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Crystal Structure of Human Glutathione S-Transferase A3-3 and Mechanistic Implications for Its High Steroid Isomerase Activity†,‡

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Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, Maryland 21702, Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, and Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205
Cite this: Biochemistry 2004, 43, 50, 15673–15679
Publication Date (Web):November 17, 2004
https://doi.org/10.1021/bi048757g
Copyright © 2004 American Chemical Society
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    Abstract

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    The crystal structure of human class alpha glutathione (GSH) S-transferase A3-3 (hGSTA3-3) in complex with GSH was determined at 2.4 Å. Despite considerable amino acid sequence identity with other human class alpha GSTs (e.g., hGSTA1-1), hGSTA3-3 is unique due to its exceptionally high steroid double bond isomerase activity for the transformation of Δ5-androstene-3,17-dione (Δ5-AD) to Δ4-androstene-3,17-dione. A comparative analysis of the active centers of hGSTA1-1 and hGSTA3-3 reveals that residues in positions 12 and 208 may contribute to their disparate isomerase activity toward Δ5-AD. Substitution of these two residues of hGSTA3-3 with the corresponding residues in hGSTA1-1 followed by kinetic characterization of the wild-type and the mutant enzymes supported this prediction. On the basis of our model of the hGSTA3-3·GSH·Δ5-AD ternary complex and available biochemical data, we propose that the thiolate group of deprotonated GSH (GS-) serves as a base to initiate the reaction by accepting a proton from the steroid and the nonionized hydroxyl group of catalytic residue Y9 (HO−Y9) functions as part of a proton-conducting wire to transfer a proton back to the steroid. Residue R15 may function to stabilize the deprotonated thiolate group of GSH (GS-), and a GSH-bound water molecule may donate a hydrogen bond to the 3-keto group of Δ5-AD and thus help the thiolate of GS- to initiate the proton transfer and the subsequent stabilization of the reaction intermediate.

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    †

     This work was supported in part by USPHS Grants CA076348 (to S.V.S.), awarded by the National Cancer Institute, and ES07804 (to P.Z.) and ES009140 (to S.V.S. and P.Z.), awarded by the National Institute of Environmental Health Sciences.

    ‡

     The atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession code 1TDI.

    §

     National Cancer Institute.

    ‖

     University of Pittsburgh.

    ⊥

     Current address: Neopharma Inc.

    #

     University of Arkansas for Medical Sciences.

    *

     Address correspondence to this author. Tel:  (301) 846-5035. Fax:  (301) 846-6073. E-mail:  [email protected].

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    2. Diogo Vila-Viçosa, Vitor H. Teixeira, Hugo A. F. Santos, and Miguel Machuqueiro . Conformational Study of GSH and GSSG Using Constant-pH Molecular Dynamics Simulations. The Journal of Physical Chemistry B 2013, 117 (25) , 7507-7517. https://doi.org/10.1021/jp401066v
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    10. Jin-Seok Noh, Joo-Won Lee, Ji-Eun Cha, Kwang-Hoon Kong. Engineering High Catalytic Efficiency of the Steroid Isomerase Activity of Human Glutathione S-transferase P1-1. Bulletin of the Korean Chemical Society 2013, 34 (2) , 645-648. https://doi.org/10.5012/bkcs.2013.34.2.645
    11. Baojian Wu, Dong Dong. Human cytosolic glutathione transferases: structure, function, and drug discovery. Trends in Pharmacological Sciences 2012, 33 (12) , 656-668. https://doi.org/10.1016/j.tips.2012.09.007
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    16. Kaspars Tars, Birgit Olin, Bengt Mannervik. Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class Glutathione Transferases. Journal of Molecular Biology 2010, 397 (1) , 332-340. https://doi.org/10.1016/j.jmb.2010.01.023
    17. Yusuke Iwasaki, Yusuke Saito, Yuki Nakano, Keisuke Mochizuki, Osamu Sakata, Rie Ito, Koichi Saito, Hiroyuki Nakazawa. Chromatographic and mass spectrometric analysis of glutathione in biological samples. Journal of Chromatography B 2009, 877 (28) , 3309-3317. https://doi.org/10.1016/j.jchromb.2009.07.001
    18. Larry J. Jolivette, Sean Ekins. Methods for Predicting Human Drug Metabolism. 2007, 131-176. https://doi.org/10.1016/S0065-2423(06)43005-5
    19. Françoise Raffalli-Mathieu, David Persson, Bengt Mannervik. Differences between bovine and human steroid double-bond isomerase activities of Alpha-class glutathione transferases selectively expressed in steroidogenic tissues. Biochimica et Biophysica Acta (BBA) - General Subjects 2007, 1770 (1) , 130-136. https://doi.org/10.1016/j.bbagen.2006.06.015
    20. Sharad Singhal, Kenneth Drake, Sushma Yadav, Jyotsana Singhal, Sanjay Awasthi. Enzymology of Glutathione S-Transferases. 2006, 339-358. https://doi.org/10.1201/9781420004489.ch16
    21. Piotr Zimniak. Substrates and Reaction Mechanisms of Glutathione Transferases. 2006, 71-101. https://doi.org/10.1201/9781420004489.ch5
    22. Hui Xiao, Shivendra Singh. Specificity of Glutathione S-Transferases in the Glutathione Conjugation of Carcinogenic Diol Epoxides. 2006, 103-128. https://doi.org/10.1201/9781420004489.ch6
    23. Shu-Chuan Jao, Jessica Chen, Kelvin Yang, Wen-Shan Li. Design of potent inhibitors for Schistosoma japonica glutathione S-transferase. Bioorganic & Medicinal Chemistry 2006, 14 (2) , 304-318. https://doi.org/10.1016/j.bmc.2005.07.077
    24. Françoise Raffalli‐Mathieu, Bengt Mannervik. Human Glutathione Transferase A3‐3 Active as Steroid Double‐Bond Isomerase. 2005, 265-278. https://doi.org/10.1016/S0076-6879(05)01017-7
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