Protein Variants | Comment | Organism |
---|---|---|
E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | KM-value for N-acetyl-D-neuraminic acid is 5.9fold higher than the wild-type value, turnover-number for N-acetyl-D-neuraminic acid is 14% of the wild-type value, KM-value for N-acetyl-L-neuraminic acid is 7.6fold higher than the wild-type value, turnover-number for N-acetyl-L-neuraminic acid is 100fold higher than the wild-type value, KM-value for D-3-deoxy-manno-2-octulosonic acid is 83% of the wild-type value, turnover-number for D-3-deoxy-manno-2-octulosonic acid is 7.4fold higher than wild-type value, KM-value for L-3-deoxy-manno-2-octulosonic acid is 37% of the wild-type value, turnover-number for L-3-deoxy-manno-2-octulosonic acid is 9.5fold higher than wild-type value. kcat/KM for N-acetyl-D-neuraminic acid is 40.4fold lower than the wild-type value, kcat/KM for N-acetyl-D-neuraminic acid is 20fold higher than the wild-type value, kcat/KM for L-3-deoxy-manno-2-octulosonic acid is 24.7fold higher than the wild-type value | Escherichia coli |
additional information | directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase | Escherichia coli |
Y98H/F115L | KM-value for N-acetyl-D-neuraminic acid is 81% of the wild-type value, turnover-number for N-acetyl-D-neuraminic acid isnearly identical to wild-type value, KM-value for D-3-deoxy-manno-2-octulosonic acid is 89% of the wild-type value, turnover-number for D-3-deoxy-manno-2-octulosonic acid is 1.25fold higher than wild-type value, KM-value for L-3-deoxy-manno-2-octulosonic acid is 1.9fold higher than the wild-type value, turnover-number for L-3-deoxy-manno-2-octulosonic acid is identical to wild-type value. kcat/KM for N-acetyl-D-neuraminic acid is 1.1fold higher than the wild-type ratio, kcat/Km for D-3-deoxy-manno-2-octulosonic acid is 1.4fold higher than the wild-type ratio, kcat/KM for L-3-deoxy-manno-2-octulosonic acid is 1.8fold lower than the wild-type ratio | Escherichia coli |
Y98H/F115L/N153Y/V251I/V265I/Y281C | KM-value for N-acetyl-D-neuraminic acid is 3.9fold higher than the wild-type value, turnover-number for N-acetyl-D-neuraminic acid is 16% of the wild-type value, KM-value for N-acetyl-L-neuraminic acid is 2.6fold higher than the wild-type value, turnover-number for N-acetyl-L-neuraminic acid is 100fold higher than the wild-type value, KM-value for D-3-deoxy-manno-2-octulosonic acid is 46% of the wild-type value, turnover-number for D-3-deoxy-manno-2-octulosonic acid is 4.36fold higher than wild-type value, KM-value for L-3-deoxy-manno-2-octulosonic acid is 32% of the wild-type value, turnover-number for L-3-deoxy-manno-2-octulosonic acid is 3.8fold higher than wild-type value. kcat/Km for N-acetyl-D-neuraminic acid is 23.8fold lower than the wild-type value, kcat/Km for N-acetyl-D-neuraminic acid is 20fold higher than the wild-type value, kcat/Km for D-3-deoxy-manno-2-octulosonic acid is 9.3fold higher than the wild-type value | Escherichia coli |
Y98H/F115L/V251I | KM-value for N-acetyl-D-neuraminic acid is 1.5fold higher than the wild-type value, turnover-number for N-acetyl-D-neuraminic acid is 92% of the wild-type value, KM-value for N-acetyl-L-neuraminic acid is 84fold higher than the wild-type value, turnover-number for N-acetyl-L-neuraminic acid is 22.5fold higher than the wild-type value, KM-value for D-3-deoxy-manno-2-octulosonic acid is nearly identical to the wild-type value, turnover-number for D-3-deoxy-manno-2-octulosonic acid is 2.3fold higher than wild-type value, KM-value for L-3-deoxy-manno-2-octulosonic acid is 1.2fold higher than the wild-type value, turnover-number for L-3-deoxy-manno-2-octulosonic acid is 1.7fold higher than wild-type value. kcat/KM for N-acetyl-D-neuraminic acid is 1.6fold lower than the wild-type value, kcat/KM for N-acetyl-D-neuraminic acid is 5fold lower than the wild-type value, kcat/KM for D-3-deoxy-manno-2-octulosonic acid is 2.2fold higher than the wild-type value, kcat/KM for L-3-deoxy-manno-2-octulosonic acid is 1.4fold higher than the wild-type value | Escherichia coli |
Y98H/F115L/V251I/V265I | KM-value for N-acetyl-D-neuraminic acid is 4.5fold higher than the wild-type value, turnover-number for N-acetyl-D-neuraminic acid is 45% of the wild-type value, KM-value for N-acetyl-L-neuraminic acid is 28.9fold higher than the wild-type value, turnover-number for N-acetyl-L-neuraminic acid is 34fold higher than the wild-type value, KM-value for D-3-deoxy-manno-2-octulosonic acid is 25% of the wild-type value, turnover-number for D-3-deoxy-manno-2-octulosonic acid is 2.6fold higher than wild-type value, KM-value for L-3-deoxy-manno-2-octulosonic acid is 36% of the wild-type value, turnover-number for L-3-deoxy-manno-2-octulosonic acid is 2.3fold higher than wild-type value. kcat/Km for N-acetyl-D-neuraminic acid is 22.4fold lower than the wild-type value, kcat/Km for N-acetyl-D-neuraminic acid is 2fold higher than the wild-type value | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.1 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L | Escherichia coli | |
2.4 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
2.6 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli | |
2.7 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
2.8 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
3 | 3.5 | N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
3.5 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
3.9 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli | |
4.4 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli | |
6.3 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
7.5 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli | |
9.2 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli | |
10.1 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
11.4 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
12.1 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
12.3 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L | Escherichia coli | |
13.8 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli | |
14.1 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L | Escherichia coli | |
14.5 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli | |
15.3 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
36.2 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
127 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
369.4 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-bromo-3-deoxy-D-mannose + pyruvate | wild-type enzyme: 5% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 5% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 10% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 10% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 10% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
D-3-deoxy-manno-2-octulosonic acid | - |
Escherichia coli | D-arabinose + pyruvate | - |
r | |
D-arabinose + pyruvate | wild-type enzyme: 20% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 40% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 65% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 40% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 85% of the activity with N-acetyl-D-mannosamine | Escherichia coli | D-3-deoxy-manno-2-octulosonic acid | - |
r | |
D-glucose + pyruvate | wild-type enzyme: 40% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 40% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 20% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 25% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 20% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
D-gulose + pyruvate | wild-type enzyme: 10% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 110% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 90% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 70% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
D-mannosamine + pyruvate | wild-type enzyme: 80% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 90% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 165% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 190% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 170% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
D-mannose + pyruvate | wild-type enzyme: 30% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 15% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 70% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 80% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 75% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
D-talose + pyruvate | wild-type enzyme: 60% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 50% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 105% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 80% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 70% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
D-xylose + pyruvate | wild-type enzyme: 45% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 40% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 55% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 25% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 25% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
L-3-deoxy-manno-2-octulosonic acid | - |
Escherichia coli | L-arabinose + pyruvate | - |
r | |
L-arabinose + pyruvate | wild-type enzyme: 10% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 35% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 70% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 40% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 80% of the activity with N-acetyl-D-mannosamine | Escherichia coli | L-3-deoxy-manno-2-octulosonic acid | - |
r | |
L-gulose + pyruvate | wild-type enzyme: 70% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 35% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 15% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 15% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 15% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
L-mannose + pyruvate | wild-type enzyme: 80% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 110% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 95% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 95% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 95% of the activity with N-acetyl-D-mannosamine | Escherichia coli | ? | - |
? | |
N-acetyl-D-mannosamine + pyruvate | - |
Escherichia coli | N-acetyl-D-neuraminic acid | - |
r | |
N-acetyl-D-neuraminic acid | - |
Escherichia coli | N-acetyl-D-mannosamine + pyruvate | - |
r | |
N-acetyl-L-mannosamine + pyruvate | wild-type enzyme: 0.2% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I: 0.5% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/V251I/V265I: 1% of the activity with N-acetyl-D-mannosamine, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C: 2% of the activity with N-acetyl-D-mannosamine, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y: 2% of the activity with N-acetyl-D-mannosamine | Escherichia coli | N-acetyl-L-neuraminic acid | - |
r | |
N-acetyl-L-neuraminic acid | - |
Escherichia coli | N-acetyl-L-mannosamine + pyruvate | - |
r |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0002 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli | |
0.0045 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli | |
0.0068 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
0.02 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
0.02 | - |
N-acetyl-L-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
1.1 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli | |
1.1 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L | Escherichia coli | |
1.2 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli | |
1.5 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
1.5 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L | Escherichia coli | |
1.7 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
1.9 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli | |
2.2 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
2.5 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
2.8 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli | |
3.2 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I | Escherichia coli | |
4.2 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
5.1 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C | Escherichia coli | |
8.9 | - |
D-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
9.7 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I | Escherichia coli | |
10.3 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, Y98H/F115L | Escherichia coli | |
10.4 | - |
L-3-deoxy-manno-2-octulosonic acid | pH 7.5, 25°C, mutant enzyme E60A/Y98H/F115L/N153Y/D150G/V251I/V265I/Y281C/N153Y | Escherichia coli | |
10.5 | - |
N-acetyl-D-neuraminic acid | pH 7.5, 25°C, wild-type enzyme | Escherichia coli |