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Literature summary for 3.2.1.179 extracted from

  • Nakamichi, Y.; Oiki, S.; Mikami, B.; Murata, K.; Hashimoto, W.
    Conformational change in the active site of streptococcal unsaturated glucuronyl hydrolase through site-directed mutagenesis at Asp-115 (2016), Protein J., 35, 300-309 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene ugl, recombinant expression of wild-type enzyme and enzyme mutants in Escherichia coli strain HMS174(DE3)pLysS Streptococcus agalactiae

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme mutants D115N and D115N/K370S, sitting drop vapor diffusion, 20 mg/ml and 40 mg/ml protein solution, respectively, is mixed with an equal volumes of reservoir solution. The reservoir solution for mutant D115N crystallization contains 1.5 M ammonium sulfate and 0.1 M Tris-HCl, pH 9.0, D115N crystals are grown at 20°C for a week. The reservoir solution for mutant D115N/K370S crystallization contains 40% ethylene glycol, 0.5 mM dithiothreitol, and 0.1 M Tris-HCl, pH 7.0, D115N/K370S is crystallized at 20°C for 2 months. X-ray diffraction structure determination and analysis at 2.00 A and 1.79 A resolution, respectively Streptococcus agalactiae

Protein Variants

Protein Variants Comment Organism
D115N site-directed mutagenesis, the mutant shows only negligible enzyme activity Streptococcus agalactiae
D115N/K370S site-directed mutagenesis, the mutant shows only negligible enzyme activity Streptococcus agalactiae

Inhibitors

Inhibitors Comment Organism Structure
chondroitin sulfate C 20% inhibition of enzyme SagUGL Streptococcus agalactiae
glycine over 95% inhibition Streptococcus agalactiae
hyaluronan 24% inhibition of enzyme SagUGL Streptococcus agalactiae
additional information poor effects by gellan, xanthan, and heparin Streptococcus agalactiae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
beta-D-4-deoxy-Delta4-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp + H2O Streptococcus agalactiae
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5-dehydro-4-deoxy-D-glucuronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp
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?

Organism

Organism UniProt Comment Textmining
Streptococcus agalactiae A0A0E1EQ72
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Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type enzyme and enzyme mutants from Escherichia coli strain HMS174(DE3)pLysS Streptococcus agalactiae

Reaction

Reaction Comment Organism Reaction ID
beta-D-4-deoxy-Delta4-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp + H2O = 5-dehydro-4-deoxy-D-glucuronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp the enzyme triggers a hydration reaction acting on the C-C double bond between the C-4 and C-5 positions of the unsaturated uronic acid residue in GAG disaccharide using the catalytic Asp residue (Asp-II) as a general acid and base catalyst to protonate the C-4 atom of unsaturated uronic acid residue and to deprotonate the water molecule. The deprotonated water molecule attacks the C-5 atom of the unsaturated uronic acid residue to yield unstable hemiketal, followed by its spontaneous conversion to 4-deoxy-L-threo-5-hexosulose-uronic acid and an amino sugar via hemiacetal formation and cleavage of the glycoside bond Streptococcus agalactiae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
beta-D-4-deoxy-Delta4-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp + H2O
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Streptococcus agalactiae 5-dehydro-4-deoxy-D-glucuronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp
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?
additional information glycosaminoglycans such as hyaluronan and heparin are depolymerized by polysaccharide lyase and the resulting unsaturated disaccharides are degraded to unsaturated uronic acids and amino sugars by enzyme UG, reaction mechanism of UGL, overview. Gellan gum, xanthan gum, hyaluronan, chondroitin sulfate C, and heparin are used as glucuronate-containing polysaccharides. SagUGL preferentially degrades unsaturated hyaluronan and chondroitin sulfate disaccharides with 1,3-glycosidic bonds Streptococcus agalactiae ?
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?

Synonyms

Synonyms Comment Organism
SagUGL
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Streptococcus agalactiae
UGL
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Streptococcus agalactiae
unsaturated glucuronyl hydrolase
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Streptococcus agalactiae

General Information

General Information Comment Organism
malfunction SagUGL mutants D115N and D115N/K370S show negligible enzyme activity. The side chain of Asn115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp175, by formation of hydrogen bonds. This interaction between Asn115 and Asp175 probably prevents the mutants from triggering the enzyme reaction using Asp175 as an acid catalyst Streptococcus agalactiae
additional information two Asp residues, Asp115 and Asp175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp115 of SagUGL) also affects the enzyme activity Streptococcus agalactiae