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Literature summary for 1.97.1.4 extracted from

  • 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 (2017), J. Am. Chem. Soc., 139, 11803-11813 .
    View publication on PubMedView publication on EuropePMC
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Cloned(Commentary)

Cloned (Comment) Organism
gene pflA, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Escherichia coli

Protein Variants

Protein Variants Comment Organism
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 Escherichia coli
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 Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ conserved cysteines coordinate three irons of a [4Fe-4S] cluster, while SAM coordinates the fourth iron through its amino and carboxylate moieties Escherichia coli
K+ the presence and identity of the bound monovalent cation, requiring a K+ ion bound in the active site for optimal activity Escherichia coli
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 Escherichia coli
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 Escherichia coli
[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 Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P0A9N4
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-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3) Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
S-adenosyl-L-methionine + 5-deazariboflavin + [formate C-acetyltransferase]-glycine
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 Escherichia coli 5'-deoxyadenosine + L-methionine + 5-deazariboflavin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical
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 ?

Synonyms

Synonyms Comment Organism
PFL-AE
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 Escherichia coli
Pyruvate formate-lyase activating enzyme
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
 assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
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 Escherichia coli

General Information

General Information Comment Organism
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 Escherichia coli
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 Escherichia coli