Reconstitution and Characterization of the Polynuclear Iron-Sulfur Cluster in Pyruvate Formate-lyase-activating Enzyme

Abstract

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 3Ser 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.Pyruvate formate-lyase (PFL) 1 is a key enzyme of the anaerobic glucose fermentation in Escherichia coli and other microorganisms, catalyzing the CoA-dependent cleavage of pyruvate to acetyl-CoA and formate (1). In its active form, this enzyme contains a stable glycyl radical (Gly-734) required for catalysis (2). Studies of mutants and substrate analogs propose that on substrate binding, the spin is transferred from Gly-734 to the reaction center (Cys-418/Cys-419), where a thiyl radical initiates the homolytic cleavage of the pyruvate C-C bond (3, 4).The PFL radical is produced post-translationally by abstraction of the H si atom from the Gly-734 methylene group (5). This occurs by the action of pyruvate formate-lyase-activating enzyme (PFL activase), which employs adenosylmethionine (AdoMet) and dihydroflavodoxin (or artificial 1eϪ donors) as co-substrates, yielding 5Ј-deoxyadenosine and methionine as stoichiometric co-products (Equation 1).Because the abstracted H atom is recovered in the 5Ј-CH 3 of deoxyadenosine, a 5Ј-deoxyadenosyl radical intermediate has been proposed as the actual H abstracting species in this system. How this nucleoside radical is generated from AdoMet is an intriguing problem, requiring in the first place PFL activase to be fully characterized at the molecular level. Previous work has identified PFL activase as a monomer of 28 kDa, and its primary structure, as deduced from the DNAnucleotide sequence (246 amino acids), has been established by N-and C-terminal amino acid sequencing (6, 7). The enzyme has long since been recognized to require Fe 2ϩ for catalytic activity (6), and iron contents in the order of one iron/polypeptide chain were determined in purified prepara...

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

“…1) and size exclusion chromatography (Fig. 2) (13)(14)(15)(16). This conclusion is supported by previous chemical (17) and atomic absorption analyses (data not shown) that have indicated a stoichiometry of between 1 and 2 iron or sulfur atoms per (10His)SplB subunit.…”

“…3B) Reversal of SP to two thymines in DNA by SP lyase is absolutely dependent on S-AdoMet (17,18). A common theme in the reaction pathway of radical SAM enzymes is cleavage of S-AdoMet by electron donation from a [4Fe-4S] cluster to generate methionine and a 5Ј-adenosyl radical (12,(13)(14)(15)(16), which would be predicted to result in a reduction in EPR signal strength. To test this notion, purified (10His)SplB was treated on ice for 20 min with 10 mM dithionite and 1 mM S-AdoMet before EPR spectroscopy.…”

“…The region from C91 to C98 was similar to the signature cysteine clusters present in the recently dubbed ''radical SAM'' protein superfamily (11), which includes enzymes such as anaerobic ribonucleotide reductase (RNR), pyruvate formate-lyase (PFL), lysine-2,3-aminomutase (KAM), and biotin synthase (BioB) (12). In radical SAM enzymes, the clustered cysteines serve as ligands in the formation of iron-sulfur ([Fe-S]) centers, and these enzymes use S-adenosylmethionine (S-AdoMet) as a cofactor to generate an adenosyl radical (13)(14)(15)(16). We constructed a version of SplB protein containing an N-terminal tag of 10 histidines [(10His)SplB] and subsequently determined that (10His)SplB (i) contained both iron and acid-labile sulfur in a stoichiometric ratio of 1-2 atoms of iron or sulfur per (10His)SplB subunit (17); (ii) exhibited a UV-visible absorption spectrum consistent with other [Fe-S] proteins (17); (iii) required both anaerobic reducing conditions and S-AdoMet for activity in vitro (17,18); and (iv) was inactive for SP cleavage unless its (10His) tag was first removed proteolytically (17,18).…”

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein

“…12), which are compatible with the presence of [4Fe-4S] 2ϩ clusters (see refs. [29][30][31][32]. Additional evidence for the presence of such a prosthetic group in an artificially restored form of IspG has been provided recently (25), together with a radiochemical demonstration that the reconstructed holoprotein can be activated efficiently, as can a number of other proteins with similar prosthetic groups (33)(34)(35), by the semiquinone radical generated via photoreduction of deazaflavins in the presence of Tris⅐HCl, thiols, and other electron donors (36).…”

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

“…PFL is a glycyl radical enzyme that catalyzes the conversion of pyruvate to formate and acetyl-CoA (28). Introduction of the radical into PFL occurs anaerobically by the PFL-activating enzyme, an iron-sulfur protein that uses S-adenosylmethionine and reduced flavodoxin as cosubstrates (57). Through direct interaction of the coenzyme with the [4Fe-4S] cluster, the PFL activase carries out the reductive cleavage of S-adenosylmethionine, producing a highly reactive 5Ј-deoxyadenosyl radical that abstracts a hydrogen atom from the Gly-734 residue in E. coli PFLB, thus introducing a free radical into the polypeptide chain (36,58).…”