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Literature summary extracted from

  • Hsu, C.C.; Hong, Z.; Wada, M.; Franke, D.; Wong, C.H.
    Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase (2005), Proc. Natl. Acad. Sci. USA, 102, 9122-9126.
    View publication on PubMedView publication on EuropePMC

Protein Variants

EC Number Protein Variants Comment Organism
4.1.3.3 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
4.1.3.3 additional information directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase Escherichia coli
4.1.3.3 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
4.1.3.3 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
4.1.3.3 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
4.1.3.3 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.1.3.3 2.1
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L Escherichia coli
4.1.3.3 2.4
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 2.6
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli
4.1.3.3 2.7
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 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
4.1.3.3 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
4.1.3.3 3.5
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 3.9
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli
4.1.3.3 4.4
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli
4.1.3.3 6.3
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 7.5
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli
4.1.3.3 9.2
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli
4.1.3.3 10.1
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 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
4.1.3.3 12.1
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 12.3
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L Escherichia coli
4.1.3.3 13.8
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli
4.1.3.3 14.1
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L Escherichia coli
4.1.3.3 14.5
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli
4.1.3.3 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
4.1.3.3 36.2
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 127
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 369.4
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli

Organism

EC Number Organism UniProt Comment Textmining
4.1.3.3 Escherichia coli
-
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.1.3.3 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 ?
-
?
4.1.3.3 D-3-deoxy-manno-2-octulosonic acid
-
Escherichia coli D-arabinose + pyruvate
-
r
4.1.3.3 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
4.1.3.3 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 ?
-
?
4.1.3.3 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 ?
-
?
4.1.3.3 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 ?
-
?
4.1.3.3 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 ?
-
?
4.1.3.3 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 ?
-
?
4.1.3.3 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 ?
-
?
4.1.3.3 L-3-deoxy-manno-2-octulosonic acid
-
Escherichia coli L-arabinose + pyruvate
-
r
4.1.3.3 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
4.1.3.3 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 ?
-
?
4.1.3.3 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 ?
-
?
4.1.3.3 N-acetyl-D-mannosamine + pyruvate
-
Escherichia coli N-acetyl-D-neuraminic acid
-
r
4.1.3.3 N-acetyl-D-neuraminic acid
-
Escherichia coli N-acetyl-D-mannosamine + pyruvate
-
r
4.1.3.3 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
4.1.3.3 N-acetyl-L-neuraminic acid
-
Escherichia coli N-acetyl-L-mannosamine + pyruvate
-
r

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.1.3.3 0.0002
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli
4.1.3.3 0.0045
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli
4.1.3.3 0.0068
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 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
4.1.3.3 0.02
-
N-acetyl-L-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 1.1
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli
4.1.3.3 1.1
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L Escherichia coli
4.1.3.3 1.2
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli
4.1.3.3 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
4.1.3.3 1.5
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L Escherichia coli
4.1.3.3 1.7
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 1.9
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli
4.1.3.3 2.2
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 2.5
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 2.8
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli
4.1.3.3 3.2
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I/V265I Escherichia coli
4.1.3.3 4.2
-
L-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 5.1
-
D-3-deoxy-manno-2-octulosonic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/N153Y/V251I/V265I/Y281C Escherichia coli
4.1.3.3 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
4.1.3.3 9.7
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, mutant enzyme Y98H/F115L/V251I Escherichia coli
4.1.3.3 10.3
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, Y98H/F115L Escherichia coli
4.1.3.3 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
4.1.3.3 10.5
-
N-acetyl-D-neuraminic acid pH 7.5, 25°C, wild-type enzyme Escherichia coli