Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

Olof Björnberg, Thibault Viennet, Nicklas Skjoldager, Aida Curovic, Kristian Fog Nielsen, Birte Svensson, Per Hägglund

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    Abstract

    Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax = 460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.
    Original languageEnglish
    JournalBiochemistry
    Volume54
    Issue number8
    Pages (from-to)1628-1637
    Number of pages10
    ISSN0006-2960
    DOIs
    Publication statusPublished - 2015

    Cite this

    Björnberg, O., Viennet, T., Skjoldager, N., Curovic, A., Nielsen, K. F., Svensson, B., & Hägglund, P. (2015). Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation. Biochemistry, 54(8), 1628-1637. https://doi.org/10.1021/bi5013639
    Björnberg, Olof ; Viennet, Thibault ; Skjoldager, Nicklas ; Curovic, Aida ; Nielsen, Kristian Fog ; Svensson, Birte ; Hägglund, Per. / Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation. In: Biochemistry. 2015 ; Vol. 54, No. 8. pp. 1628-1637.
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    title = "Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation",
    abstract = "Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax = 460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.",
    author = "Olof Bj{\"o}rnberg and Thibault Viennet and Nicklas Skjoldager and Aida Curovic and Nielsen, {Kristian Fog} and Birte Svensson and Per H{\"a}gglund",
    year = "2015",
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    Björnberg, O, Viennet, T, Skjoldager, N, Curovic, A, Nielsen, KF, Svensson, B & Hägglund, P 2015, 'Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation', Biochemistry, vol. 54, no. 8, pp. 1628-1637. https://doi.org/10.1021/bi5013639

    Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation. / Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas; Curovic, Aida; Nielsen, Kristian Fog; Svensson, Birte; Hägglund, Per.

    In: Biochemistry, Vol. 54, No. 8, 2015, p. 1628-1637.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

    AU - Björnberg, Olof

    AU - Viennet, Thibault

    AU - Skjoldager, Nicklas

    AU - Curovic, Aida

    AU - Nielsen, Kristian Fog

    AU - Svensson, Birte

    AU - Hägglund, Per

    PY - 2015

    Y1 - 2015

    N2 - Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax = 460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.

    AB - Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax = 460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.

    U2 - 10.1021/bi5013639

    DO - 10.1021/bi5013639

    M3 - Journal article

    C2 - 25675241

    VL - 54

    SP - 1628

    EP - 1637

    JO - Biochemistry

    JF - Biochemistry

    SN - 0006-2960

    IS - 8

    ER -

    Björnberg O, Viennet T, Skjoldager N, Curovic A, Nielsen KF, Svensson B et al. Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation. Biochemistry. 2015;54(8):1628-1637. https://doi.org/10.1021/bi5013639