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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P0A9N4 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3) | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
S-adenosyl-L-methionine + 5-deazariboflavin + [formate C-acetyltransferase]-glycine | - |
Escherichia coli | 5'-deoxyadenosine + L-methionine + 5-deazariboflavin semiquinone + [formate C-acetyltransferase]-glycin-2-yl radical | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PFL-AE | - |
Escherichia coli |
Pyruvate formate-lyase activating enzyme | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.6 | - |
assay at | Escherichia coli |
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 | 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 |