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Literature summary extracted from

  • Timms, N.; Windle, C.L.; Polyakova, A.; Ault, J.R.; Trinh, C.H.; Pearson, A.R.; Nelson, A.; Berry, A.
    Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with gamma-thialysine by using a chemical mutagenesis strategy (2013), ChemBioChem, 14, 474-481.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.1.3.3 gene nanA, recombinant expression of His6-tagged wild-type and mutant enzymes from plasmid pKK223-3 in Escherichia coli strain BL21(DE3) Staphylococcus aureus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.1.3.3 purified recombinant wild-type enzyme and mutants K165C variant and K165-gamma-thialysine, alone or in complex with pyruvate, hanging drop vapour diffusion method, mixing of 0.002 ml of protein solution containing 8 mg/ml protein in 50 mM, pH 7.4, with 0.002 ml of reservoir solution containing 100 mM Tris-HCl, pH 7.0-8.5, 200 mM NaCl, and 18-28% w/v PEG 3350, pyruvate complexes of the wild-type and K165-gamma-thialysine mutant enzymes crystals are soaked in the mother liquor containing 100 mM sodium pyruvate and 15% v/v PEG 400 for 1 min before being sequentially transferred to mother liquor with 5% increments in PEG 400 concentration. The final soak contains the mother liquor containing 100 mM sodium pyruvate and 25% v/v PEG 400, 18°C, X-ray diffraction structure determination and analysis at about 2.0 A resolution, molecular replacement Staphylococcus aureus

Protein Variants

EC Number Protein Variants Comment Organism
4.1.3.3 C118A site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 C118S site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 C238A site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 C238S site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 C270A site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 C270S site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 C82A site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 C82S site-directed mutagenesis, the mutant shows kinetic properties identical to the wild-type enzyme Staphylococcus aureus
4.1.3.3 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 Staphylococcus aureus
4.1.3.3 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 Staphylococcus aureus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.1.3.3 0.013
-
 N-acetylneuraminate purified recombinant mutant K165C, pH 7.4, 30°C Staphylococcus aureus
4.1.3.3 0.015
-
 N-acetylneuraminate purified recombinant mutant K165-gamma-thialysine, pH 6.8, 30°C Staphylococcus aureus
4.1.3.3 0.023
-
 N-acetylneuraminate purified recombinant mutant K165-gamma-thialysine, pH 7.4, 30°C Staphylococcus aureus
4.1.3.3 0.036
-
 N-acetylneuraminate purified recombinant wild-type enzyme, pH 7.4, 30°C Staphylococcus aureus
4.1.3.3 0.04
-
 N-acetylneuraminate purified recombinant wild-type enzyme, pH 6.8, 30°C Staphylococcus aureus

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.1.3.3 33992
-
 x * 33992, recombinant His6-tagged wild-type enzyme, mass spectrometry Staphylococcus aureus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.1.3.3 N-acetylneuraminate Staphylococcus aureus
-
 N-acetyl-D-mannosamine + pyruvate
-
 r
4.1.3.3 N-acetylneuraminate Staphylococcus aureus NCTC 8325
-
 N-acetyl-D-mannosamine + pyruvate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
4.1.3.3 Staphylococcus aureus Q2G160 gene nanA
-
4.1.3.3 Staphylococcus aureus NCTC 8325 Q2G160 gene nanA
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.1.3.3 recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Staphylococcus aureus

Renatured (Commentary)

EC Number Renatured (Comment) Organism
4.1.3.3 recombinant wild-type enzyme with various concentrations of urea in Tris·HCl buffer, 50 mM, pH 7.4, the refolded enzyme shows higher activity than the native wild-type enzyme Staphylococcus aureus

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.1.3.3 4.33
-
 purified recombinant wild-type enzyme, pH 7.4, 30°C Staphylococcus aureus
4.1.3.3 6
-
 purified recombinant refolded wild-type enzyme, pH 7.4, 30°C Staphylococcus aureus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.1.3.3 N-acetylneuraminate
-
 Staphylococcus aureus N-acetyl-D-mannosamine + pyruvate
-
 r
4.1.3.3 N-acetylneuraminate
-
 Staphylococcus aureus NCTC 8325 N-acetyl-D-mannosamine + pyruvate
-
 r

Subunits

EC Number Subunits Comment Organism
4.1.3.3 ? x * 33992, recombinant His6-tagged wild-type enzyme, mass spectrometry Staphylococcus aureus

Synonyms

EC Number Synonyms Comment Organism
4.1.3.3 class I NAL
-
 Staphylococcus aureus
4.1.3.3 N-Acetylneuraminic acid lyase
-
 Staphylococcus aureus
4.1.3.3 NAL
-
 Staphylococcus aureus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.1.3.3 30
-
 assay at Staphylococcus aureus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.1.3.3 0.013
-
 N-acetylneuraminate purified recombinant mutant K165C, pH 7.4, 30°C Staphylococcus aureus
4.1.3.3 0.015
-
 N-acetylneuraminate purified recombinant mutant K165-gamma-thialysine, pH 6.8, 30°C Staphylococcus aureus
4.1.3.3 0.023
-
 N-acetylneuraminate purified recombinant mutant K165-gamma-thialysine, pH 7.4, 30°C Staphylococcus aureus
4.1.3.3 0.036
-
 N-acetylneuraminate purified recombinant wild-type enzyme, pH 7.4, 30°C Staphylococcus aureus
4.1.3.3 0.04
-
 N-acetylneuraminate purified recombinant wild-type enzyme, pH 6.8, 30°C Staphylococcus aureus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.1.3.3 6.8
-
 mutant K165-gamma-thialysine Staphylococcus aureus
4.1.3.3 7.4
-
 wild-type enzyme Staphylococcus aureus

pH Range

EC Number pH Minimum pH Maximum Comment Organism
4.1.3.3 5 10 pH-activity profiles of the wild-type enzyme, overview Staphylococcus aureus
4.1.3.3 6 9 pH-activity profiles of the K165-gamma-thialysine mutant enzyme, overview Staphylococcus aureus