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Literature summary for 4.2.2.3 extracted from

  • Park, D.; Jagtap, S.; Nair, S.K.
    Structure of a PL17 family alginate lyase demonstrates functional similarities among exotype depolymerases (2014), J. Biol. Chem., 289, 8645-8655.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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 [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

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Saccharophagus degradans Q21FJ0
-
-
Saccharophagus degradans DSM 17024 Q21FJ0
-
-

Purification (Commentary)

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

Reaction Comment Organism Reaction ID
R2-beta-D-mannuronic acid-R1 = R2-OH + 4-deoxy-alpha-L-erythro-hex-4-enopyranuronosyl-R1 substrate binding, strutural change and exolytic mechanism of alginate depolymerization by Alg17c, overview Saccharophagus degradans

Substrates and Products (Substrate)

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 ?
-
?
sodium alginate
-
Saccharophagus degradans DSM 17024 ?
-
?

Subunits

Subunits Comment Organism
homodimer structure analysis, SDS-PAGE and analytical gel filtration, overview Saccharophagus degradans

Synonyms

Synonyms Comment Organism
Alg17C
-
Saccharophagus degradans
alginate lyase
-
Saccharophagus degradans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Saccharophagus degradans

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Saccharophagus degradans

General Information

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 [mM/s]

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