Structure of human estrogenic 17β-hydroxysteroid dehydrogenase at 2.20 å resolution

Ghosh, Debashis; Плетнев, В. З.; Zhu, D.-W.; Wawrzak, Z.; Duax, William L.; Pangborn, W.; Labrie, Fernand; Lin, Sheng‐Xiang

doi:10.1016/s0969-2126(01)00183-6

Cited by 258 publications

(204 citation statements)

References 39 publications

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“…In addition, structural comparisons of 3a,20P-hydroxysteroid dehydrogenase, dihydropteridine reductase, and the epimerase have revealed a common core of approximately 180 structurally equivalent amino acid residues (Holm et al, 1994). Recently, the X-ray crystallographic analyses of 7a-hydroxysteroid dehydrogenase from E. coli, mouse lung carbonyl reductase, and 170-hydroxysteroid dehydrogenase have also revealed that these proteins have similar structures to that observed for the epimerase (Ghosh et al, 1995;Tanaka et al, 1996aTanaka et al, , 1996b. Although these various enzymes are involved in a wide range of metabolic processes, it has been suggested that their reaction mechanisms may be similar (Person et al, 1995).…”

Section: Comparison Of Udp-galactose 4-epimerase With Dihydropteridinmentioning

confidence: 95%

High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol

1996

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UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-glucose and UDP-galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between P-NADH and a 4-keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 8, resolution with crystals grown in the presence of a substrate analogue, UDP-phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372-381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high-resolution X-ray crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol. The model was refined to 1.8 8, resolution with a final overall R-factor of 18.6%. This high-resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP-phenol and UDPglucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP-galactose 4-epimerase belongs to a family of enzymes known as the short-chain dehydrogenases, which contain a characteristic Tyr-Lys couple thought to be important for catalysis. The epimerase/NADH/UDP-phenol model presented here represents a well-defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr-Lys couple in the reaction mechanism.Keywords: epimerases; galactose metabolism; short-chain dehydrogenases; substrate analogues; X-ray crystallography The use of substrate analogues has had profound impact on our understanding of enzyme structure and function, as evidenced, for example, by the elegant X-ray crystallographic studies of thermolysin. By studying the three-dimensional structures of thermolysin complexed with various substrate or transition state analogues, it has been possible to propose, in combination with other biochemical information, a detailed catalytic mechanism for the enzyme (Matthews, 1988). As in any X-ray crystallographic investigation, however, the results must be approached with caution, because the compounds employed in such studies are not true substrates or intermediates. For example, in the recent structural investigation of the enzyme phosphotriesterase complexed with the substrate analogue diethyl 4-methylbenzylphosphonate, it appears that the inhibitor does not bind in a manner imitating true substrate binding (Vanhooke et al., 1996). This issue of potential artifactual binding has been a concern in the structural investigations of UDP-galactose 4-epimerase from Escherichia coli. As part of the Leloir pathway for the metabolism

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Section: Comparison Of Udp-galactose 4-epimerase With Dihydropteridinmentioning

confidence: 95%

High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol

1996

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“…The structure of 17␤-HSD1 from human placenta was first determined at 2.2 Å resolution (16). More recently, the structure of the complex of 17␤-HSD1 with E 2 , as well as the enzyme structure in the presence of E 2 and NADP, have been reported (17,18).…”

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confidence: 99%

Dehydroepiandrosterone and Dihydrotestosterone Recognition by Human Estrogenic 17β-Hydroxysteroid Dehydrogenase

Han

Campbell²,

Gangloff

et al. 2000

Journal of Biological Chemistry

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Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17␤-hydroxysteroid dehydrogenase (17␤-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein.Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. The K m of estrone is more than 1000-fold lower than that of DHEA and the K m of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17␤-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 3 Val variant showed a significantly decreased K m for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a ␤-oriented form, which approaches the transition-state conformation for DHEA reduction.

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“…3) are conserved in most of these proteins. These residues include the almost invariant tyrosine (Y-152 of SDH) and lysine residues of the consensus sequence Y-X-X-X-K, which are essential for catalysis (9,14,30) and which are located in the active site (15,16,43), and the glycine residues of the G-X-X-X-G-X-G segment (G-12 of SDH), which are characteristic of the NAD ϩ -binding domain (32,45) in the N-terminal part.…”

Section: Resultsmentioning

confidence: 99%

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

1997

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The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(؉) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.The purple, nonsulfur bacterium Rhodobacter sphaeroides Si4 is capable of growing on a variety of sugar alcohols (polyols), including D-mannitol and D-glucitol (sorbitol). In the presence of either substrate, R. sphaeroides Si4 concomitantly induces two NAD ϩ -dependent enzymes: mannitol dehydrogenase (MDH; EC 1.1.1.67 [38]) and sorbitol dehydrogenase (SDH; EC 1.1.1.14 [36]). SDH and MDH are involved in R. sphaeroides Si4 D-glucitol metabolism, but due to its low K m for D-glucitol, SDH is the predominant enzyme for the metabolization of this substrate at low concentrations (36). Both enzymes were purified and characterized. SDH is a dimeric enzyme with a subunit molecular mass of 29,000 Da and is active on sorbitol and galactitol, with sorbitol being the preferred substrate. MDH is a monomeric enzyme with a subunit molecular mass of 51,404 Da and is active on D-mannitol, sorbitol, and D-arabitol, with a preference for D-mannitol. Because of the potential of MDH for biotechnological applications (37,38,41), the corresponding gene (mtlK) has been cloned, sequenced (39), and overexpressed (35).In this communication, we demonstrate that the smoS gene, which encodes SDH, is located within a previously unrecognized polyol operon. The gene coding for an ATP-binding protein of an ATP-binding cassette (ABC) transport system (smoK) and the structural genes smoS and mtlK have been identified. On the basis of sequence homology data, SDH can be attributed to the short-chain dehydrogenase/reductase (SDR) protein family (21). Finally, overexpression of SDH in Escherichia coli provides sufficient amounts of the recombinant enzyme for crystallization and structure analysis studies. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains used are listed in Table 1. R. sphaeroides Si4 is an isolate of this laboratory (31). E. coli XL1-Blue MRF...

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Structure of human estrogenic 17β-hydroxysteroid dehydrogenase at 2.20 å resolution

Cited by 258 publications

References 39 publications

High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol

High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol

Dehydroepiandrosterone and Dihydrotestosterone Recognition by Human Estrogenic 17β-Hydroxysteroid Dehydrogenase

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

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