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4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction

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Abstract

4-Hydroxyphenylacetate decarboxylase (4Hpad) is an Fe/S cluster containing glycyl radical enzyme (GRE), which catalyses the last step of tyrosine fermentation in clostridia, generating the bacteriostatic p-cresol. The respective activating enzyme (4Hpad-AE) displays two cysteine-rich motifs in addition to the classical S-adenosylmethionine (SAM) binding cluster (RS cluster) motif. These additional motifs are also present in other glycyl radical activating enzymes (GR-AE) and it has been postulated that these orthologues may use an alternative SAM homolytic cleavage mechanism, generating a putative 3-amino-3-carboxypropyl radical and 5′-deoxy-5′-(methylthio)adenosine but not a 5′-deoxyadenosyl radical and methionine. 4Hpad-AE produced from a codon-optimized synthetic gene binds a maximum of two [4Fe–4S]2+/+ clusters as revealed by EPR and Mössbauer spectroscopy. The enzyme only catalyses the turnover of SAM under reducing conditions, and the reaction products were identified as 5′-deoxyadenosine (quenched form of 5′-deoxyadenosyl radical) and methionine. We demonstrate that the 5′-deoxyadenosyl radical is the activating agent for 4Hpad through p-cresol formation and correlation between the production of 5′-deoxyadenosine and the generation of glycyl radical in 4Hpad. Therefore, we conclude that 4Hpad-AE catalyses a classical SAM-dependent glycyl radical formation as reported for GR-AE without auxiliary clusters. Our observation casts doubt on the suggestion that GR-AE containing auxiliary clusters catalyse the alternative cleavage reaction detected for glycerol dehydratase activating enzyme.

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Abbreviations

4Hpad:

4-Hydroxyphenylacetate decarboxylase

4Hpad-AE:

4-Hydroxyphenylacetate decarboxylase activating enzyme

4Hpad-AE*:

4-Hydroxyphenylacetate decarboxylase activating enzyme treated with Fe/S cluster proteins (12.0 ± 0.3 mol Fe per mole of protein)

Gdh-AE:

Glycerol dehydratase activating enzyme

GR-AE:

Glycyl radical activating enzyme

GRE:

Glycyl radical enzyme

HPLC:

High-performance liquid chromatography

ISC:

Fe/S cluster

ITC:

Isothermal titration calorimetry

Nrd-AE:

Glycyl radical activating enzyme for class III ribonucleotide reductase

Pfl-AE:

Glycyl radical activating enzyme for pyruvate formate lyase

PITC:

Phenylisothiocyanate

RS cluster:

S-Adenosylmethionine-binding [4Fe–4S]2+/1+ cluster

SAM:

S-Adenosylmethionine

TLC:

Thin-layer chromatography

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Acknowledgments

The Deutsche Forschungsgemeinschaft (grants MA 3348/2-1 and MA 3348/2-2) supported this work. Silke Steinborn and Rainer Dietrich (Strukturbiologie/Biochemie group) are acknowledged for excellent technical assistance. We thank Angelica Woyda (Analytische Chemie und Umweltchemie, Humboldt-Universität zu Berlin) for collecting electrospray ionization mass spectrometry data, Bern Miniert (MPI-CEC) for recording the Mössbauer spectra and Tobias Werther for help in the analysis of the ITC experiments. We are grateful to Thorsten Selmer (Fachhochschule Aachen, Germany) for the 4Hpad clone and all members of the Strukturbiologie/Biochemie group for helpful discussions. We also acknowledge the reviewers for valuable suggestions and Wolfgang Buckel (MPI für Terrestrische Mikrobiologie, Marburg, Germany) for critical reading of the manuscript.

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Authors and Affiliations

  1. Institut für Biologie, Strukturbiologie/Biochemie-Radikalenzyme, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099, Berlin, Germany

    Brinda Selvaraj & Berta M. Martins

  2. Core Facility for Protein Spectroscopy, Institute of Cytobiology and Cytopathology, Philipps University Marburg, Robert-Koch-Strasse 6, 35032, Marburg, Germany

    Antonio J. Pierik

  3. MPI für Chemische Energie Konversion (MPI-CEC), Stiftstrasse 34-36, 45470, Mülheim, Germany

    Eckhard Bill

Authors
  1. Brinda Selvaraj
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  2. Antonio J. Pierik
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  3. Eckhard Bill
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  4. Berta M. Martins
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Correspondence to Berta M. Martins.

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Selvaraj, B., Pierik, A.J., Bill, E. et al. 4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction. J Biol Inorg Chem 18, 633–643 (2013). https://doi.org/10.1007/s00775-013-1008-2

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