Identification of two histidyl residues in the active site of human placental estradiol 17.beta.-dehydrogenase

Chin, Chang Chen; Murdock, Gary L.; Warren, James C.

doi:10.1021/bi00257a012

Cited by 17 publications

(7 citation statements)

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“…His 221 , first identified by affinity labeling studies (27)(28)(29), was thought to be involved in the specific binding of the steroid. Indeed, the construction of an H221A mutant led to an enzyme displaying a higher K m and lower V max relative to the wild type (16).…”

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Unusual Charge Stabilization of NADP+ in 17β-Hydroxysteroid Dehydrogenase

Mazza

Breton

Housset

et al. 1998

Journal of Biological Chemistry

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Type 1 17␤-hydroxysteroid dehydrogenase (17␤-HSD1), a member of the short chain dehydrogenase reductase (SDR) family, is responsible for the synthesis of 17␤-estradiol, the biologically active estrogen involved in the genesis and development of human breast cancers. Here, we report the crystal structures of the H221L 17␤-HSD1 mutant complexed to NADP ؉ and estradiol and the H221L mutant/NAD ؉ and a H221Q mutant/estradiol complexes. These structures provide a complete picture of the NADP The 17␤-estradiol (E 2 ) is known to promote the genesis and development of human breast cancers (1, 2). Its presence in tumor cells comes from in situ synthesis (3), and its concentration is significantly increased in malignant breast tissues (4 -6). Type 1 17␤-hydroxysteroid dehydrogenase (17␤-HSD1) 1 catalyzes the reversible transformation of estrone (E 1 ) into the biologically active estradiol (E 2 ) (7). Thus, preventing the formation of E 2 by a specific inhibition of 17␤-HSD1 activity should be of paramount importance for cancer therapy.The 17␤-HSD1 (M r ϭ 34,900, 327 residues) is active as a homodimer (8) Based on amino acid sequence alignments, 17␤-HSD1 was thought to belong to the group of NAD(H)-preferring enzymes (22). However, biochemical studies (7) and the structure of the 17␤-HSD1⅐E 2 ⅐NADP ϩ ternary complex (25) have shown that 17␤-HSD1 is able to bind both NAD(H) and NADP(H). 17␤-HSD1 appears to be unique among the SDR family because it lacks both the aspartic acid residue at position 36 (Leu 36 in 17␤-HSD1), characteristic of NAD(H) preferring enzymes, and the basic residue located in the consensus sequence of the dinucleotide binding motif Gly-Xaa-Xaa-Xaa-Gly-Xaa-Gly (which is replaced by Ser 12 in 17␤-HSD1). This motif forms an ionic interaction with the ribose 2Ј-phosphate and is characteristic of NADP(H)-preferring enzymes.His 221 , first identified by affinity labeling studies (27-29), was thought to be involved in the specific binding of the steroid. Indeed, the construction of an H221A mutant led to an enzyme displaying a higher K m and lower V max relative to the wild type (16). Furthermore, the crystal structure of the enzyme complexed with E 2 (25, 26) revealed that His 221 is directly involved in the specific binding of the steroid.Here, we report the construction of H221L and H221Q mutants, their characterization by enzymatic assays, their crystallization, and the determination of the structures of the binary complexes H221Q⅐E 2 and H221L⅐NAD ϩ and the ternary complex H221L⅐NADP ϩ ⅐E 2 at 2.7, 3.0, and 2.7 Å resolution, respectively. We show for the first time a well ordered conformation for the 191-199 loop and speculate about its role in cofactor binding and hydride transfer. Moreover, the specificity of the enzyme for estrogens is reassessed. EXPERIMENTAL PROCEDURESMaterials-Spodoptera frugiperda, purified AcNPV DNA (Autographa Californica nuclear polyhedrosis virus), and transfer vector pVL1393 were purchased from Invitrogen Corporation; Grace's insect cell culture medium, yeastolate, lactalbu...

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confidence: 99%

Unusual Charge Stabilization of NADP+ in 17β-Hydroxysteroid Dehydrogenase

Mazza

Breton

Housset

et al. 1998

Journal of Biological Chemistry

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“…The topography of the active site has been studied by affinity labeling with various steroid bromoacetates. Steroids bearing the reagent group at the 3-, 12/3-or 16a-position affinity alkylate histidine residues and yield AT-(carboxymethyl)histidine or A™-(carboxymethyl)histidine on subsequent acid hydrolysis of the enzyme.Previous work (Chin et al, 1982) has demonstrated that the 3-methoxyestriol 16-(bromoacetate) and 12/3-hydroxy-4estrene-3,17-dione 12-(bromoacetate) label different histidine residues to form AT-(carboxymethyl)histidine. However, these data did not exclude the possibility of also labeling a single histidine at different positions in the imidazole ring by these two steroid derivatives.…”

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confidence: 99%

“…Previous work (Chin et al, 1982) has demonstrated that the 3-methoxyestriol 16-(bromoacetate) and 12/3-hydroxy-4estrene-3,17-dione 12-(bromoacetate) label different histidine residues to form AT-(carboxymethyl)histidine. However, these data did not exclude the possibility of also labeling a single histidine at different positions in the imidazole ring by these two steroid derivatives.…”

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Human placental estradiol 17.beta.-dehydrogenase: sequence of a histidine-bearing peptide inthe catalytic region

Murdock¹,

Chin²,

Warren³

1986

Biochemistry

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The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12 beta-hydroxy-4-estrene-3,17-dione 12-(bromo[2-14C]acetate) and 3-methoxyestriol 16-(bromo[2-14C]acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12 beta and 16 alpha derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl)cysteine and histidine residues to form either N tau- or N pi-(carboxymethyl)histidine. The inactivated enzyme samples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive N tau- and N phi-(carboxymethyl)histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that the two histidine residues identified in the present study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17 beta-dehydrogenase.

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“…Our recent study, using a combined method of enzymology and molecular biology, concluded for the first time that 17/3-HSD is formed by two identical 34.5 kDa subunits (Lin et al, 1992). Besides the subunit structure, some studies on the enzyme active site or binding site using chemical modification have been reported (Chin, Murdock & Warren, 1982;Murdock, Chin & Warren, 1986). Nevertheless, the information above is still incomplete and determination of the enzyme structure is required for a proper understanding of its functions.…”

Section: Introductionmentioning

confidence: 99%

Crystal growth of human estrogenic 17β-hydroxysteroid dehydrogenase

Zhu¹,

Lee²,

Labrie³

et al. 1994

Acta Cryst D

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Estrogenic 17fl-hydroxysteroid dehydrogenase from human placenta, an enzyme of low solubility, has been crystallized in the complex form with its cofactor NADP ÷. These are the first crystals with X-ray diffraction quality for structure analysis from any human steroid-converting enzyme. The crystals were grown by vapor diffusion in the presence of 0.06% /3-octylglucoside, using polyethylene glycol 4000 as the precipitant (27-28%) and one of several different salts at pH 7.5 and room temperature. Crystals grown with magnesium chloride diffract up to 2.4A. The most important steps leading to the rapid success of the crystallization of this labile enzyme were the following: preparation of a highly active and homogeneous enzyme protein using a rapid procedure; the choice of a suitable enzyme buffer system and a detergent favorable to maintaining high activity and solubility for the enzyme; and a combined screening procedure. The present study could be useful for the successful crystal growth of other hydrophobic or membrane-bound proteins.

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Identification of two histidyl residues in the active site of human placental estradiol 17.beta.-dehydrogenase

Cited by 17 publications

References 12 publications

Unusual Charge Stabilization of NADP+ in 17β-Hydroxysteroid Dehydrogenase

Unusual Charge Stabilization of NADP+ in 17β-Hydroxysteroid Dehydrogenase

Human placental estradiol 17.beta.-dehydrogenase: sequence of a histidine-bearing peptide inthe catalytic region

Crystal growth of human estrogenic 17β-hydroxysteroid dehydrogenase

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