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Literature summary for 4.2.2.1 extracted from

  • Nukui, M.; Taylor, K.B.; McPherson, D.T.; Shigenaga, M.K.; Jedrzejas, M.J.
    The function of hydrophobic residues in the catalytic cleft of Streptococcus pneumoniae hyaluronate lyase. Kinetic characterization of mutant enzyme forms (2003), J. Biol. Chem., 278, 3079-3088.
    View publication on PubMed
Search for more literature for 4.2.2.1

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type and mutant enzymes in Escherichia coli BL21(DE3) Streptococcus pneumoniae

Crystallization (Commentary)

Crystallization (Comment) Organism
mutant enzymes at 5.1-6.2 mg/ml concentration, hanging drop vapour diffusion method, equal volumes of protein and reservoir solutions, the latter containing 60-65% saturated ammonium sulfate, 0.2 M sodium chloride, 2% dioxane, 500 mM sodium citrate, pH 6.0, X-ray diffraction structure determination and analysis at 1.5-2.3 A resolution Streptococcus pneumoniae

Protein Variants

Protein Variants Comment Organism
F343V site-directed mutagensis, residue of the hydrophobic patch involved in substrate positioning during catalysis, reduced activity, crystallization for structure determination Streptococcus pneumoniae
W291A/W292A site-directed mutagensis, residues of the hydrophobic patch involved in substrate positioning during catalysis, inactive mutant, crystallization for structure determination Streptococcus pneumoniae
W291A/W292A/F343V site-directed mutagensis, residues of the hydrophobic patch involved in substrate positioning during catalysis, inactive mutant, crystallization for structure determination Streptococcus pneumoniae
W292A site-directed mutagensis, residue of the hydrophobic patch involved in substrate positioning during catalysis, highly reduced activity, crystallization for structure determination Streptococcus pneumoniae
W292A/F343V site-directed mutagensis, residues of the hydrophobic patch involved in substrate positioning during catalysis, highly reduced activity, crystallization for structure determination Streptococcus pneumoniae

Inhibitors

Inhibitors Comment Organism Structure
NaCl slight inhibition, wild-type and mutant enzymes Streptococcus pneumoniae

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics Streptococcus pneumoniae
0.08
-
 hyaluronan mutant F343V, pH 6.0, 37°C Streptococcus pneumoniae
0.09
-
 hyaluronan wild-type enzyme, pH 6.0, 37°C Streptococcus pneumoniae
0.6
-
 hyaluronan mutant W292A, pH 6.0, 37°C Streptococcus pneumoniae
3.8
-
 hyaluronan mutant W292A/F343V, pH 6.0, 37°C Streptococcus pneumoniae

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane cell surface Streptococcus pneumoniae 16020
-

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+
-
 Streptococcus pneumoniae

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
83027
-
 x * 83208, mutant W292A, mass spectrometry, x * 83273, mutant F343V, mass spectrometry, x * 83097, mutant W291A/W292A, mass spectrometry, x * 83102, mutant W292A/F343V, mass spectrometry, x * 83027, mutant W291A/W292A/F343V, mass spectrometry Streptococcus pneumoniae
83097
-
 x * 83208, mutant W292A, mass spectrometry, x * 83273, mutant F343V, mass spectrometry, x * 83097, mutant W291A/W292A, mass spectrometry, x * 83102, mutant W292A/F343V, mass spectrometry, x * 83027, mutant W291A/W292A/F343V, mass spectrometry Streptococcus pneumoniae
83102
-
 x * 83208, mutant W292A, mass spectrometry, x * 83273, mutant F343V, mass spectrometry, x * 83097, mutant W291A/W292A, mass spectrometry, x * 83102, mutant W292A/F343V, mass spectrometry, x * 83027, mutant W291A/W292A/F343V, mass spectrometry Streptococcus pneumoniae
83208
-
 x * 83208, mutant W292A, mass spectrometry, x * 83273, mutant F343V, mass spectrometry, x * 83097, mutant W291A/W292A, mass spectrometry, x * 83102, mutant W292A/F343V, mass spectrometry, x * 83027, mutant W291A/W292A/F343V, mass spectrometry Streptococcus pneumoniae
83273
-
 x * 83208, mutant W292A, mass spectrometry, x * 83273, mutant F343V, mass spectrometry, x * 83097, mutant W291A/W292A, mass spectrometry, x * 83102, mutant W292A/F343V, mass spectrometry, x * 83027, mutant W291A/W292A/F343V, mass spectrometry Streptococcus pneumoniae

Organism

Organism UniProt Comment Textmining
Streptococcus pneumoniae Q54873
-
-

Reaction

Reaction Comment Organism Reaction ID
[hyaluronate]n = (4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine-[hyaluronate]n-m-1 + 2 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine + hyaluronate processive reaction mode, catalytic mechanism, catalytic residues are Asn349, His399, and Tyr408, residues responsible for product release are Glu388, Asp398, and Thr400, the socalled negative patch, residues responsible for correct substrate positioning are Trp291, Trp292, and Phe343, the socalled hydrophobic patch, substrate binding structure of wild-type and mutant enzymes Streptococcus pneumoniae
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.19
-
 mutant W292A/F343V, in absence of NaCl Streptococcus pneumoniae
4.3
-
 mutant W292A, in absence of NaCl Streptococcus pneumoniae
68.9
-
 mutant F343V, in absence of NaCl Streptococcus pneumoniae
231.4
-
 wild-type enzyme, in absence of NaCl Streptococcus pneumoniae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
hyaluronan from human umbilical cord, degradation, from the reducing to the nonreducing end Streptococcus pneumoniae 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine unsaturated disaccharide units, product identification ?

Subunits

Subunits Comment Organism
? x * 83208, mutant W292A, mass spectrometry, x * 83273, mutant F343V, mass spectrometry, x * 83097, mutant W291A/W292A, mass spectrometry, x * 83102, mutant W292A/F343V, mass spectrometry, x * 83027, mutant W291A/W292A/F343V, mass spectrometry Streptococcus pneumoniae

Synonyms

Synonyms Comment Organism
spnHL
-
 Streptococcus pneumoniae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Streptococcus pneumoniae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
 assay at Streptococcus pneumoniae