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Information on EC 4.1.3.3 - N-acetylneuraminate lyase and Organism(s) Staphylococcus aureus and UniProt Accession Q2G160

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EC Tree
     4 Lyases
         4.1 Carbon-carbon lyases
             4.1.3 Oxo-acid-lyases
                4.1.3.3 N-acetylneuraminate lyase
IUBMB Comments
This enzyme is involved in the degradation of N-acetylneuraminate. It is specific for the open form of the sugar. It also acts on N-glycoloylneuraminate and on O-acetylated sialic acids, other than 4-O-acetylated derivatives.
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Staphylococcus aureus
UNIPROT: Q2G160
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The taxonomic range for the selected organisms is: Staphylococcus aureus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
n-acetylneuraminate lyase, sialic acid aldolase, n-acetylneuraminic acid aldolase, n-acetylneuraminic acid lyase, neu5ac aldolase, n-acetyl-d-neuraminic acid aldolase, neuac lyase, n-acetylneuraminate pyruvate lyase, n-acetylneuraminate pyruvate-lyase, neuraminic acid aldolase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N-Acetylneuraminic acid lyase
-
Acetylneuraminate pyruvate-lyase
-
-
-
-
Lyase, acetylneuraminate
-
-
-
-
N-Acetylneuraminate aldolase
-
-
-
-
N-Acetylneuraminate lyase
-
-
-
-
N-acetylneuraminate pyruvate lyase
-
-
-
-
N-Acetylneuraminate pyruvate-lyase
-
-
-
-
N-Acetylneuraminic acid aldolase
-
-
-
-
N-Acetylneuraminic acid lyase
-
-
-
-
N-Acetylneuraminic aldolase
-
-
-
-
N-Acetylneuraminic lyase
-
-
-
-
NALase
-
-
-
-
NANA lyase
-
-
-
-
NeuAc aldolase
-
-
-
-
Neuraminate aldolase
-
-
-
-
Neuraminic acid aldolase
-
-
-
-
Neuraminic aldolase
-
-
-
-
NPL
-
-
-
-
Sialate lyase
-
-
-
-
Sialic acid aldolase
-
-
-
-
Sialic aldolase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
aceneuramate pyruvate-lyase (N-acetyl-D-mannosamine-forming)
This enzyme is involved in the degradation of N-acetylneuraminate. It is specific for the open form of the sugar. It also acts on N-glycoloylneuraminate and on O-acetylated sialic acids, other than 4-O-acetylated derivatives.
CAS REGISTRY NUMBER
COMMENTARY hide
9027-60-5
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
N-acetylneuraminate
N-acetyl-D-mannosamine + pyruvate
show the reaction diagram
N-acetyl-D-mannosamine + pyruvate
N-acetylneuraminate
show the reaction diagram
the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-D-mannosamine and pyruvate
-
-
r
N-acetylneuraminate
N-acetyl-D-mannosamine + pyruvate
show the reaction diagram
-
-
-
r
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
N-acetylneuraminate
N-acetyl-D-mannosamine + pyruvate
show the reaction diagram
N-acetyl-D-mannosamine + pyruvate
N-acetylneuraminate
show the reaction diagram
the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-D-mannosamine and pyruvate
-
-
r
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2R)-sialic acid alditol
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.013 - 3.2
N-acetylneuraminate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.013 - 22.1
N-acetylneuraminate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.39
(2R)-sialic acid alditol
at 30°C, pH not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.33
purified recombinant wild-type enzyme, pH 7.4, 30°C
6
purified recombinant refolded wild-type enzyme, pH 7.4, 30°C
11
purified recombinant enzyme, pH 8.0, temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
mutant K165-gamma-thialysine
7.4
wild-type enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
pH-activity profiles of the wild-type enzyme, overview
6 - 9
pH-activity profiles of the K165-gamma-thialysine mutant enzyme, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
129
calculated from amino acid sequence
33992
x * 33992, recombinant His6-tagged wild-type enzyme, mass spectrometry
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 33992, recombinant His6-tagged wild-type enzyme, mass spectrometry
homotetramer
4 * 33000, calculated from amino acid sequence
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ligand-free and (2R)-sialic acid alditol-bound enzyme, hanging drop vapor diffusion method, using
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
purified recombinant enzyme, hanging drop vapour diffusion method, 0.002-0.0025 ml of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, is mixed with 0.0017-0.0022 ml of reservoir solution containing 25% w/v PEG 3350, 200 mM ammonium sulfate, 100 mM Bis-Tris pH 5.5, equilibration against 1 ml of reservoir solution, 8-20°C, method optimization, X-ray diffraction structure determination and analysis at 1.7 A resolution, molecular replacement
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydrophobic interaction chromatography, and gel filtration to over 95% purity
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene nanA, recombinant expression of His6-tagged wild-type and mutant enzymes from plasmid pKK223-3 in Escherichia coli strain BL21(DE3)
gene nanA, DNA and amino acid sequence determination and analysis, sequence comparison, expression in Escherichia coli strain BL21(DE3)
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
North, R.A.; Kessans, S.A.; Atkinson, S.C.; Suzuki, H.; Watson, A.J.; Burgess, B.R.; Angley, L.M.; Hudson, A.O.; Varsani, A.; Griffin, M.D.; Fairbanks, A.J.; Dobson, R.C.
Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus
Acta Crystallogr. Sect. F
69
306-312
2013
Staphylococcus aureus (Q6GK01), Staphylococcus aureus, Staphylococcus aureus MRSA252 (Q6GK01)
Manually annotated by BRENDA team
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
ChemBioChem
14
474-481
2013
Staphylococcus aureus (Q2G160), Staphylococcus aureus, Staphylococcus aureus NCTC 8325 (Q2G160)
Manually annotated by BRENDA team
North, R.A.; Watson, A.J.; Pearce, F.G.; Muscroft-Taylor, A.C.; Friemann, R.; Fairbanks, A.J.; Dobson, R.C.
Structure and inhibition of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus
FEBS Lett.
590
4414-4428
2016
Staphylococcus aureus (Q2G160), Staphylococcus aureus, Staphylococcus aureus NCTC 8325 (Q2G160)
Manually annotated by BRENDA team