Application | Comment | Organism |
---|---|---|
additional information | strategy for identifying target sites for focussed saturation mutagenesis of transketolase, for the incremental modification of substrate specificity. Shell active-site residues that are phylogenetically variant can be randomly mutated to improve the overall activity but not specificity of transketolase. Residues with low sequence entropy that no longer interact with either of the smaller target substrates can similarly provide non-specific activity improvements in at least half of the mutants. The other half of these mutants show a preference for the hydroxylated substrate. Residues with low sequence entropy that interact directly with altered regions of the target substrate are most likely to improve the substrate specificity | Escherichia coli |
Cloned (Comment) | Organism |
---|---|
expressed from the self-promoting tktA gene in the plasmid pQR711 in Escherichia coli TOP10 or XL10 cells. Both wild-type and mutant D469T overexpressed from pQR412 | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
A29D | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
A29E | specific activity with propionaldehyde as substrate is 3.4fold greater than for wild-type | Escherichia coli |
D259A | specific activity with propionaldehyde as substrate is 2.3fold greater than for wild-type | Escherichia coli |
D259G | specific activity with propionaldehyde as substrate is 1.7fold greater than for wild-type | Escherichia coli |
D259Stop | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
D469A | specific activity with propionaldehyde as substrate is 4.3fold greater than for wild-type | Escherichia coli |
D469S | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
D469T | specific activity with propionaldehyde as substrate is 4.9fold greater than for wild-type. 8.5fold improvement in specificity towards propionaldehyde relative to glycolaldehyde | Escherichia coli |
D469Y | specific activity with propionaldehyde as substrate is 4.4fold greater than for wild-type. 64fold improvement in specificity towards propionaldehyde relative to glycolaldehyde | Escherichia coli |
H100A | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
H100I | has the same specific activity with propionaldehyde as substrate as the wild-type. Does not improve specific activity towards propionaldehyde | Escherichia coli |
H100V | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
H26A | specific activity with propionaldehyde as substrate is 2.3fold greater than for wild-type | Escherichia coli |
H26K | specific activity with propionaldehyde as substrate is 1.2fold greater than for wild-type | Escherichia coli |
H26T | specific activity with propionaldehyde as substrate is 2.2fold greater than for wild-type. 8.5fold improvement in specificity towards propionaldehyde relative to glycolaldehyde | Escherichia coli |
H26V | has the same specific activity with propionaldehyde as substrate as the wild-type | Escherichia coli |
H461Q | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
H461S | specific activity with propionaldehyde as substrate is 1.4fold greater than for wild-type | Escherichia coli |
H461Y | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
R358I | specific activity with propionaldehyde as substrate is 1.9fold greater than for wild-type | Escherichia coli |
R358P | specific activity with propionaldehyde as substrate is 1.5fold greater than for wild-type | Escherichia coli |
R520G | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
R520I | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
R520P | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
R520Stop | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
R520V | specific activity with propionaldehyde as substrate is 4.7fold greater than for wild-type | Escherichia coli |
S188Q | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
S188R | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
S188T | specific activity with propionaldehyde as substrate is lower than for wild-type | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
55 | - |
propionaldehyde | mutant D469T, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli | |
140 | - |
propionaldehyde | wild-type, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
wild-type and mutant D469T | Escherichia coli |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.0012 | - |
mutant R520I, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.0014 | - |
mutant S188T, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.0018 | - |
mutant S188R, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.002 | - |
mutant D259Stop, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.007 | - |
mutant H461Y, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.008 | - |
mutant H100A and mutant H100V, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.0086 | - |
mutant R520G, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.009 | - |
mutant R520P, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.013 | - |
mutant H461Q, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.02 | - |
mutant D469S, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.022 | - |
mutant S188Q, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.025 | - |
mutant H26V, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.027 | - |
mutant A29D, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.028 | - |
mutant H100I and mutant H26V, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.029 | - |
wild-type, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.036 | - |
mutant H26K, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.04 | - |
mutant H461S, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0. Mutant D469Y, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehydee, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.043 | - |
mutant R358P, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.05 | - |
mutant D259G, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.055 | - |
mutant R358I, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.063 | - |
mutant H26T, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.066 | - |
mutant D259A and mutant H26A, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.09 | - |
mutant H100V, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.1 | - |
mutant A29E, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.11 | - |
mutant S188R, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.12 | - |
mutant D259Stop, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.125 | - |
mutant D469A, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.127 | - |
mutant D469Y, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.13 | - |
mutant H100A and mutant S188T, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.14 | - |
mutant D469T, mutant R520V and mutant R520Stop, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.14 | - |
mutant R520Stop, in the presence of 50 mM Li-hydroxypyruvate, 50 mM propionaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.16 | - |
mutant H26T, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.26 | - |
mutant H26A, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.31 | - |
mutant H26K, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.37 | - |
mutant D469T, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.42 | - |
mutant R520I, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.43 | - |
mutant D469S, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.48 | - |
mutant H461Y, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.52 | - |
mutant H461Q, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.61 | - |
mutant D469A, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehydee, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
0.88 | - |
mutant H100I, in the presence of 50 mM Li-hydroxypyruvate, 50 mM glycolaldehyde, and 50 mM Tris-HCl, 2.4 mM thiamine diphosphate, 9 mM MgCl2, pH 7.0 | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
hydroxypyruvate + glycolaldehyde | - |
Escherichia coli | L-erythrulose + ? | - |
? | |
Li-hydroxypyruvate + propionaldehyde | - |
Escherichia coli | 1,3-dihydroxypentan-2-one + ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
TktA | - |
Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
thiamine diphosphate | - |
Escherichia coli |