Cloned (Comment) | Organism |
---|---|
subcloning of His-tagged wild-type and mutant enzymes in Escherichia coli strain DH5-alpha, expression in Escherichia coli strain BL21(DE3) Rosetta, the enzymes are secreted from periplasmic space | Saccharophagus degradans |
Crystallization (Comment) | Organism |
---|---|
purified recombinant soluble His-tagged wild-type and selenomethionine-labeled enzyme, and two mutant enzymes H202L and Y258A, and Y258A DELTAMMG variant, free or in complex with an alginate trisaccharide, hanging drop vapour diffusion method, mixing of 15 mg/ml protein in in 20 mM HEPES, pH 7.5, 100 mM KCl, with 0.1 M Tris, pH 8.0, 5% 2-methyl-2,4-pentanediol, 10% PEG 6000, 3 days, 16°C, X-ray diffraction structure determination and analysis at 1.85 A, 1.7 A, 2.45 A, and 1.9 A resolution, respectively | Saccharophagus degradans |
Protein Variants | Comment | Organism |
---|---|---|
H202L | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Saccharophagus degradans |
H415A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Saccharophagus degradans |
N201A | site-directed mutagenesis, inactive mutant | Saccharophagus degradans |
Q149A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Saccharophagus degradans |
R260A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Saccharophagus degradans |
R438A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Saccharophagus degradans |
Y258A | site-directed mutagenesis, inactive mutant | Saccharophagus degradans |
Y450A | site-directed mutagenesis, inactive mutant | Saccharophagus degradans |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0101 | - |
sodium alginate | pH 7.5, 30°C, mutant Q149A | Saccharophagus degradans | |
0.0121 | - |
sodium alginate | pH 7.5, 30°C, mutant R260A | Saccharophagus degradans | |
0.0216 | - |
sodium alginate | pH 7.5, 30°C, H415A | Saccharophagus degradans | |
0.0223 | - |
sodium alginate | pH 7.5, 30°C, wild-type enzyme | Saccharophagus degradans | |
0.0311 | - |
sodium alginate | pH 7.5, 30°C, mutant R438A | Saccharophagus degradans | |
0.0916 | - |
sodium alginate | pH 7.5, 30°C, mutant H202L | Saccharophagus degradans |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | relevance of this zinc ion in catalytic activity, metal-binding protein ligands are His533, Gln551, and Lys573. The metal ion establishes the orientation of His415 and Arg438, which would otherwise be mobile, to set these residues for interactions with substrate | Saccharophagus degradans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Saccharophagus degradans | the enzyme Alg17c is an exolytic alginate lyase, structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization, overview | ? | - |
? | |
additional information | Saccharophagus degradans DSM 17024 | the enzyme Alg17c is an exolytic alginate lyase, structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization, overview | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharophagus degradans | Q21FJ0 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant soluble His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Rosetta culture supernatant by nickel affinity chromatography and gel filtration | Saccharophagus degradans |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
[alpha-1,4-L-guluronate]n = alpha-L-guluronate + 4-deoxy-alpha-L-erythro-hex-4-enuronosyl-[alpha-1,4-L-guluronate]n-2 | substrate binding, structural change and exolytic mechanism of alginate depolymerization by Alg17c, overview | Saccharophagus degradans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | the enzyme Alg17c is an exolytic alginate lyase, structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization, overview | Saccharophagus degradans | ? | - |
? | |
additional information | the enzyme is active on poly-MM, poly-GG, and poly-MG substrates. Exolytic depolymerization of these polysaccharides by alginate lyase yields a monosaccharide and a product containing a DELTA-(4,5)-unsaturated uronic acid moiety. A mixture of alginate di-, tri-, and tetrasaccharides are processed into mono- and disaccharides in the presence of Alg17c. An alginate trisaccharide represents the minimal length substrate for Alg17c, complete processing only of the tri- and tetrasaccharide substrates, substrate specificity and binding structure, Fourier electron density map, overview | Saccharophagus degradans | ? | - |
? | |
additional information | the enzyme Alg17c is an exolytic alginate lyase, structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization, overview | Saccharophagus degradans DSM 17024 | ? | - |
? | |
additional information | the enzyme is active on poly-MM, poly-GG, and poly-MG substrates. Exolytic depolymerization of these polysaccharides by alginate lyase yields a monosaccharide and a product containing a DELTA-(4,5)-unsaturated uronic acid moiety. A mixture of alginate di-, tri-, and tetrasaccharides are processed into mono- and disaccharides in the presence of Alg17c. An alginate trisaccharide represents the minimal length substrate for Alg17c, complete processing only of the tri- and tetrasaccharide substrates, substrate specificity and binding structure, Fourier electron density map, overview | Saccharophagus degradans DSM 17024 | ? | - |
? | |
sodium alginate | - |
Saccharophagus degradans | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | structure analysis, SDS-PAGE and analytical gel filtration, overview | Saccharophagus degradans |
Synonyms | Comment | Organism |
---|---|---|
Alg17C | - |
Saccharophagus degradans |
alginate lyase | - |
Saccharophagus degradans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Saccharophagus degradans |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0149 | - |
sodium alginate | pH 7.5, 30°C, mutant R260A | Saccharophagus degradans | |
0.0156 | - |
sodium alginate | pH 7.5, 30°C, mutant Q149A | Saccharophagus degradans | |
0.018 | - |
sodium alginate | pH 7.5, 30°C, mutant R438A | Saccharophagus degradans | |
0.057 | - |
sodium alginate | pH 7.5, 30°C, H415A | Saccharophagus degradans | |
10.9 | - |
sodium alginate | pH 7.5, 30°C, mutant H202L | Saccharophagus degradans | |
56.9 | - |
sodium alginate | pH 7.5, 30°C, wild-type enzyme | Saccharophagus degradans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Saccharophagus degradans |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the polysaccharide lyase PL7 family. Structure and beta-elimination mechanism for glycolytic bond cleavage by Alg17c are similar to those observed for family 15 polysaccharide lyases and other lyases, evolutionary relationships and structure-based hierarchy in the classification, overview | Saccharophagus degradans |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.58 | - |
sodium alginate | pH 7.5, 30°C, mutant R438A | Saccharophagus degradans | |
1.3 | - |
sodium alginate | pH 7.5, 30°C, mutant R260A | Saccharophagus degradans | |
1.6 | - |
sodium alginate | pH 7.5, 30°C, mutant Q149A | Saccharophagus degradans | |
2.6 | - |
sodium alginate | pH 7.5, 30°C, H415A | Saccharophagus degradans | |
120 | - |
sodium alginate | pH 7.5, 30°C, mutant H202L | Saccharophagus degradans | |
2570 | - |
sodium alginate | pH 7.5, 30°C, wild-type enzyme | Saccharophagus degradans |