Hydrogen Exchange of the Glycyl Radical of Pyruvate Formate-Lyase Is Catalyzed by Cysteine 419

Parast, Camran V.; Wong, Kenny K.; Lewisch, Sandra A.; Kozarich, John W.; Peisach, Jack; Magliozzo, Richard S.

doi:10.1021/bi00008a001

Cited by 78 publications

(125 citation statements)

References 17 publications

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“…H/D exchange at Gly-734 requires that this residue be in close proximity to Cys-419, which mediates the exchange via reversible H atom abstraction (supplemental Scheme S3). When PFL is activated with catalytic amounts of PFL-AE, the observed H/D exchange is consistent with that previously reported (13). However, as the ratio of PFL-AE to PFL is increased to 0.1 and 1.0, the extent of H/D exchange at short incubation times decreases, as indicated by the decreased collapse of the Gly-734 radical doublet signal to a singlet.…”

Section: Discussionsupporting

confidence: 91%

“…(2, 3, 13). The active site residue Cys-419 has been demonstrated to be essential for the H/D exchange at Gly-734 because the mutant C419S-PFL does not undergo H/D exchange (13). These results provide evidence that the Gly-734 radical in activated PFL abstracts H ⅐ from Cys-419 to generate a thiyl radical, which can subsequently abstract a hydrogen from Gly-734 to regenerate the glycyl radical; this reversible H ⅐ abstraction involving a species with an exchangeable hydrogen (Cys-419) results in deuterium exchange into the Gly-734 (supplemental Scheme S3).…”

Section: Stability Of the Pfl Glycyl Radical Is Affected By The Presementioning

confidence: 99%

“…The doublet signal collapses to a singlet when activated PFL is diluted in D 2 O, pointing to an exchange of the remaining ␣-H on Gly-734 with solvent (2,12). The unexpected exchange of deuterium from D 2 O into Gly-734 is mediated by the active site residue Cys-419, as demonstrated by the lack of exchange in the mutant C419S-PFL (13). These results provide evidence that the role for Cys-419 in the H/D exchange at Gly-734 is analogous to its role in the catalytic mechanism; specifically, the H/D exchange at Gly-734 is a result of a reversible exchange of an H ⅐ between Gly-734 and Cys-419.…”

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confidence: 93%

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Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

Peng

Veneziano

Gillispie

et al. 2010

Journal of Biological Chemistry

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Section: Discussionsupporting

confidence: 91%

Section: Stability Of the Pfl Glycyl Radical Is Affected By The Presementioning

confidence: 99%

mentioning

confidence: 93%

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Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

Peng

Veneziano

Gillispie

et al. 2010

Journal of Biological Chemistry

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“…The L. lactis sequence ISCCVSP (residues 414 to 420 [ Fig. 4]) is highly conserved and includes two adjacent Cys residues that have been implicated in the cleavage of the C-C bond of pyruvate and hydrogen exchange of the glycyl radical of the enzyme (25).…”

Section: Resultsmentioning

confidence: 99%

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

Arnau¹,

Jørgensen²,

Madsen³

et al. 1997

J Bacteriol

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The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.Pyruvate has a key role in the metabolic network of Lactococcus lactis. In this industrially relevant organism, sugars are metabolized predominantly to generate energy and as a carbon source. The catabolism of sugars leads to pyruvate, whose further metabolism determines the nature of the fermentation (see reference 5 for a review). Lactate is the major end product responsible for acidification, while other minor metabolites, mainly diacetyl, are responsible for aroma in fermented milk products. Several genes involved in pyruvate metabolism have been studied for their role in the production of diacetyl (Fig.

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“…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).…”

mentioning

confidence: 99%

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

Külzer¹,

Pils²,

Kappl³

et al. 1998

Journal of Biological Chemistry

143

124

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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...

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Hydrogen Exchange of the Glycyl Radical of Pyruvate Formate-Lyase Is Catalyzed by Cysteine 419

Cited by 78 publications

References 17 publications

Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

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

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