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Literature summary for 2.4.1.10 extracted from

  • Ortiz-Soto, M.E.; Rivera, M.; Rudino-Pinera, E.; Olvera, C.; Lopez-Munguia, A.
    Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties (2008), Protein Eng. Des. Sel., 21, 589-595.
    View publication on PubMed
Search for more literature for 2.4.1.10

Activating Compound

Activating Compound Comment Organism Structure
levan 1.4fold increase in Vmax of wild-type in the presence of 15 g/l of levan is observed Bacillus subtilis
levan 3.7fold increase in Vmax of S164A in the presence of 15 g/l of levan is observed Bacillus subtilis
additional information levan has no effect on Y429N, a mutant that has lost the fructan-synthesizing activity Bacillus subtilis

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Bacillus subtilis

Crystallization (Commentary)

Crystallization (Comment) Organism
mutant enzyme S164A, 12 days of micro-dialysis of the purified protein (8 g/l) against deionized water, PDB accession code 2VDT Bacillus subtilis

Protein Variants

Protein Variants Comment Organism
A344P site directed mutagenesis, same behavior like the wild-type Bacillus subtilis
F414W site directed mutagenesis, same behavior like the wild-type Bacillus subtilis
G361F site-directed mutagenesis, less stable than the wild-type, synthesizes mainly oligosaccharides, still catalyzes the synthesis of low amounts of polymer, pH-optimum 6, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
H243L site-directed mutagenesis, less stable than the wild-type, pH-optimum 6, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
I341V site-directed mutagenesis, pH-optimum 6 Bacillus subtilis
R360K site-directed mutagenesis, pH-optimum 6, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
R360S site-directed mutagenesis, pH-optimum 6, decrease in activity, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
R433A site-directed mutagenesis, synthesizes only oligosaccharides, pH-optimum 6-7, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
S164A site-directed mutagenesis, S164A is catalytically important, as it maintains the nucleophile in an appropriate position regarding the sucrose molecule. S164A results in a 12fold more stable and less hydrolytic enzyme than the wild-type, with a half-life of 628.0 (+51.0) min, pH-optimum 6, decrease in activity, slightly higher affinity for sucrose Bacillus subtilis
S164K inactive Bacillus subtilis
Y429 site-directed mutagenesis, Y429 plays an indirect but important role in catalysis and acceptor specificity, as this is a key residue coordinating the sucrose position in the levansucrase binding pocket through a complex water network Bacillus subtilis
Y429A site-directed mutagenesis Bacillus subtilis
Y429N site-directed mutagenesis, synthesizes only oligosaccharides, pH-optimum 5-6, decrease in activity, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information mutant enzyme S164K is inactive Bacillus subtilis
additional information
-
 additional information mutants A344P and F414W have the same behavior like the wild-type Bacillus subtilis
2.5
-
 sucrose mutant enzyme S164A, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
7.4
-
 sucrose mutant enzyme I341V, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
8
-
 sucrose wild-type, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
10.5
-
 sucrose mutant enzyme H243L, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
29.3
-
 sucrose mutant enzyme R433A, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
30
-
 sucrose mutant enzyme R369K, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
154
-
 sucrose mutant enzyme R360S, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
297.3
-
 sucrose mutant enzyme G361F, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
319.4
-
 sucrose mutant enzyme Y429N, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis

Organism

Organism UniProt Comment Textmining
Bacillus subtilis P05655 expression in Escherichia coli
-

Purification (Commentary)

Purification (Comment) Organism
ion exchange chromatography Bacillus subtilis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information mutant enzymes Y429N and R433A no longer produce levan but exclusively oligosaccharides Bacillus subtilis ?
-
 ?
sucrose + D-maltose
-
 Bacillus subtilis ?
-
 ?
sucrose + D-xylose
-
 Bacillus subtilis beta-D-fructofuranosyl-beta-D-xylopyranoside + D-glucose
-
 ?
sucrose + levan mutant enzymes H243L and S164A synthesize either high or low levan molecular weight Bacillus subtilis ?
-
 ?

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 mutants A344P and F414W have the same behavior like the wild-type Bacillus subtilis
40
-
 t1/2: 16.0 min mutant enzym R433A, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: 17.21 min mutant enzym R360S, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: 31.4 min mutant enzym I341V, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: 42.11 min mutant enzym R360K, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: 47.04 min mutant enzym Y429N, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: 52.0 min wild-type, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: 628.0 min mutant enzym S164A, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: 7.9 min mutant enzym H243L, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
40
-
 t1/2: less than 1min mutant enzym G361F, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
 additional information mutants A344P and F414W have the same behavior like the wild-type Bacillus subtilis
6.3
-
 sucrose mutant enzyme Y429N Bacillus subtilis
6.4
-
 sucrose mutant enzyme S164A Bacillus subtilis
13.5
-
 sucrose mutant enzyme R360S Bacillus subtilis
57.4
-
 sucrose mutant enzyme G361F Bacillus subtilis
87.5
-
 sucrose mutant enzyme R433A Bacillus subtilis
141.5
-
 sucrose mutant enzyme H243L Bacillus subtilis
164.6
-
 sucrose wild-type enzyme Bacillus subtilis
166.3
-
 sucrose mutant enzyme I341V Bacillus subtilis
170.2
-
 sucrose mutant enzyme R369K Bacillus subtilis