Information on EC 1.97.1.4 - [formate-C-acetyltransferase]-activating enzyme

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.97.1.4
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RECOMMENDED NAME
GeneOntology No.
[formate-C-acetyltransferase]-activating enzyme
REACTION
REACTION DIAGRAM
COMMENTARY hide
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
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
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 hide
206367-15-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
induction of transcription by anaerobiosis and darkness, but little induction on protein level
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Manually annotated by BRENDA team
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Q6RFH6
SwissProt
Manually annotated by BRENDA team
enzyme activity is present in late log and stationary growth phase of cells grown on cellobiose
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Manually annotated by BRENDA team
enzyme activity is present in late log and stationary growth phase of cells grown on cellobiose
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Manually annotated by BRENDA team
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Swissprot
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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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
malfunction
mutation of either pflB, which codes for PFL, or pflA, which codes for pyruvate formate lyase activating enzyme, results in abrogation of mixed acid fermentation on galactose, and leads to a decrease in pneumococcal virulence
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
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
show the reaction diagram
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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
show the reaction diagram
S-Adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
?
show the reaction diagram
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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
show the reaction diagram
S-adenosyl-L-methionine + NADH + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + NAD+ + formate acetyltransferase-glycine-2-yl-radical
show the reaction diagram
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40% of activity with reduced methyl viologen
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?
S-adenosyl-L-methionine + NADPH + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + NADP+ + formate acetyltransferase-glycine-2-yl-radical
show the reaction diagram
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95% of activity with reduced methyl viologen
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-
?
S-adenosyl-L-methionine + reduced methyl viologen + formate acetyltransferase-glycine
5'-deoxyadenosine + methionine + methyl viologen + formate acetyltransferase-glycine-2-yl-radical
show the reaction diagram
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
S-Adenosyl-L-methionine + dihydroflavodoxin + formate acetyltransferase-glycine
?
show the reaction diagram
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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
show the reaction diagram
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ferredoxin
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can partly substitute for flavodoxin
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flavodoxin
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preferred cofactor
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S-adenosyl-L-methionine
[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
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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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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
K+
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the presence and identity of the bound monovalent cation, requiring a K+ ion bound in the active site for optimal activity
Na+
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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
additional information
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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
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
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 hide
LITERATURE
IMAGE
oxamate
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pyruvate or oxamate are required for optimal activation
pyruvate
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pyruvate or oxamate are required for optimal activation
additional information
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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 hide
LITERATURE
IMAGE
0.0012
inactive pyruvate formate-lyase
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0.0028
S-adenosyl-L-methionine
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additional information
additional information
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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 hide
LITERATURE
43
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25°C, pH 8.1
105
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pH 6.8, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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assay at
7.6
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assay at
7.7
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20% of activity at pH 6.8
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Escherichia coli (strain K12);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28035
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x * 28035, calculation from nucleotide sequence
29500
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1 * 29500, SDS-PAGE
29900
1 * 29900, calculated, 1 * 30000, SDS-PAGE
30000
gel filtration
34000
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gel filtration
40000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 28035, calculation from nucleotide sequence
dimer
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2 * 28000
monomer
additional information
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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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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, presence of Fe(SO4)2(NH4)2 and dithiothreitol, relatively stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)
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using strictly anaerobic conditions
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
clone encodes a putative protein of 8532 amino acids
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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
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gene pflA, ligands CcpA, GlnR, and GntR interact with the putative promoters of pflA and pflB, CcpA and GlnR bind to promoters containing cre sites
gene pflA, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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over-expressed in Escherichia coli
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overexpression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
analysis of the transcriptional regulation of gene pflA, overview
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C102S
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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
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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
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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
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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
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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
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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
D104A
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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
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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
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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in dental plaque, under anaerobic conditions, enzyme is always kept active
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