on wild-type human 3␤-HSD1 and the 3␤-HSD1 mutants-C72S, C72F, C111S, C111A, C83S and C83A.…”

Section: Discussioncontrasting

confidence: 74%

“…This evaluation of the roles of key Cys residues in human 3␤-HSD1 has explained the reasons for the historically divergent values of Michaelis-Menten constants that have been reported in the literature for this enzyme [2,[6][7][8]. The importance of an intersubunit disulfide linkage to utilization of substrate and cofactor has been characterized, and the role of a Cys residue in the orientation of cofactor by a member of the SCOR family of enzymes has been elucidated.…”

Section: Discussionmentioning

confidence: 95%

“…In an early report [6], the Michaelis-Menten kinetic constants of substrates (DHEA, pregnenolone) for microsomal 3␤-HSD1 from human placenta, were obtained in buffer containing ␤-mercaptoethanol (BME), and the K m values for these substrates were 0.2-0.4 M, 10-fold lower than the K m values of 2-4 M reported by other investigators for microsomal [7] or purified 3␤-HSD1 [2] prepared from human placenta in the absence of BME. An elegant study using microsomal human placental 3␤-HSD in the presence of H 2 O 2 or BME showed that the enzyme utilized substrates with dramatically lower K m values when Cys residues were in the reduced form due to BME [8].…”

Section: Introductionmentioning

confidence: 91%

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Structure/function of human type 1 3β-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

Thomas

Huether

Mack

et al. 2007

The Journal of Steroid Biochemistry and Molecular Biology

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The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.

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Section: Discussioncontrasting

confidence: 74%

Section: Discussionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 91%

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Structure/function of human type 1 3β-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

Thomas

Huether

Mack

et al. 2007

The Journal of Steroid Biochemistry and Molecular Biology

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“…The conformational integrity of the 283-310 deletion mutant is supported because NADH allosterically activates the isomerase activity of the mutant in the same manner that was demonstrated for the wild-type enzyme (Thomas et al 1995). An active, cytosolic form of the enzyme does not support the need for a proper phospholipid, membrane environment, which was suggested by studies in which treatment with phospholipase A inactivated microsomal and mitochondrial 3 -HSD (Ferre et al 1975).…”

Section: Discussionmentioning

confidence: 99%

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

Jl¹,

Evans²,

Blanco³

et al. 1999

Journal of Molecular Endocrinology

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Human 3 -hydroxysteroid dehydrogenase/steroid 5 -4 -isomerase (3 -HSD/isomerase) is a bifunctional, single enzyme protein that is membranebound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3 -HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/ mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3 -HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3 -HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3 -HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH 2 -terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/ quaternary structure of this key steroidogenic enzyme.

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“…3b-HSD is present in the adrenal gland, testis, ovary and placenta, as well as in a large number of peripheral intracrine tissues, including the prostate, breast, liver and skin (Ferre et al 1975;Lacoste et al 1990). It catalyzes the final step in progesterone biosynthesis in the ovary and is required for testosterone production in the testis.…”

Section: Introductionmentioning

confidence: 98%

Immunohistochemical localisation of 3?-hydroxysteroid dehydrogenase in Sarcocystis spp.

Yarım¹,

Yıldız

Kabakci

et al. 2004

Parasitol Res

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3Beta-hydroxysteroid-dehydrogenase (3beta-HSD) is an isoenzyme that catalyses an essential step in the synthesis of all classes of active steroid hormones. The presence of steroid hormones of the vertebrate type in invertebrates is acknowledged in addition to a group of steroid-like hormones called ecdysteroids that were present in arthropods and helminths. In the present study, 3beta-HSD was detected in the bradyzoites enclosed in sarcocysts of Sarcocystis spp. with immunohistochemistry. The results suggest that self-originating steroid hormones may play important roles in the development of Sarcocystis spp., and possibly in the regulation of the reciprocal immune interaction between the host and these parasites.

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Human placental Δ5-3β hydroxysteroid dehydrogenase activity (Δ5-33 HSDH): Intracellular distribution, kinetic properties, retroinhibition and influence of membrane delipidation

Cited by 77 publications

References 16 publications

Structure/function of human type 1 3β-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

Structure/function of human type 1 3β-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

Immunohistochemical localisation of 3?-hydroxysteroid dehydrogenase in Sarcocystis spp.

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