Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form

The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5'-deoxyadenosyl radical intermediate produced by the activase has been sug...

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Bibliographic Details
Published in:The Journal of biological chemistry 1998-02, Vol.273 (9), p.4897-4903
Main Authors: Külzer, R, Pils, T, Kappl, R, Hüttermann, J, Knappe, J
Format: Article
Language:English
Subjects:
Acetyltransferases
Amino Acid Sequence
Apoproteins - metabolism
Conserved Sequence
Cysteine - genetics
Electron Spin Resonance Spectroscopy
Enzyme Activation
Enzymes - biosynthesis
Enzymes - genetics
Escherichia coli
Escherichia coli - genetics
Iron - metabolism
Iron-Sulfur Proteins - biosynthesis
Iron-Sulfur Proteins - genetics
Ligands
Molecular Sequence Data
Mutagenesis, Site-Directed
Recombinant Proteins - biosynthesis
S-Adenosylhomocysteine - metabolism
S-Adenosylmethionine - metabolism
Spectrophotometry
Sulfides - metabolism
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Summary:The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5'-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S]+) from axial to rhombic symmetry.
ISSN:0021-9258
DOI:10.1074/jbc.273.9.4897