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 (Comment) | Organism |
---|---|
expression in Escherichia coli | Bacillus subtilis |
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 | 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 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 | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | P05655 | expression in Escherichia coli | - |
Purification (Comment) | Organism |
---|---|
ion exchange chromatography | Bacillus subtilis |
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 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 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 |