Cloned (Comment) | Organism |
---|---|
cloning of inactive enzyme variant SPNH5 from strain HSC5 and of active variant SPNJ4, expression of C-terminally HA-tagged and His6-tagged variant SPNJ4 in Escherichia coli strain TOP10 | Streptococcus pyogenes |
Protein Variants | Comment | Organism |
---|---|---|
D330G | mutation of the inactive enzyme variant SPNH5 causes only a slight increase in activity, but the mutation in SPNJ4 causes an increase in activity | Streptococcus pyogenes |
K289R | swap at residue 289 of SPNH5 does not increase activity of the inactive enzyme variant but reduces actiivty of variant SPNJ4 | Streptococcus pyogenes |
K289R/D330G | the mutation increases SPNH5 activity to the level of about 30% activity of active enzyme variant SPNJ4, but reduces activity of variant SPNJ4 | Streptococcus pyogenes |
L199I/Q253H/L280V | swaps of the 3 N-terminal residues of the enzymatic domain of variant SPNJ4 do not affect activity | Streptococcus pyogenes |
R289K/G330D/I374V | the mutations introduced into enzyme variant SPNJ4 result in an inactivation of the enzyme | Streptococcus pyogenes |
R289K/I374V | swaps of the the 7th and 9th polymorphic residues of variant SPNJ4 result in less activation compared to mutation D330G alone | Streptococcus pyogenes |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.175 | - |
NAD+ | enzyme variant SPNJ4, pH 7.4, 37°C | Streptococcus pyogenes | |
2.14 | - |
NAD+ | enzyme variant SPNJ4 mutant R289K, pH 7.4, 37°C | Streptococcus pyogenes |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
extracellular | the enzyme is secreted from the bacterial cell and translocated into the host epithelial cell cytosol by a process termed cytolysin-mediated translocation. Translocation across the host cell membrane requires a second streptococcal protein, the cholesterol-dependent cytolysin streptolysin O. Both active and inactive SPN haplotypes undergo cytolysin-mediated translocation.The enzyme has multiple domains, including an N-terminal domain required for translocation and a C-terminal enzymatic domain | Streptococcus pyogenes | - |
- |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
48600 | - |
x * 48600, recombinant C-terminally HA-tagged and His6-tagged enzyme variant SPNJ4 from Escherichia coli strain TOP10, SDS-PAGE | Streptococcus pyogenes |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
NAD+ + H2O | Streptococcus pyogenes | - |
ADP-D-ribose + nicotinamide | - |
? | |
NAD+ + H2O | Streptococcus pyogenes JRS4 | - |
ADP-D-ribose + nicotinamide | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptococcus pyogenes | - |
M serotype 6 | - |
Streptococcus pyogenes JRS4 | - |
M serotype 6 | - |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally HA-tagged and His6-tagged enzyme variant SPNJ4 from Escherichia coli strain TOP10 | Streptococcus pyogenes |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
NAD+ + H2O | - |
Streptococcus pyogenes | ADP-D-ribose + nicotinamide | - |
? | |
NAD+ + H2O | - |
Streptococcus pyogenes JRS4 | ADP-D-ribose + nicotinamide | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 48600, recombinant C-terminally HA-tagged and His6-tagged enzyme variant SPNJ4 from Escherichia coli strain TOP10, SDS-PAGE | Streptococcus pyogenes |
More | the enzyme has multiple domains, including an N-terminal domain required for translocation and a C-terminal enzymatic domain | Streptococcus pyogenes |
Synonyms | Comment | Organism |
---|---|---|
(Streptococcus pyogenes NAD+ glycohydrolase) | - |
Streptococcus pyogenes |
NAD+ glycohydrolase | - |
Streptococcus pyogenes |
SPN | - |
Streptococcus pyogenes |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Streptococcus pyogenes |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.021 | - |
NAD+ | enzyme variant SPNJ4 mutant R289N, pH 7.4, 37°C | Streptococcus pyogenes | |
0.023 | - |
NAD+ | enzyme variant SPNJ4 mutant R289K, pH 7.4, 37°C | Streptococcus pyogenes | |
0.037 | - |
NAD+ | enzyme variant SPNJ4, pH 7.4, 37°C | Streptococcus pyogenes | |
0.063 | - |
NAD+ | enzyme variant SPNJ4 mutant R289N/D330G, pH 7.4, 37°C | Streptococcus pyogenes | |
0.105 | - |
NAD+ | enzyme variant SPNJ4 mutant R289K/D330G, pH 7.4, 37°C | Streptococcus pyogenes | |
0.192 | - |
NAD+ | enzyme variant SPNJ4 mutant D330G/I374V, pH 7.4, 37°C | Streptococcus pyogenes | |
0.66 | - |
NAD+ | enzyme variant SPNJ4 mutant D330G, pH 7.4, 37°C | Streptococcus pyogenes |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Streptococcus pyogenes |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme is evolving and has diverged into NAD+ glycohydrolase-inactive variants that correlate with tissue tropism. Of a total 454 amino acids, the activity-deficient variants differ at only nine highly conserved positions | Streptococcus pyogenes |
additional information | no one single residue can account for the inability of the deficient variants to cleave the glycosidic bond of beta-NAD+ into nicotinamide and ADP-ribose. Reciprocal changes at 3 specific residues are required to both abolish activity of the proficient version and restore full activity to the deficient variant | Streptococcus pyogenes |
physiological function | the enzyme is secreted from the bacterial cell and translocated into the host cell cytosol where it contributes to cell death | Streptococcus pyogenes |