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Literature summary for 2.4.1.4 extracted from

  • Verges, A.; Cambon, E.; Barbe, S.; Moulis, C.; Remaud-Simeon, M.; Andre, I.
    Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase (2017), Protein Sci., 26, 566-577 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.4.1.4

Protein Variants

Protein Variants Comment Organism
R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437R/N439D compared to the wild-type enzyme, and in agreement with the loss of polymerase activity, the mutant enzyme incorporates higher amounts of glucosyl units in maltose (24.8% versus 5.8% for wild-type enzyme) and in maltotriose (18.6% versus 2.9% for wild-type enzyme). The mutant enzyme incorporates 23% glucosyl units into erlose (alpha-D-glucopyranosyl-(1->4)-alpha-D-glucopyranosyl-(1->2)-beta-D-fructose). No glucosyl units are incorporated into erlose (alpha-D-glucopyranosyl-(1->4)-alpha-D-glucopyranosyl-(1->2)-beta-D-fructose)by the wild-type enzyme. Panose (alpha-D-glucopyranosyl-(1->6)-alpha-D-glucopyranosyl-(1->4)-alpha-D-glucose) is produced by the mutant enzyme, 13.9% of the glucosyl units are incorporated into this trisaccharide. No panose is produced by the wild-type enzyme. The Tm-value is slightly lowered compared to wild-type enzyme (-3°C) Neisseria polysaccharea
R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R compared to the wild-type enzyme, and in agreement with the loss of polymerase activity, the mutant enzyme incorporates higher amounts of glucosyl units in maltose (15.3% versus 5.8% for wild-type enzyme) and in maltotriose (23% versus 2.9% for wild-type enzyme). The mutant enzyme incorporates nearly 46% of the glucosyl residues in turanose, versus only 19% for the wild-type enzyme. Panose (alpha-D-glucopyranosyl-(1->6)-alpha-D-glucopyranosyl-(1->4)-alpha-D-glucose) is produced by the mutant enzyme, 8.5% of the glucosyl units are incorporated into this trisaccharide. No panose is produced by the wild-type enzyme. The Tm-value is slightly lowered compared to wild-type enzyme (-1.9°C) Neisseria polysaccharea
R226L/I228V/F229A/A289I/F290Y/E300I/V331T compared to the wild-type enzyme, and in agreement with the loss of polymerase activity, the mutant enzyme incorporates higher amounts of glucosyl units in maltose (27.3% versus 5.8% for wild-type enzyme) and in maltotriose (20.5% versus 2.9% for wild-type enzyme). With the mutant enzyme 20% glucosyl units are incorporated into erlose (alpha-D-glucopyranosyl-(1->4)-alpha-D-glucopyranosyl-(1->2)-beta-D-fructose). No glucosyl units are incorporated into erlose by the wild-type enzyme. 1.6% panose is produced by the mutants enzyme and it is not produced by the wild-type enzyme. The Tm-value is slightly lowered compared to wild-type enzyme (-3°C) Neisseria polysaccharea

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
50.2
-
 sucrose 30°C, pH 7.0, wild-type enzyme Neisseria polysaccharea
78.43
-
 sucrose 30°C, pH 7.0, mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R Neisseria polysaccharea

Organism

Organism UniProt Comment Textmining
Neisseria polysaccharea Q9ZEU2
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
sucrose compared to the wild-type enzyme, and in agreement with their loss of polymerase activity, all three mutant enzymes incorporate higher amounts of glucosyl units in maltose (27.3% (mutant enzyme R226L/I228V/F229A/A289I/F290Y/E300I/V331T); 24.8% (mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437R/N439D), 15.3% (mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R), versus 5.8% for wild-type enzyme) and in maltotriose (20.5% (mutant R226L/I228V/F229A/A289I/F290Y/E300I/V331T); 18.6% (mutant R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437R/N439D), 23% (mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R), versus 2.9% for wild-type enzyme). Compared to the others, the mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R is more specialized in turanose production, incorporating nearly 46% of the glucosyl residues in turanose, versus only 19% for the wild-type enzyme. With mutants enzyme R226L/I228V/F229A/A289I/F290Y/E300I/V331T and mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437R/N439D, 20% and 23% glucosyl units are incorporated into erlose (alpha-D-glucopyranosyl-(1->4)-alpha-D-glucopyranosyl-(1->2)-beta-D-fructose), respectively. Much lower values are observed with mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R (only 1.4%) and none for the wild-type enzyme. Panose (alpha-D-glucopyranosyl-(1->6)-alpha-D-glucopyranosyl-(1->4)-alpha-D-glucose) is mainly produced by mutants R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437R/N439D and R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R, 13.9% and 8.5% of the glucosyl units incorporated into this trisaccharide, respectively. In comparison, the value goes down to 1.6% with mutant enzyme R226L/I228V/F229A/A289I/F290Y/E300I/V331T and it is not produced by the wild-type enzyme Neisseria polysaccharea maltose + maltotriose + turanose + erlose
-
 ?

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
46
-
 Tm-value, mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437R/N439D Neisseria polysaccharea
46
-
 Tm-value, mutant enzyme R226L/I228V/F229A/A289I/F290Y/E300I/V331T Neisseria polysaccharea
47.1
-
 Tm-value, mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R Neisseria polysaccharea
49
-
 wild-type enzyme Neisseria polysaccharea

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.3
-
 sucrose 30°C, pH 7.0, wild-type enzyme Neisseria polysaccharea
4.5
-
 sucrose 30°C, pH 7.0, mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R Neisseria polysaccharea

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.012
-
 sucrose 30°C, pH 7.0, mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437R/N439D Neisseria polysaccharea
0.015
-
 sucrose 30°C, pH 7.0, mutant enzyme R226L/I228V/F229A/A289I/F290Y/E300I/V331T Neisseria polysaccharea
0.026
-
 sucrose 30°C, pH 7.0, wild-type enzyme Neisseria polysaccharea
0.058
-
 sucrose 30°C, pH 7.0, mutant enzyme R226K/I228V/A289I/F290Y/E300I/V331T/G396S/T398V/Q437S/N439D/C445R Neisseria polysaccharea