4.1.3.3: N-acetylneuraminate lyase

increased Km

D187N

decreased Vmax, increased Km

E188D

cleaves 5-N-acetyl-9-O-acetylneuraminic acid as good as N-acetylneuraminic acid

E188Q

cleaves 5-N-acetyl-9-O-acetylneuraminic acid as good as N-acetylneuraminic acid

K161R

decreased Vmax

Y133C

significant decrease in enzyme activity

Y133F

significant decrease in enzyme activity

Y133H

significant decrease in enzyme activity

Y133W

significant decrease in enzyme activity

D191P

ratio of turnover-number to KM-value for (2S,4S,5R,6R)-5-(acetylamino)-6-[(dipropylamino)carbonyl]-2,4-dihydroxytetrahydro-pyran-2-carboxylic acid is 1.4fold higher than the wild-type ratio

666805

E192A

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192C

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192D

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192F

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192G

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192H

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192I

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192K

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192L

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192M

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192N

3 entries

E192P

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192Q

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192R

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192S

2 entries

E192T

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192V

2 entries

E192W

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

E192Y

strong interaction of Glu192 (wild type) with hydroxyl groups at C8 and C9 of N-acetylneuraminic acid

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

I229D

the mutant shows decreased kcat compared to the wild type enzyme

I229N

the mutant shows increased kcat compared to the wild type enzyme

I229R

the mutant shows increased kcat compared to the wild type enzyme

L142R

increased activity towards L-aspartate-beta-semialdehyde

653594

L142R/Y190D/E192A

less efficient than wild-type enzyme

653594

L171D

the mutant is insoluble

L171D/L199D/I229D

the mutant is insoluble

L171D/L199N/I229D

the mutant is insoluble

L171N

the mutant is insoluble

L171N/I229N

the mutant shows decreased kcat compared to the wild type enzyme

L171R

the mutant shows decreased kcat compared to the wild type enzyme

L171R/L199N/I229R

the mutant is insoluble

L171R/L199R/I229R

the mutant is insoluble

L199D

the mutant is insoluble

L199N

the mutant shows increased kcat compared to the wild type enzyme

L199N/I229D

the mutant shows decreased kcat compared to the wild type enzyme

L199N/I229N

the mutant shows decreased kcat compared to the wild type enzyme

L199N/I229R

the mutant shows increased kcat compared to the wild type enzyme

L199R

the mutant is insoluble

L199R/I229N

the mutant shows increased kcat compared to the wild type enzyme

S208G

ratio of turnover-number to KM-value for N-acetyl-D-neuraminic acid is 2.5fold lower than the wild-type ratio, ratio of turnover-number to KM-value for (2S,4S,5R,6R)-5-(acetylamino)-6-[(dipropylamino)carbonyl]-2,4-dihydroxytetrahydro-pyran-2-carboxylic acid is 1.5fold higher than the wild-type ratio

666805

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

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

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

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

L171N/I229N

Escherichia coli W3110 / ATCC 27325

the mutant shows decreased kcat compared to the wild type enzyme

L199N/I229D

Escherichia coli W3110 / ATCC 27325

the mutant shows decreased kcat compared to the wild type enzyme

L199N/I229N

Escherichia coli W3110 / ATCC 27325

the mutant shows decreased kcat compared to the wild type enzyme

L199N/I229R

Escherichia coli W3110 / ATCC 27325

the mutant shows increased kcat compared to the wild type enzyme

L199R/I229N

Escherichia coli W3110 / ATCC 27325

the mutant shows increased kcat compared to the wild type enzyme

E192N

the turnover-number for sialic acid is 65% of the wild-type value, the Km-value for sialic acid is 8.6fold higher than the wild-type value, the turnover-number for (2R,3R,4R)-6-[(aminooxy)carbonyl]-3,4,6-trihydroxy-N-propyltetrahydro-2H-pyran-2-carboxamide is 57% of the wild-type value, the KM-value for (2R,3R,4R)-6-[(aminooxy)carbonyl]-3,4,6-trihydroxy-N-propyltetrahydro-2H-pyran-2-carboxamide is 28.2fold lower than the wild-type value. The mutant enzyme is most efficient in catalysis of the synthesis of the tertiary amides (4S,5R,6R)-6-[(diethylamino)carbonyl]-2,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, (4R,5R,6R)-6-[(diethylamino)carbonyl]-2,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, (4S,5R,6R)-2,4,5-trihydroxy-6-{[methyl(propyl)amino]carbonyl}tetrahydro-2H-pyran-2-carboxylic acid, (4R,5R,6R)-2,4,5-trihydroxy-6-{[methyl(propyl)amino]carbonyl}tetrahydro-2H-pyran-2-carboxylic acid, (4S,5R,6R)-6-[(dipropylamino)carbonyl]-2,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, (4R,5R,6R)-6-[(dipropylamino)carbonyl]-2,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, (4S,5R,6R)-6-[(dibutylamino)carbonyl]-2,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, (4R,5R,6R)-6-[(dibutylamino)carbonyl]-2,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, (4S,5R,6R)-2,4,5-trihydroxy-6-(pyrrolidin-1-ylcarbonyl)tetrahydro-2H-pyran-2-carboxylic acid, (4R,5R,6R)-2,4,5-trihydroxy-6-(pyrrolidin-1-ylcarbonyl)tetrahydro-2H-pyran-2-carboxylic acid, (4S,5R,6R)-2,4,5-trihydroxy-6-(piperidin-1-ylcarbonyl)tetrahydro-2H-pyran-2-carboxylic acid, (4R,5R,6R)-2,4,5-trihydroxy-6-(piperidin-1-ylcarbonyl)tetrahydro-2H-pyran-2-carboxylic acid, (4S,5R,6R)-2,4,5-trihydroxy-6-(morpholin-4-ylcarbonyl)tetrahydro-2H-pyran-2-carboxylic acid, (4R,5R,6R)-2,4,5-trihydroxy-6-(morpholin-4-ylcarbonyl)tetrahydro-2H-pyran-2-carboxylic acid

663630

F252A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

F252Y

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

S47A

site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme

S47C

site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme

S47T

site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme

T167A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

T167S

site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme

T48A

site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme

T48S

site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme

Y110A

site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme

Y110F

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Y137A

site-directed mutagenesis, inactive mutant

Y137F

site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme

Limosilactobacillus antri

G210S/Y213G

C8P490

the mutant shows reduced activity compared to the wild type enzyme

749150

G211S

Limosilactobacillus antri

C8P490

the mutant shows reduced activity compared to the wild type enzyme

749150

P193Y

Limosilactobacillus antri

C8P490

the mutant shows reduced activity compared to the wild type enzyme

749150

G210S/Y213G

Limosilactobacillus antri DSMZ 16041

the mutant shows reduced activity compared to the wild type enzyme

G211S

Limosilactobacillus antri DSMZ 16041

the mutant shows reduced activity compared to the wild type enzyme

P193Y

Limosilactobacillus antri DSMZ 16041

the mutant shows reduced activity compared to the wild type enzyme

W189A

Mycoplasma sp.

A0A2P1G7E2

the kcat/Km value of the mutant is about 3fold higher than those of the wild type enzyme

748534

W189F

Mycoplasma sp.

A0A2P1G7E2

the kcat/Km value of the mutant is about 10fold higher than those of the wild type enzyme

748534

W189Y

Mycoplasma sp.

A0A2P1G7E2

the kcat/Km value of the mutant is about 7fold higher than those of the wild type enzyme

748534

K164A

Pasteurella multocida

Q9CKB0

site-directed mutagenesis, determiniation of three structures of the K164A variant: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid and N-glycolylneuraminic acid. Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide, every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme

727035

C118A

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C118S

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C238A

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C238S

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C270A

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C270S

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C82A

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C82S

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

K165C

site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site and complete conversion of the cysteine into gamma-thialysine through dehydroalanine as by chemical mutagenesis, ESI-mass spectrometry and kinetic characterisation, the K165C variant is severely impaired in catalysis, kcat/Km is reduced 720fold compared with wild-type

K165Dha

site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site and complete conversion of the cysteine into gamma-thialysine, Dha, through dehydroalanine as by chemical mutagenesis, ESI-mass spectrometry and kinetic characterisation, the enzyme containing gamma-thialysine regains 17-30% of the wild-type activity

C238S

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C270S

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C82A

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

C82S

site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme

K165C