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Literature summary for 3.2.2.5 extracted from

  • Chandrasekaran, S.; Ghosh, J.; Port, G.C.; Koh, E.I.; Caparon, M.G.
    Analysis of polymorphic residues reveals distinct enzymatic and cytotoxic activities of the Streptococcus pyogenes NAD+ glycohydrolase (2013), J. Biol. Chem., 288, 20064-20075.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

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 [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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Streptococcus pyogenes
-
M serotype 6
-
Streptococcus pyogenes JRS4
-
M serotype 6
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally HA-tagged and His6-tagged enzyme variant SPNJ4 from Escherichia coli strain TOP10 Streptococcus pyogenes

Substrates and Products (Substrate)

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

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

Synonyms Comment Organism
(Streptococcus pyogenes NAD+ glycohydrolase)
-
Streptococcus pyogenes
NAD+ glycohydrolase
-
Streptococcus pyogenes
SPN
-
Streptococcus pyogenes

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Streptococcus pyogenes

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
assay at Streptococcus pyogenes

General Information

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