Cloned (Comment) | Organism |
---|---|
protein expression in Escherichia coli BL21-Gold(DE3) in the presence of 100 microgram/ml Carbenicillin, 30°C | Escherichia phage K1F |
Crystallization (Comment) | Organism |
---|---|
crystal structure of the catalytic domain of endoN from caliphate K1F reveals a functional trimer, folding is mediated by an intramolecular C-terminal chaperone domain | Escherichia phage K1F |
Protein Variants | Comment | Organism |
---|---|---|
Q853A | binding site mutation, active within control range with soluble polysialic acid | Escherichia phage K1F |
R596A/R647A | control active site mutation without affecting maturation or binding, EC50: 1.9 nM polysialic acid | Escherichia phage K1F |
R596A/R647A/Q853A | active site mutation plus binding site mutation, EC50: 5.0 nM surface bound polysialic acid | Escherichia phage K1F |
R596A/R647A/R837A | active site mutation plus binding site mutation, 5-fold increased EC50: 11 nM polysialic acid | Escherichia phage K1F |
R596A/R647A/R837A/S848A | active site mutation plus binding site mutation, EC50: 30 nM surface bound polysialic acid, increased EC50: 30 nM polysialic acid | Escherichia phage K1F |
R596A/R647A/S848A | active site mutation plus binding site mutation, only binding site mutant with SDS resistance which is a criterion for kinetic stabilization of the enzyme, EC50: 4.1 nM surface bound polysialic acid | Escherichia phage K1F |
R596A/R647A/S848A/Q853A | active site mutation plus binding site mutation, increased EC50: 6.2 nM polysialic acid | Escherichia phage K1F |
R596A/R647A/S911A | active site mutation plus cleavage site mutation, tremendously increased EC50: 360 nM polysialic acid | Escherichia phage K1F |
R837A | binding site mutation, increased molar activity with soluble polysialic acid | Escherichia phage K1F |
R837A/Q853A | binding site mutation, insoluble enzyme | Escherichia phage K1F |
R837A/S848A | binding site mutation, increased molar activity with soluble polysialic acid | Escherichia phage K1F |
R837A/S848A/Q853A | binding site mutation, insoluble enzyme | Escherichia phage K1F |
S848A | binding site mutation, active within control range with soluble polysialic acid | Escherichia phage K1F |
S848A/Q853A | binding site mutation, active within control range with soluble polysialic acid | Escherichia phage K1F |
S911A | cleavage site mutation: prevents proteolysis of the C-terminal domain that functions as an intramolecular chaperone and is normally released during enzyme maturation. Substrate binding is reduced similarly to binding site mutations. Altered activities: 3times higher activity with soluble polycialic acid as substrate, 190fold reduced activity with immobilized polysialic acid as substrate, no difference in activity with minimal substrate tetrameric sialic acid | Escherichia phage K1F |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.85 | - |
tetrameric silica acid | identical kinetic parameters for wild type and cleavage site mutant S911A | Escherichia phage K1F |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
polysialic acid capsules of bacteria + H2O | Escherichia phage K1F | minimum substrate is tetrameric polysialic acid, processive enzyme activity on oligomers larger than that, confirmation by H NMR spectroscopy and anion-exchange chromatography | oligomers of alpha 2,8-linked polysialic acid | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia phage K1F | - |
- |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
proteolytic modification | proteolysis of the C-terminal domain that functions as an intramolecular chaperone and is released during enzyme maturation of wild-type enzyme | Escherichia phage K1F |
Purification (Comment) | Organism |
---|---|
description in Schwarzer, D. et al. (2007) J.Biol. Chem. 282, 2821-2831 | Escherichia phage K1F |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
longchain alpha 2,8-linked polysialic acid + H2O | minimum substrate is tetrameric polysialic acid, processive enzyme activity on oligomers larger than that, confirmation by H NMR spectroscopy and anion-exchange chromatography | Escherichia phage K1F | oligomers of alpha 2,8-linked polysialic acid | major product is an oligomer consisting of 3 monomers in wild-type and cleavage mutant S911A, in binding site mutant R837A/S848A more random oligomers are produced | ? | |
polysialic acid capsules of bacteria + H2O | minimum substrate is tetrameric polysialic acid, processive enzyme activity on oligomers larger than that, confirmation by H NMR spectroscopy and anion-exchange chromatography | Escherichia phage K1F | oligomers of alpha 2,8-linked polysialic acid | - |
? | |
tetrameric silica acid + H2O | - |
Escherichia phage K1F | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
endo-N-acetylneuraminidase | - |
Escherichia phage K1F |
endoNF | from coliphage K1F, lacking N-terminal capsid binding domain | Escherichia phage K1F |
endosialidase | - |
Escherichia phage K1F |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
4.37 | - |
tetrameric silica acid | identical kinetic parameters for wild type and cleavage site mutant S911A | Escherichia phage K1F |