Literature summary for 4.1.3.3 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.

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

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

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

Purification (Comment)	Organism
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)

Renatured (Comment)	Organism
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]

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

Substrates and Products (Substrate)

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

Subunits

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

Synonyms

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

pH Range

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