Altered substrate specificity in flavocytochrome b(2)

structural insights into the mechanism of L-lactate dehydrogenation

Christopher G Mowat, Annemarie Wehenkel, Amanda J Green, Malcolm D Walkinshaw, Graeme A Reid, Stephen K Chapman

    Research output: Contribution to journalArticle

    Abstract

    Flavocytochrome b(2) from Saccharomyces cerevisiae is a L-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301,.829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate L-mandelate than it is With L-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 Angstrom resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 Angstrom resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.

    Original languageEnglish
    Pages (from-to)9519-9526
    Number of pages8
    JournalBiochemistry
    Volume43
    Issue number29
    DOIs
    Publication statusPublished - 27 Jul 2004

    Keywords

    • ESCHERICHIA-COLI
    • RESOLUTION
    • MUTANT
    • (S)-MANDELATE DEHYDROGENASE
    • PSEUDOMONAS-PUTIDA
    • FLAVOPROTEIN
    • AMINO-ACID OXIDASE
    • YEAST FLAVOCYTOCHROME-B2
    • EXPRESSION
    • ACTIVE-SITE

    Cite this

    Mowat, C. G., Wehenkel, A., Green, A. J., Walkinshaw, M. D., Reid, G. A., & Chapman, S. K. (2004). Altered substrate specificity in flavocytochrome b(2): structural insights into the mechanism of L-lactate dehydrogenation. Biochemistry, 43(29), 9519-9526. https://doi.org/10.1021/bi049263m
    Mowat, Christopher G ; Wehenkel, Annemarie ; Green, Amanda J ; Walkinshaw, Malcolm D ; Reid, Graeme A ; Chapman, Stephen K. / Altered substrate specificity in flavocytochrome b(2) : structural insights into the mechanism of L-lactate dehydrogenation. In: Biochemistry. 2004 ; Vol. 43, No. 29. pp. 9519-9526.
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    abstract = "Flavocytochrome b(2) from Saccharomyces cerevisiae is a L-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301,.829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate L-mandelate than it is With L-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 Angstrom resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 Angstrom resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.",
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    Mowat, CG, Wehenkel, A, Green, AJ, Walkinshaw, MD, Reid, GA & Chapman, SK 2004, 'Altered substrate specificity in flavocytochrome b(2): structural insights into the mechanism of L-lactate dehydrogenation', Biochemistry, vol. 43, no. 29, pp. 9519-9526. https://doi.org/10.1021/bi049263m

    Altered substrate specificity in flavocytochrome b(2) : structural insights into the mechanism of L-lactate dehydrogenation. / Mowat, Christopher G; Wehenkel, Annemarie ; Green, Amanda J ; Walkinshaw, Malcolm D; Reid, Graeme A; Chapman, Stephen K.

    In: Biochemistry, Vol. 43, No. 29, 27.07.2004, p. 9519-9526.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Altered substrate specificity in flavocytochrome b(2)

    T2 - structural insights into the mechanism of L-lactate dehydrogenation

    AU - Mowat, Christopher G

    AU - Wehenkel, Annemarie

    AU - Green, Amanda J

    AU - Walkinshaw, Malcolm D

    AU - Reid, Graeme A

    AU - Chapman, Stephen K

    PY - 2004/7/27

    Y1 - 2004/7/27

    N2 - Flavocytochrome b(2) from Saccharomyces cerevisiae is a L-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301,.829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate L-mandelate than it is With L-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 Angstrom resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 Angstrom resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.

    AB - Flavocytochrome b(2) from Saccharomyces cerevisiae is a L-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301,.829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate L-mandelate than it is With L-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 Angstrom resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 Angstrom resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.

    KW - ESCHERICHIA-COLI

    KW - RESOLUTION

    KW - MUTANT

    KW - (S)-MANDELATE DEHYDROGENASE

    KW - PSEUDOMONAS-PUTIDA

    KW - FLAVOPROTEIN

    KW - AMINO-ACID OXIDASE

    KW - YEAST FLAVOCYTOCHROME-B2

    KW - EXPRESSION

    KW - ACTIVE-SITE

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    JO - Biochemistry

    JF - Biochemistry

    SN - 0006-2960

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