Literature summary for 3.2.1.39 extracted from

Labourel, A.; Jam, M.; Jeudy, A.; Hehemann, J.H.; Czjzek, M.; Michel, G.
The beta-glucanase ZgLamA from Zobellia galactanivorans evolved a bent active site adapted for efficient degradation of algal laminarin (2014), J. Biol. Chem., 289, 2027-2042.

Cloned(Commentary)

Cloned (Comment)	Organism
gene Zobellia_2431, DNA and amino acid sequence determination and analysis, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Zobellia galactanivorans

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His-tagged wild-type and mutant enzymes, hanging drop vapour diffusion method, mixing of 0.002 ml of 10 mg/ml protein with 0.002 ml reservoir solution containing 24% PEG 3350 and 100 mM sodium citrate, pH 5.2, 4°C, crystals of enzyme mutant E269S in complex with laminarin oligosaccharides are obtained from 0.002 ml of 13.3 mg/ml protein and 5 mM of purified hexasaccharides,with 0.001 ml of reservoir solution containing 100 mM sodium malonate, imidazole, and boric acid (MIB-buffer), pH 4.0, and 19% of PEG 1500 in hanging drops at 20°C. Single crystals of mutant E269S in complex with MLG trisaccharides are obtained from mixing 0.002 ml 11.7 mg/ml of protein, 0.04% w/v of MLG degradation products with 0.001 ml of reservoir solution containing 100 mM MIB buffer, pH 4.0, 17% of PEG 1500, and 10% of glycerol in hanging drops at 12°C, X-ray diffraction structure determination and analysis at 1.13-1.45 A resolution, molecular replacement method	Zobellia galactanivorans

Crystallization (Comment)

Organism

purified recombinant His-tagged wild-type and mutant enzymes, hanging drop vapour diffusion method, mixing of 0.002 ml of 10 mg/ml protein with 0.002 ml reservoir solution containing 24% PEG 3350 and 100 mM sodium citrate, pH 5.2, 4°C, crystals of enzyme mutant E269S in complex with laminarin oligosaccharides are obtained from 0.002 ml of 13.3 mg/ml protein and 5 mM of purified hexasaccharides,with 0.001 ml of reservoir solution containing 100 mM sodium malonate, imidazole, and boric acid (MIB-buffer), pH 4.0, and 19% of PEG 1500 in hanging drops at 20°C. Single crystals of mutant E269S in complex with MLG trisaccharides are obtained from mixing 0.002 ml 11.7 mg/ml of protein, 0.04% w/v of MLG degradation products with 0.001 ml of reservoir solution containing 100 mM MIB buffer, pH 4.0, 17% of PEG 1500, and 10% of glycerol in hanging drops at 12°C, X-ray diffraction structure determination and analysis at 1.13-1.45 A resolution, molecular replacement method

Zobellia galactanivorans

Protein Variants

Protein Variants	Comment	Organism
E269S	site-directed mutagenesis, nucleophile replacement, inactive mutant	Zobellia galactanivorans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
laminarin + H2O	Zobellia galactanivorans	-	?	-	?
laminarin + H2O	Zobellia galactanivorans DSM 12802	-	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Zobellia galactanivorans	G0L5X4	gene Zobellia_2431	-
Zobellia galactanivorans DSM 12802	G0L5X4	gene Zobellia_2431	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration	Zobellia galactanivorans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
Glc-beta-(1->4)-Glc-beta-(1->3)-Glc-beta-(1->4)-Glc + H2O	-	Zobellia galactanivorans	D-glucose + Glc-beta-(1->4)-Glc-beta-(1->3)-Glc	containing one beta-1,4-linkage separated by one beta-1,3-linkage, i.e. G4G3G, which is not further degraded	?
Glc-beta-(1->4)-Glc-beta-(1->3)-Glc-beta-(1->4)-Glc + H2O	-	Zobellia galactanivorans DSM 12802	D-glucose + Glc-beta-(1->4)-Glc-beta-(1->3)-Glc	containing one beta-1,4-linkage separated by one beta-1,3-linkage, i.e. G4G3G, which is not further degraded	?
laminarin + H2O	-	Zobellia galactanivorans	?	-	?
laminarin + H2O	the wild-type enzyme displays a bent topology adapted to the binding of helical-shaped laminarin	Zobellia galactanivorans	?	-	?
laminarin + H2O	-	Zobellia galactanivorans DSM 12802	?	-	?
laminarin + H2O	the wild-type enzyme displays a bent topology adapted to the binding of helical-shaped laminarin	Zobellia galactanivorans DSM 12802	?	-	?
laminaritriose + H2O	the smallest oligosaccharide that can be degraded by the enzyme	Zobellia galactanivorans	D-glucose + laminaribiose	-	?
laminaritriose + H2O	the smallest oligosaccharide that can be degraded by the enzyme	Zobellia galactanivorans DSM 12802	D-glucose + laminaribiose	-	?
additional information	the enzyme specifically cleaves beta-1,4-linkages next to beta-1,3-linkages and is active on linear 1,3-beta-D-glucans with 3-6 monomers and on a glucan tetrasaccharide containing two beta-1,4-linkages separated by one beta-1,3-linkage. The activity towards beta-1,4-linkages is much lower than towards beta-1,3-linkages. Enzyme-substrate complex structure analysis using wild-type and mutant enzymes with glucan oligomers, binding structure and substrate recognition, overview	Zobellia galactanivorans	?	-	?
additional information	the enzyme specifically cleaves beta-1,4-linkages next to beta-1,3-linkages and is active on linear 1,3-beta-D-glucans with 3-6 monomers and on a glucan tetrasaccharide containing two beta-1,4-linkages separated by one beta-1,3-linkage. The activity towards beta-1,4-linkages is much lower than towards beta-1,3-linkages. Enzyme-substrate complex structure analysis using wild-type and mutant enzymes with glucan oligomers, binding structure and substrate recognition, overview	Zobellia galactanivorans DSM 12802	?	-	?

Synonyms

Synonyms	Comment	Organism
beta-glucanase	-	Zobellia galactanivorans
LamA	-	Zobellia galactanivorans
laminarinase	-	Zobellia galactanivorans
ZgLamA	-	Zobellia galactanivorans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	40	assay at	Zobellia galactanivorans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.5	-	assay at	Zobellia galactanivorans

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the glycohydrolase family GH16	Zobellia galactanivorans
additional information	involvement of Glu269 in the catalytic machinery of the enzyme	Zobellia galactanivorans