Cloned (Comment) | Organism |
---|---|
gene ugl, recombinant expression of wild-type enzyme and enzyme mutants in Escherichia coli strain HMS174(DE3)pLysS | Streptococcus agalactiae |
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 | 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 | 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 | 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 | - |
5-dehydro-4-deoxy-D-glucuronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptococcus agalactiae | A0A0E1EQ72 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type enzyme and enzyme mutants from Escherichia coli strain HMS174(DE3)pLysS | Streptococcus agalactiae |
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 | 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 | - |
Streptococcus agalactiae | 5-dehydro-4-deoxy-D-glucuronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp | - |
? | |
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 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
SagUGL | - |
Streptococcus agalactiae |
UGL | - |
Streptococcus agalactiae |
unsaturated glucuronyl hydrolase | - |
Streptococcus agalactiae |
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 |