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Information on EC 1.97.1.4 - [formate-C-acetyltransferase]-activating enzyme and Organism(s) Escherichia coli and UniProt Accession P0A9N4
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1.97.1.4 [formate-C-acetyltransferase]-activating enzymeIUBMB Comments
An iron-sulfur protein. A single glycine residue in EC 2.3.1.54, formate C-acetyltransferase, is oxidized to the corresponding radical by transfer of H from its CH2 to AdoMet with concomitant cleavage of the latter. The reaction requires Fe2+. The first stage is reduction of the AdoMet to give methionine and the 5'-deoxyadenosin-5'-yl radical, which then abstracts a hydrogen radical from the glycine residue.
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Word Map
- 1.97.1.4
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glycyl
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5\'-deoxyadenosyl
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iron-sulfur
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organometallic
- adomet
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oxygen-sensitive
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homolytic
-
endor
- adenosylmethionine
- 5'-deoxyadenosine
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h-atom
-
sulfonium
-
knappe
-
deoxyadenosyl
- medicine
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Synonyms
pfl-ae, pyruvate formate-lyase activating enzyme, pyruvate formate lyase activating enzyme, pfl activase, pfl-activating enzyme, pyruvate formate-lyase-activating enzyme, pfl activating enzyme, pyruvate formate-lyase activase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Activase, pyruvate formate-lyase
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Formate acetyltransferase activase
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Formate-lyase-activating enzyme
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PFL activase
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PFL-activating enzyme
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PFL-AE
PFL-glycine:S-adenosyl-L-methionine H transferase (flavodoxin-oxidizing, S-adenosyl-L-methionine-cleaving)
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Pyruvate formate-lyase activase
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Pyruvate formate-lyase activating enzyme
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PFL-AE
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine = 5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine = 5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
the glycyl radical in pyruvate formate-lyase is produced by stereospecific abstraction of the pro-S hydrogen of Gly734 by the 5'-deoxyadenosine radical generated in the active center of the enzyme
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S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine = 5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
mechanism, a deoxyadenosyl radical intermediate, generated by the reductive cleavage of S-adenosylmethionine serves as the actual H atom abstracting species
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S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine = 5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
4Fe-4S cluster is involved in catalysis by coordinating S-adenosyl-L-methionine
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
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reduction
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SYSTEMATIC NAME
IUBMB Comments
[formate C-acetyltransferase]-glycine dihydroflavodoxin:S-adenosyl-L-methionine oxidoreductase (S-adenosyl-L-methionine cleaving)
An iron-sulfur protein. A single glycine residue in EC 2.3.1.54, formate C-acetyltransferase, is oxidized to the corresponding radical by transfer of H from its CH2 to AdoMet with concomitant cleavage of the latter. The reaction requires Fe2+. The first stage is reduction of the AdoMet to give methionine and the 5'-deoxyadenosin-5'-yl radical, which then abstracts a hydrogen radical from the glycine residue.
CAS REGISTRY NUMBER
COMMENTARY
206367-15-9
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + 5-deazariboflavin + [formate C-acetyltransferase]-glycine
5'-deoxyadenosine + L-methionine + 5-deazariboflavin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
-
-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine
5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
S-Adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
?
-
-
-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + [pyruvate formate-lyase]-glycine
methionine + 5'-deoxyadenosine + [pyruvate formate-lyase]-glycine radical + flavodoxin
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-
-
-
r
S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine
5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
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-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine
5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
PFL dimer, photoreduced enzyme PFL-AE
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-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
-
-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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-
-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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-
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-
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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formate acetyltransferase-glycine is the inactive form of the enzyme
formate acetyltransferase-glycine-2-yl-radical is the active form of the enzyme
?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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the glycyl radical in pyruvate formate-lyase is produced by stereospecific abstraction of the pro-S hydrogen of Gly734 by the 5'-deoxyadenosine radical generated in the active center of the enzyme
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?
S-adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + flavodoxin + formate acetyltransferase-glycine-2-yl-radical
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the interaction with substrate formate C-acetyltransferase is very slow and rate-limited by large conformational changes. The enzyme binds S-adenosyl-L-methionine with the same affinity of about 0.006 mM regardless of the presence or absence of formate C-acetyltransferase. Activation of formate C-acetyltransferase in the presence of its substrate pyruvate or the analogue oxamate results in stoichiometric conversion of the [4Fe-4S]1+ cluster to the glycyl radical on formate C-acetyltransferase, however 3.7-fold less activation is achieved in the absence of these small molecules. Formate C-acetyltransferase, formate C-acetyltransferase activating enzyme, and S-adenosyl-L-methionine are essentially fully bound in vivo, whereas electron donor proteins are partially bound
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-
?
additional information
?
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in vivo concentrations of the entire PFL system is calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and S-adenosyl-L-methionine are essentially fully bound in vivo, whereas electron donor proteins are partially bound
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-
?
additional information
?
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PFL-AE utilizes S-adenosylmethionine (SAM) and reduced flavodoxin as the cosubstrates to generate a 5'-deoxyadenosyl radical, which then activates PFL by abstracting a hydrogen atom from residue G734
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-
?
additional information
?
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usage of an S-adenosyl-L-methionine binding assay to accurately determine the equilibrium constants for S-adenosyl-L-methionine binding to enzyme PFL-AE alone and in complex with substrate PFL, activation of PFL in the presence of its substrate pyruvate or the analogue oxamate results in stoichiometric conversion of the [4Fe-4S]1+ cluster to the glycyl radical on PFL. 3.7fold less activation is achieved in the absence of these small molecules, demonstrating that pyruvate or oxamate are required for optimal activation. For the assay, the enzyme PFL-AE is attached to a CM5 sensor chip using standard thiol coupling procedures
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?
additional information
?
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the enzyme also activates an enzyme which has both pyruvate formate-lyase activity and 2-ketobutyrate formate-lyase activity
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?
additional information
?
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the enzyme also activates an enzyme which has both pyruvate formate-lyase activity and 2-ketobutyrate formate-lyase activity
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?
additional information
?
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a DELTAAla-containing peptide which lacks hydrogens at the 734-Calpha atom is recognized by the enzyme and is able to trap covalently the nucleophilic 5-deoxyadenosine radical
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-
?
additional information
?
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pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase by generating a catalytically essential radical on Gly-734 of pyruvate formate-lyase. PFL-AE shifts the closed/open formation of pyruvate formate-lyase to the open conformation, in which Gly-734 is more solvent-exposed and accessible to the PFL-AE active site
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-
?
additional information
?
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pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase by generating a catalytically essential radical on Gly-734 of pyruvate formate-lyase. PFL-AE shifts the closed/open formation of pyruvate formate-lyase to the open conformation, in which Gly-734 is more solvent-exposed and accessible to the PFL-AE active site
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + dihydroflavodoxin + [formate C-acetyltransferase]-glycine
5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
-
-
-
?
S-Adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
?
-
-
-
-
?
additional information
?
-
in vivo concentrations of the entire PFL system is calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and S-adenosyl-L-methionine are essentially fully bound in vivo, whereas electron donor proteins are partially bound
-
-
?
additional information
?
-
-
pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase by generating a catalytically essential radical on Gly-734 of pyruvate formate-lyase. PFL-AE shifts the closed/open formation of pyruvate formate-lyase to the open conformation, in which Gly-734 is more solvent-exposed and accessible to the PFL-AE active site
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-
?
additional information
?
-
pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase by generating a catalytically essential radical on Gly-734 of pyruvate formate-lyase. PFL-AE shifts the closed/open formation of pyruvate formate-lyase to the open conformation, in which Gly-734 is more solvent-exposed and accessible to the PFL-AE active site
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
[4Fe-4S]-center
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an increase in pre-edge intensity is due to additional contributions from sulfide and thiolate of the Fe4S4 cluster into the C-S sigma* orbital. There is a backbonding interaction between the Fe4S4 cluster and C-S sigma* orbitals of S-adenosyl-L-methionine in this inner sphere complex. This backbonding is enhanced in the reduced form and this configurational interaction between the donor and acceptor orbitals facilitates the electron transfer from the cluster to S-adenosyl-L-methionine, that otherwise has a large outer sphere electron transfer barrier. The energy of the reductive cleavage of the C-S bond is sensitive to the dielectric of the protein in the immediate vicinity of the site as a high dielectric stabilizes the more charge separated reactant increasing the reaction barrier
additional information
reduced flavodoxin serves as an electron donor and SAM as a cosubstrate for PFL-AE to generate a 5'-deoxyadenosyl radical, which is responsible for PFL activation
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S-adenosyl-L-methionine
pyruvate formate-lyase activating enzyme is a radical SAM enzyme. Conserved cysteines coordinate three irons of a [4Fe-4S] cluster, while SAM coordinates the fourth iron through its amino and carboxylate moieties
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
conserved cysteines coordinate three irons of a [4Fe-4S] cluster, while SAM coordinates the fourth iron through its amino and carboxylate moieties
K+
the presence and identity of the bound monovalent cation, requiring a K+ ion bound in the active site for optimal activity
Na+
Na+ as the most likely ion present in the solved enzyme structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23Na in the solution state of the as isolated enzyme
[4Fe-4S] cluster
Cobalt
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Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometry
copper
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Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometry
Iron
additional information
enzyme PFL-AE binds a catalytically essential monovalent cation at its active site. PFL-AE is thus a type I M+-activated enzyme whose M+ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster. PFL-AE in the absence of any simple monovalent cations has little or no activity, and among monocations, going down Group 1 of the periodic table from Li+ to Cs+, PFL-AE activity sharply maximizes at K+ and NH4+. Cation binding site structure, e.g. with Mg2+, Cs+, Ca2+, Tl+, Li+, Zn2+, K+, NH4+, and Na+, overview. Modeling of different cations bound to the cation binding site of the enzyme, negative Fo-Fc electron density appears when the site is modeled as potassium or calcium, more extensive positive Fo-Fc electron density is present in the site when modeled with water than when modeled with sodium or magnesium. Residue D104 is important for cation binding
[4Fe-4S] cluster
conserved cysteines coordinate three irons of a [4Fe-4S] cluster, while SAM coordinates the fourth iron through its amino and carboxylate moieties. In the absence of SAM, the signal from the [4Fe-4S]+ cluster changes with the presence and identity of the cation
[4Fe-4S] cluster
the [4Fe-4S] cluster of enzyme PFL-AE is coordinated by the cysteines of a conserved CX3CX2C motif, with the fourth unique iron coordinated by S-adenosyl-L-methionine. PFL-AE contains six cysteine residues (Cys12, Cys29, Cys33, Cys36, Cys94, Cys102) and only Cys29, Cys33, and Cys36 are involved in coordinating the iron sulfur cluster
Iron
-
[4Fe-4S]2+clusters at the subunit interface can undergoe reversible oxidative conversion to [2Fe-2S]2+clusters under conditions of incomplete anaerobicity
Iron
-
binds one Fe(II) per protein monomer. Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometry
Iron
-
anaerobically purified enzyme, 4FE-4S cluster in a diamagnetic 2+ oxidation state
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
peptides
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peptides homologous to the Gly734 site of pyruvate formate-lyase that are active as substrates
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
contains a covalently bound chromophoric factor which has an optical absorptiion peak at 388 nm
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
kinetics and equilibrium constant for the enzyme's interaction with substrate PFL, the interaction is very slow and rate-limited by large conformational changes, circular dichroism study
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
pyruvate formate-lyase activating enzyme (PFL-AE) is a member of the large and diverse radical S-adenosyl-L-methionine (SAM) superfamily, members of which use an iron-sulfur cluster and SAM to initiate difficult radical transformations in all kingdoms of life. Radical SAM enzymes share a common CX3CX2C motif or variation thereof, and the conserved cysteines coordinate three irons of a [4Fe-4S] cluster, while SAM coordinates the fourth iron through its amino and carboxylate moieties
metabolism
enzyme is activated by pyruvate formate-lyase-activating enzyme by generating a catalytically essential radical on residue Gly734. In the open conformation of the enzyme, the Gly734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. The presence of the activating enzyme increases the proportion of enzyme in the open conformation. The activating enzyme accesses residue Gly734 for direct hydrogen atom abstraction by binding to the Gly734 loop in the open conformation, thereby shifting the closed open equilibrium of the enzyme to the right
physiological function
physiological function
physiological function
PFL-AE is a radical S-adenosyl-L-methionine enzyme that utilizes an iron-sulfur cluster and S-adenosyl-L-methionine to activate pyruvate formate lyase (PFL) via pro-S hydrogen abstraction from Gly734
physiological function
pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-L-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate lyase
physiological function
pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase
physiological function
-
holo-flavodoxin is capable of associating with NADP+-dependent flavodoxin oxidoreductase and pyruvate formate-lyase activating enzyme, whereas there is no detectable interaction between apo-flavodoxin. Holo-flavodoxin interacts with pyruvate formate-lyase activating enzyme with a dissociation constant of 23.3 microM
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
28000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
model in which the enzyme can exist in either a closed conformation, with residue Gly734 buried in the active site and harboring a stable glycyl radical, or an open conformation, with Gly734 more solvent-exposed and accessible to the activating enzyme's active site
additional information
model in which the enzyme can exist in either a closed conformation, with residue Gly734 buried in the active site and harboring a stable glycyl radical, or an open conformation, with Gly734 more solvent-exposed and accessible to the activating enzyme's active site
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
modeling of the complex between pyruvate formate-lyase activating enzyme and flavodoxin. In the pyruvate formate-lyase activating enzyme/flavodoxin complex, FMN is located 10.7 A from the [4Fe-4S] cluster in pyruvate formate-lyase activating enzyme. The flavodoxin binding site on pyruvate formate-lyase activating enzyme is the only location other than the pyruvate formate-lyase binding site where the [4Fe-4S] cluster is close to the surface of the enzyme, which would be necessary for efficient electron transfer
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D104A
site-directed mutagenesis, mutation of the cation binding site, the D104A variant has very low activity in presence of KCl compared to the wild-type, S-adenosyl-L-methionine does not bind well in this variant
D129A
site-directed mutagenesis, mutation of the cation binding site, the mutant retains the ability to bind cations, the variant binds M+ and SAM in a manner similar to wild-type
C102S
-
mutant enzymes C12S, C94S, C102S display full holoactivase activity, albeit absolute values are slightly lower, by a factor of 2 than the value of the wild type enzyme. Mutant enzymes C29S, C33S and C36S are catalytically incompetent
C12S
-
mutant enzymes C12S, C94S, C102S display full holoactivase activity, albeit absolute values are slightly lower, by a factor of 2 than the value of the wild type enzyme. Mutant enzymes C29S, C33S and C36S are catalytically incompetent
C29S
-
mutant enzymes C12S, C94S, C102S display full holoactivase activity, albeit absolute values are slightly lower, by a factor of 2 than the value of the wild type enzyme. Mutant enzymes C29S, C33S and C36S are catalytically incompetent
C33S
-
mutant enzymes C12S, C94S, C102S display full holoactivase activity, albeit absolute values are slightly lower, by a factor of 2 than the value of the wild type enzyme. Mutant enzymes C29S, C33S and C36S are catalytically incompetent
C36S
-
mutant enzymes C12S, C94S, C102S display full holoactivase activity, albeit absolute values are slightly lower, by a factor of 2 than the value of the wild type enzyme. Mutant enzymes C29S, C33S and C36S are catalytically incompetent
C94S
-
mutant enzymes C12S, C94S, C102S display full holoactivase activity, albeit absolute values are slightly lower, by a factor of 2 than the value of the wild type enzyme. Mutant enzymes C29S, C33S and C36S are catalytically incompetent
additional information
additional information
for activity assay, the enzyme PFL-AE is attached to a CM5 sensor chip using standard thiol coupling procedures
additional information
recombinant coexpression of the enzyme with pyruvate format lyase and flavodoxin or ferredoxin in Saccharomyces cerevisiae leads to over 20fold increased expression of endogenous formate dehydrogenases FDH1 and FDH2
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene pflA, coexpression of pyruvate formate lyase (PFL, gene pflB, UniProt ID P09373 ) with the pyruvate formate lyase activating enzyme, as well as with appropriate electron donors flavodoxin and ferredoxin (encoded by genes fldA and fdx, respectively), in a non-ethanol-producing Saccharomyces cerevisiae strain IMI076 lacking pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc- MTH1-DELTAT ura3-52), plasmid maps, overview. Reduced flavodoxin is the preferred electron donor for PFL, but coexpression of either of the electron donors has a positive effect on growth under aerobic conditions. Subcloning in Escherichia coli strain DH5alpha. The PFL pathway can be functional at aerobic growth conditions in yeast when coexpressed with appropriate electron donors
gene pflA, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Johnson, M.K.; Staples, C.R.; Duin, E.C.; Lafferty, M.E.; Duderstadt, R.E.
Novel roles for Fe-S clusters in stabilizing or generating radical intermediates
Pure Appl. Chem.
70
939-946
1998
Escherichia coli
-
Kulzer, R.; Pils, T.; Kappl, R.; Huttermann, J.; Knappe, J.
Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form
J. Biol. Chem.
273
4897-4903
1998
Escherichia coli
Frey, M.; Rothe, M.; Wagner, A.F.V.; Knappe, J.
Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom
J. Biol. Chem.
269
12432-12437
1994
Escherichia coli
Wong, K.K.; Murray, B.W.; Lewisch, S.A.; Baxter, M.K.; Ridky, T.W.; Ulissi-DeMario, L.; Kozarich, J.W.
Molecular properties of pyruvate formate-lyase activating enzyme
Biochemistry
32
14102-14110
1993
Escherichia coli
Wagner, A.F.V.; Demand, J.; Schilling, G.; Pils, T.; Knappe, J.
A dehydroalanyl residue can capture the 5'-deoxyadenosyl radical generated from S-adenosylmethionine by pyruvate formate-lyase-activating enzyme
Biochem. Biophys. Res. Commun.
254
306-310
1999
Escherichia coli
Conradt, H.; Hohmann-Berger, M.; Hohmann, H.P.; Blaschkowski, H.P.; Knappe, J.
Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties
Arch. Biochem. Biophys.
228
133-142
1984
Escherichia coli
Rdel, W.; Plaga, W.; Frank, W.; Knappe, J.
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Hesslinger, C.; Fairhurst, S.A.; Sawers, G.
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Escherichia coli
Broderick, J.B.; Henshaw, T.F.; Cheek, J.; Wojtuszewski, K.; Smith, S.R.; Trojan, M.R.; McGhan, R.M.; Kopf, A.; Kibbey, M.; Broderick, W.E.
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Escherichia coli
Krebs, C.; Broderick, W.E.; Henshaw, T.F.; Broderick, J.B.; Huynh, B.H.
Coordination of adenosylmethionine to a unique iron site of the [4Fe-4S] of pyruvate formate-lyase activating enzyme: a moessbauer spectroscopic study
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Escherichia coli
Peng, Y.; Veneziano, S.E.; Gillispie, G.D.; Broderick, J.B.
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Escherichia coli, Escherichia coli (P0A9N4)
Crain, A.V.; Broderick, J.B.
Flavodoxin cofactor binding induces structural changes that are required for protein-protein interactions with NADP(+) oxidoreductase and pyruvate formate-lyase activating enzyme
Biochim. Biophys. Acta
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Escherichia coli
Dey, A.; Peng, Y.; Broderick, W.E.; Hedman, B.; Hodgson, K.O.; Broderick, J.B.; Solomon, E.I.
S K-edge XAS and DFT calculations on SAM dependent pyruvate formate-lyase activating enzyme: nature of interaction between the Fe4S4 cluster and SAM and its role in reactivity
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Crain, A.V.; Broderick, J.B.
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Escherichia coli
Zhang, Y.; Dai, Z.; Krivoruchko, A.; Chen, Y.; Siewers, V.; Nielsen, J.
Functional pyruvate formate lyase pathway expressed with two different electron donors in Saccharomyces cerevisiae at aerobic growth
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Escherichia coli (P0A9N4)
Shisler, K.A.; Hutcheson, R.U.; Horitani, M.; Duschene, K.S.; Crain, A.V.; Byer, A.S.; Shepard, E.M.; Rasmussen, A.; Yang, J.; Broderick, W.E.; Vey, J.L.; Drennan, C.L.; Hoffman, B.M.; Broderick, J.B.
Monovalent cation activation of the radical SAM enzyme pyruvate formate-lyase activating enzyme
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Escherichia coli (P0A9N4)
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