Crystal Structures of the Multispecific 17β-Hydroxysteroid Dehydrogenase Type 5: Critical Androgen Regulation in Human Peripheral Tissues

Abstract Human type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5;AKR1C3) plays a major role in the metabolism of androgens in peripheral tissues. In prostate basal cells, this enzyme is involved in the transformation of dehydroepiandrosterone into dihydrotestosterone, the most potent androgen. It is thus a potential target for prostate cancer therapy because it is understood that the testosterone formation by this enzyme is an important factor, particularly in patients who have undergone surgical or medical castration. Here we report the first structure of a human type 5 17β-HSD in two ternary complexes, in which we found that the androstenedione molecule has a different binding position from that of testosterone. The two testosterone-binding orientations in the substrate-binding site demonstrate the structural basis of the alternative binding and multispecificity of the enzyme. Phe306 and Trp227 are the key residues involved in ligand recognition as well as product release. A safety belt in the cofactor-binding site enhances nicotinamide adenine dinucleotide phosphate binding and accounts for its high affinity as demonstrated by kinetic studies. These structures have provided a dynamic view of the enzyme reaction converting androstenedione to testosterone as well as valuable information for the development of potent enzyme inhibitors.

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Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616142114 ◽

2017 ◽

Vol 114 (7) ◽

pp. E1091-E1100 ◽

Cited By ~ 41

Author(s):

Mario D. Garcia ◽

Amanda Nouwens ◽

Thierry G. Lonhienne ◽

Luke W. Guddat

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Kinetic Studies ◽

Herbicidal Activity ◽

Structural Basis ◽

Acetohydroxyacid Synthase ◽

Branched Chain Amino Acid ◽

Application Rates ◽

Herbicide Binding ◽

Branched Chain

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted “S”-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

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Structural basis for stereospecific inhibition of ASCT2 from rational design

10.1101/2020.05.29.124305 ◽

2020 ◽

Author(s):

Rachel-Ann A. Garibsingh ◽

Elias Ndaru ◽

Alisa A. Garaeva ◽

Massimiliano Bonomi ◽

Dirk J. Slotboom ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Rational Design ◽

Md Simulations ◽

Homology Model ◽

Structural Basis ◽

Protein Targets ◽

Peripheral Tissues ◽

Ligand Discovery ◽

Multiple Conformations

ABSTRACTASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. ASCT2 is upregulated in cancer, where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides an emerging strategy for cancer therapy. Here, guided by a homology model of ASCT2 in an outward-facing conformation, we rationally designed novel inhibitors exploiting stereospecific pockets in the substrate binding site. A cryo-EM structure of ASCT2 in complex with inhibitor (Lc-BPE) validated our predictions and was subsequently refined based on computational analysis. The final structures, combined with MD simulations, show that the inhibitor samples multiple conformations in the ASCT2 binding site. Our results demonstrate the utility of combining computational modeling and cryo-EM for SLC ligand discovery, and a viable strategy for structure determination of druggable conformational states for challenging membrane protein targets.

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Phosphate Binding to Isolated Chloroplast Coupling Factor (CF1)

Zeitschrift für Naturforschung C ◽

10.1515/znc-1988-3-411 ◽

1988 ◽

Vol 43 (3-4) ◽

pp. 213-218 ◽

Cited By ~ 4

Author(s):

Bernhard Huchzermeyer

Keyword(s):

Atpase Activity ◽

Binding Site ◽

Inorganic Phosphate ◽

Coupling Factor ◽

Binding Domain ◽

Atp Binding ◽

Phosphate Binding ◽

Chloroplast Coupling Factor ◽

Single Binding Site ◽

Site Binding

A single binding site for phosphate was found on isolated chloroplast coupling factor in the absence of nucleotides. In our experiments the phosphate binding site showed a Kd of 170 μᴍ. We did not observe any differences whether the ATPase activity of CF] had been activated or not. If the enzyme was incubated with [γ-32P]ATP the amount of 32P bound per CF1 depended on the pretreatment of the enzyme: In the presence of ADP no ATP or phosphate was bound to CF,. After activation of ATPase activity one mol of ATP per mol CF, was rapidly bound and hydrolyzed while there was a slowly occurring binding of another phosphate without concomitant nucleotide binding. We conclude that there are two different types of phosphate binding observed in our experiments: 1) Inorganic phosphate can be bound by one catalytic site per mol of CF1 2) The γ-phosphate of ATP is able to bind to an ATP binding domain of the enzyme if this domain can exchange substrates with the incubation medium. This ATP binding domain appears to differ from the site binding inorganic phosphate, because at least a portion of the coupling factor contains more than one labelled phosphate during our ATPase tests.

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Synthesis of 2α.- 6α- 6β-bromoprogesterone and study of the binding site of 20β-hydroxysteroid dehydrogenase

Biochemistry ◽

10.1021/bi00764a027 ◽

1972 ◽

Vol 11 (14) ◽

pp. 2720-2726 ◽

Cited By ~ 27

Author(s):

Chang-Chen Chin ◽

James C. Warren

Keyword(s):

Binding Site ◽

Hydroxysteroid Dehydrogenase

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Kinetic Studies of Proton Transfer in the Microenvironment of a Binding Site

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1982.tb05833.x ◽

1982 ◽

Vol 121 (3) ◽

pp. 637-642 ◽

Cited By ~ 26

Author(s):

Menachem GUTMAN ◽

Dan HUPPERT ◽

Esther NACHLIEL

Keyword(s):

Proton Transfer ◽

Binding Site ◽

Kinetic Studies

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Structural Basis for Inhibition of Protein-tyrosine Phosphatase 1B by Isothiazolidinone Heterocyclic Phosphonate Mimetics

Journal of Biological Chemistry ◽

10.1074/jbc.m606873200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32784-32795 ◽

Cited By ~ 36

Author(s):

Paul J. Ala ◽

Lucie Gonneville ◽

Milton C. Hillman ◽

Mary Becker-Pasha ◽

Min Wei ◽

...

Keyword(s):

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Structural Basis ◽

Side Chain ◽

Protein Tyrosine Phosphatase 1B ◽

Phosphate Binding ◽

Protein Tyrosine ◽

Starting Point ◽

Network Of Hydrogen Bonds ◽

Orthogonal Orientation

Crystal structures of protein-tyrosine phosphatase 1B in complex with compounds bearing a novel isothiazolidinone (IZD) heterocyclic phosphonate mimetic reveal that the heterocycle is highly complementary to the catalytic pocket of the protein. The heterocycle participates in an extensive network of hydrogen bonds with the backbone of the phosphate-binding loop, Phe182 of the flap, and the side chain of Arg221. When substituted with a phenol, the small inhibitor induces the closed conformation of the protein and displaces all waters in the catalytic pocket. Saturated IZD-containing peptides are more potent inhibitors than unsaturated analogs because the IZD heterocycle and phenyl ring directly attached to it bind in a nearly orthogonal orientation with respect to each other, a conformation that is close to the energy minimum of the saturated IZD-phenyl moiety. These results explain why the heterocycle is a potent phosphonate mimetic and an ideal starting point for designing small nonpeptidic inhibitors.

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Biochemical Factors Governing the Steady-State Estrone/Estradiol Ratios Catalyzed by Human 17β-Hydroxysteroid Dehydrogenases Types 1 and 2 in HEK-293 Cells

Endocrinology ◽

10.1210/en.2008-1817 ◽

2009 ◽

Vol 150 (9) ◽

pp. 4154-4162 ◽

Cited By ~ 18

Author(s):

Daniel P. Sherbet ◽

Oleg L. Guryev ◽

Mahboubeh Papari-Zareei ◽

Dario Mizrachi ◽

Siayareh Rambally ◽

...

Keyword(s):

Nicotinamide Adenine Dinucleotide ◽

Redox State ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Glucose Deprivation ◽

Hydroxysteroid Dehydrogenase ◽

Structural Features ◽

Intact Cells ◽

Hek 293 ◽

Hydroxysteroid Dehydrogenases ◽

293 Cells

Abstract Human 17β-hydroxysteroid dehydrogenase types 1 and 2 (17βHSD1 and 17βHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17βHSD1 and 17βHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17βHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2′-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP+ ratio. The shift to E2 oxidation parallels changes in apparent Km values for purified 17βHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17βHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17βHSD2, and these mutations lower the apparent Km of recombinant 17βHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17βHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP+ ratios. In contrast, the preference of 17βHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17βHSD2 in intact cells.

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Human kidney 11β-hydroxysteroid dehydrogenase: regulation by adrenocorticotropin?

Acta Endocrinologica ◽

10.1530/eje.0.1340301 ◽

1996 ◽

Vol 134 (3) ◽

pp. 301-307 ◽

Cited By ~ 21

Author(s):

S Diederich ◽

M Quinkler ◽

K Miller ◽

P Heilmann ◽

M Schöneshöfer ◽

...

Keyword(s):

Human Kidney ◽

Kinetic Studies ◽

Hydroxysteroid Dehydrogenase ◽

Inhibitory Effect ◽

Benjamin Franklin ◽

Dependent Inhibition ◽

Direct Inhibitory Effect ◽

Dose Dependent Inhibition ◽

Endogenous Substrates ◽

Radioactive Detection

Diederich S, Quinkler M, Miller K, Heilmann P, Schöneshöfer M, Oelkers W. Human kidney 11βhydroxysteroid dehydrogenase: regulation by adrenocorticotropin? Eur J Endocrinol 1996;134:301–7. ISSN 0804–4643 In ectopic adrenocorticotropin (ACTH) syndrome (EAS) with higher ACTH levels than in pituitary Cushing's syndrome and during ACTH infusion, the ratio of cortisol to cortisone in plasma and urine is increased, suggesting inhibition of renal 11β-hydroxysteroid dehydrogenase (11β-HSD) by ACTH or by ACTH-dependent steroids. Measuring the conversion of cortisol to cortisone by human kidney slices under different conditions, we tested the possibility of 11β-HSD regulation by ACTH and corticosteroids. Slices prepared from unaffected parts of kidneys removed because of renal cell carcinoma were incubated with unlabeled or labeled cortisol, and cortisol and cortisone were quantitated after HPLC separation by UV or radioactive detection. The 11β-HSD activity was not influenced by incubation with increasing concentrations (10−12–10−9 mol/l) of ACTH (1–24 or 1–39) for 1 h. Among 12 ACTH-dependent steroids tested (10−9–10−6 mol/l), only corticosterone (IC50 = 2 × 10−7 mol/l), 18-OH-corticosterone and 11βOH-androstenedione showed a significant dose-dependent inhibition of 11β-HSD activity. The percentage conversion rate of cortisol to cortisone was concentration dependent over the whole range of cortisol concentrations tested (10−8–10−5 mol/l). A direct inhibitory effect of ACTH on 11β-HSD is, therefore, unlikely. The only steroids inhibiting the conversion of cortisol to cortisone are natural substrates for 11β-HSD Kinetic studies show a saturation of the enzyme at high cortisol concentrations. Thus, the reduced percentage renal cortisol inactivation in EAS seems to be due mainly to overload of the enzyme with endogenous substrates (cortisol, corticosterone and others) rather than to direct inhibition of 11β-HSD by ACTH or ACTHdependent steroids, not being substrates of 11β-HSD. S Diederich, Department of Endocrinology, Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany

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