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Literature summary for 4.1.3.3 extracted from

  • Daniels, A.D.; Campeotto, I.; van der Kamp, M.W.; Bolt, A.H.; Trinh, C.H.; Phillips, S.E.; Pearson, A.R.; Nelson, A.; Mulholland, A.J.; Berry, A.
    Reaction mechanism of N-acetylneuraminic acid lyase revealed by a combination of crystallography, QM/MM simulation, and mutagenesis (2014), ACS Chem. Biol., 9, 1025-1032.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 4.1.3.3

Crystallization (Commentary)

Crystallization (Comment) Organism
recombinant enzyme in complex with N-acetylneuraminate and acetyl-D-mannosamine/pyruvate, soaking in the mother liquor containing 15% w/v PEG400, then 20% w/v PEG400, and subsequently for 5 min in 25% w/v PEG400 containing 75 mM Neu5Ac, X-ray diffraction structure determination and analysis Haemophilus influenzae

Protein Variants

Protein Variants Comment Organism
F252A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Haemophilus influenzae
F252Y site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Haemophilus influenzae
S47A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Haemophilus influenzae
S47C site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Haemophilus influenzae
S47T site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme Haemophilus influenzae
T167A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Haemophilus influenzae
T167S site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme Haemophilus influenzae
T48A site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Haemophilus influenzae
T48S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Haemophilus influenzae
Y110A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Haemophilus influenzae
Y110F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Haemophilus influenzae
Y137A site-directed mutagenesis, inactive mutant Haemophilus influenzae
Y137F site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Haemophilus influenzae

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.2
-
 N-acetylneuraminate wild-type enzyme, pH 7.4, 30°C Haemophilus influenzae
3.7
-
 N-acetylneuraminate mutant Y137F enzyme, pH 7.4, 30°C Haemophilus influenzae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
N-acetylneuraminate Haemophilus influenzae
-
 N-acetyl-D-mannosamine + pyruvate
-
 r

Organism

Organism UniProt Comment Textmining
Haemophilus influenzae
-
-
-

Reaction

Reaction Comment Organism Reaction ID
aceneuramate = N-acetyl-D-mannosamine + pyruvate the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon-carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. A triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function Haemophilus influenzae
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-acetylneuraminate
-
 Haemophilus influenzae N-acetyl-D-mannosamine + pyruvate
-
 r
N-acetylneuraminate the enzyme catalyzes the reversible condensation of pyruvate with N-acetyl-D-mannosamine to yield the sialic acid N-acetylneuraminic acid Haemophilus influenzae N-acetyl-D-mannosamine + pyruvate
-
 r

Synonyms

Synonyms Comment Organism
N-Acetylneuraminic acid lyase
-
 Haemophilus influenzae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Haemophilus influenzae

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.013
-
 N-acetylneuraminate mutant Y137F enzyme, pH 7.4, 30°C Haemophilus influenzae
4.17
-
 N-acetylneuraminate wild-type enzyme, pH 7.4, 30°C Haemophilus influenzae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
 assay at Haemophilus influenzae

General Information

General Information Comment Organism
evolution N-acetylneuraminic acid lyase is a class I aldolase Haemophilus influenzae
additional information reaction mechanism by crystal structure analysis, and quantum mechanical/molecular mechanical modeling, overview Haemophilus influenzae

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0033
-
 N-acetylneuraminate mutant Y137F enzyme, pH 7.4, 30°C Haemophilus influenzae
1.9
-
 N-acetylneuraminate wild-type enzyme, pH 7.4, 30°C Haemophilus influenzae