Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding

Sarah J Thackray, Chiara Bruckmann, J L Ross Anderson, Laura P Campbell, Rong Xiao, Li Zhao, Christopher G Mowat, Farhad Forouhar, Liang Tong, Stephen K Chapman

    Research output: Contribution to journalArticle

    Abstract

    Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific hemecontaining enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe(2+)) form and in complex with its substrate L-tryptophan (L-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to L-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 angstrom resolution, respectively, in binary complexes with L-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of L-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K(d) values for L-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 mu M reduced), H55A TDO (11.8 mu M oxidized, 3.7 mu M reduced), and H55S TDO (18.4 mu M oxidized, 5.3 mu M) reduced).

    Original languageEnglish
    Pages (from-to)10677-10684
    Number of pages8
    JournalBiochemistry
    Volume47
    Issue number40
    DOIs
    Publication statusPublished - 7 Oct 2008

    Fingerprint

    Tryptophan Oxygenase
    Catalysis
    Histidine
    Tryptophan
    Catalytic Domain
    Enzymes
    Xanthomonas campestris
    Indoleamine-Pyrrole 2,3,-Dioxygenase
    Alanine
    Serine
    Hydrogen

    Keywords

    • REDOX
    • TOLERANCE
    • ENZYME
    • KYNURENINE PATHWAY
    • MECHANISM
    • EXPRESSION
    • METABOLISM
    • HUMAN INDOLEAMINE 2,3-DIOXYGENASE
    • CATABOLISM
    • CRYSTAL-STRUCTURE

    Cite this

    Thackray, S. J., Bruckmann, C., Anderson, J. L. R., Campbell, L. P., Xiao, R., Zhao, L., ... Chapman, S. K. (2008). Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding. Biochemistry, 47(40), 10677-10684. https://doi.org/10.1021/bi801202a
    Thackray, Sarah J ; Bruckmann, Chiara ; Anderson, J L Ross ; Campbell, Laura P ; Xiao, Rong ; Zhao, Li ; Mowat, Christopher G ; Forouhar, Farhad ; Tong, Liang ; Chapman, Stephen K. / Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding. In: Biochemistry. 2008 ; Vol. 47, No. 40. pp. 10677-10684.
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    abstract = "Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific hemecontaining enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe(2+)) form and in complex with its substrate L-tryptophan (L-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to L-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 angstrom resolution, respectively, in binary complexes with L-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of L-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K(d) values for L-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 mu M reduced), H55A TDO (11.8 mu M oxidized, 3.7 mu M reduced), and H55S TDO (18.4 mu M oxidized, 5.3 mu M) reduced).",
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    Thackray, SJ, Bruckmann, C, Anderson, JLR, Campbell, LP, Xiao, R, Zhao, L, Mowat, CG, Forouhar, F, Tong, L & Chapman, SK 2008, 'Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding', Biochemistry, vol. 47, no. 40, pp. 10677-10684. https://doi.org/10.1021/bi801202a

    Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding. / Thackray, Sarah J; Bruckmann, Chiara; Anderson, J L Ross; Campbell, Laura P; Xiao, Rong; Zhao, Li; Mowat, Christopher G; Forouhar, Farhad; Tong, Liang; Chapman, Stephen K.

    In: Biochemistry, Vol. 47, No. 40, 07.10.2008, p. 10677-10684.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding

    AU - Thackray, Sarah J

    AU - Bruckmann, Chiara

    AU - Anderson, J L Ross

    AU - Campbell, Laura P

    AU - Xiao, Rong

    AU - Zhao, Li

    AU - Mowat, Christopher G

    AU - Forouhar, Farhad

    AU - Tong, Liang

    AU - Chapman, Stephen K

    N1 - M1 - Article

    PY - 2008/10/7

    Y1 - 2008/10/7

    N2 - Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific hemecontaining enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe(2+)) form and in complex with its substrate L-tryptophan (L-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to L-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 angstrom resolution, respectively, in binary complexes with L-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of L-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K(d) values for L-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 mu M reduced), H55A TDO (11.8 mu M oxidized, 3.7 mu M reduced), and H55S TDO (18.4 mu M oxidized, 5.3 mu M) reduced).

    AB - Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific hemecontaining enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe(2+)) form and in complex with its substrate L-tryptophan (L-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to L-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 angstrom resolution, respectively, in binary complexes with L-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of L-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K(d) values for L-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 mu M reduced), H55A TDO (11.8 mu M oxidized, 3.7 mu M reduced), and H55S TDO (18.4 mu M oxidized, 5.3 mu M) reduced).

    KW - REDOX

    KW - TOLERANCE

    KW - ENZYME

    KW - KYNURENINE PATHWAY

    KW - MECHANISM

    KW - EXPRESSION

    KW - METABOLISM

    KW - HUMAN INDOLEAMINE 2,3-DIOXYGENASE

    KW - CATABOLISM

    KW - CRYSTAL-STRUCTURE

    U2 - 10.1021/bi801202a

    DO - 10.1021/bi801202a

    M3 - Article

    VL - 47

    SP - 10677

    EP - 10684

    JO - Biochemistry

    JF - Biochemistry

    SN - 0006-2960

    IS - 40

    ER -