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The ferredoxin-like domain of the activating enzyme is required for generating a lasting glycyl radical in 4-hydroxyphenylacetate decarboxylase

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Abstract

4-Hydroxyphenylacetate decarboxylase-activating enzyme (4Hpad-AE) uses S-adenosylmethionine (SAM or AdoMet) and a [4Fe-4S]2+/+ cluster (RS cluster) to generate a stable glycyl radical on the decarboxylase. 4Hpad-AE might bind up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert C-terminal to the RS cluster-binding motif. Except for the AEs of pyruvate formate-lyase and anaerobic ribonucleotide reductase, all glycyl radical-activating enzymes possess a similar ferredoxin-like domain, whose functional role is still poorly understood. To assess the role of the putative ferredoxin clusters from 4Hpad-AE, we combined biochemical and spectroscopic methods to characterize a truncated version of the protein (Δ66-AE) devoid of the ferredoxin-like domain. We found that Δ66-AE is stable, harbors a fully active RS cluster and can activate the decarboxylase. From the similar cleavage rates for S-adenosylmethionine of Δ66-AE and wild-type AE, we infer the reactivity of the RS cluster is unperturbed by the absence of the ferredoxin-like domain. Thus, the auxiliary clusters are not required as electron conduit to the RS cluster for effective reductive cleavage of SAM. The activation of the decarboxylase by Δ66-AE is almost as fast as with wild-type AE, but the generated glycyl radical is short living. We postulate that the ferredoxin-like domain is not required for SAM-dependent glycyl radical generation in the decarboxylase, but is necessary for producing a lasting glycyl radical.

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Abbreviations

Ferredoxin clusters:

Auxiliary [4Fe-4S] clusters coordinated by the ferredoxin-like domain

Nrd-AE:

Class-III ribonucleotide reductase-activating enzyme

EPR:

Electron paramagnetic resonance

GRE:

Glycyl radical enzyme

HPLC:

High-performance liquid chromatography

Δ66-AE:

4Hpad-AE variant devoid of the 2x[4Fe-4S] ferredoxin-like domain

4Hpad-AE:

4-Hydroxyphenylacetate decarboxylase-activating enzyme

Fe/S:

Iron–sulfur cluster

ITC:

Isothermal titration calorimetry

Pfl-AE:

Pyruvate formate-lyase-activating enzyme

RS cluster:

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

WT-AE:

Wild-type 4Hpad-AE (12.0 ± 0.3 mol Fe per mol protein)

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Acknowledgments

The Deutsche Forschungsgemeinschaft (DFG) (Grants MA 3348/2-1 and MA 3348/2-2) supported this work. We thank Thorsten Selmer (FH Aachen, Germany) for the 4Hpad clone, Silke Steinborn and Rainer Dietrich (Strukturbiologie/Biochemie) for technical assistance; Bernd Mienert (MPI-CEC) for recording the Mössbauer spectra; and all members of the Strukturbiologie/Biochemie group for helpful discussions, especially Tobias Werther for help in the analysis of the ITC experiments. We are grateful to Wolfgang Buckel (MPI für terrestrische Mikrobiologie, Marburg, Germany) for continuous support and helpful discussions. We also acknowledge the reviewers for their helpful suggestions.

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Author notes

  1. Antonio J. Pierik

    Present address: Department of Chemistry, University of Kaiserslautern, Erwin-Schrödinger-Strasse 54, 67663, Kaiserslautern, Germany

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, Philipps University, Robert-Koch-Strasse 6, 35032, Marburg, Germany

    Antonio J. Pierik

  3. MPI für Chemische Energiekonversion (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. The ferredoxin-like domain of the activating enzyme is required for generating a lasting glycyl radical in 4-hydroxyphenylacetate decarboxylase. J Biol Inorg Chem 19, 1317–1326 (2014). https://doi.org/10.1007/s00775-014-1189-3

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  • DOI: https://doi.org/10.1007/s00775-014-1189-3

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