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Literature summary extracted from

  • Thomas, F.; Lundqvist, L.C.; Jam, M.; Jeudy, A.; Barbeyron, T.; Sandstroem, C.; Michel, G.; Czjzek, M.
    Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct modes of action and exquisite adaptation to their natural substrate (2013), J. Biol. Chem., 288, 23021-23037.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.2.3 phylogenetic analysis, recombinant expression of His-tagged AlyA5 in Escherichia coli strain BL21(DE3) Zobellia galactanivorans
4.2.2.11 phylogenetic analysis, recombinant expression of His-tagged AlyA1PL7 in Escherichia coli strain BL21(DE3) Zobellia galactanivorans
4.2.2.11 phylogenetic analysis, recombinant expression of His-tagged AlyA5 in Escherichia coli strain BL21(DE3) Zobellia galactanivorans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.2.3 purified recombinant full-length dimeric AlyA5, hanging drop vapor diffusion method, 0.002 ml of 7.7 mg/ml protein solution with 0.1 mg/ml oligoglucuronate, are mixed with 0.001 ml of reservoir solution containing PEG 3350 and 0.2 M sodium/potassium tartrate, and equilibration against 0.2 ml reservoir solution, 21°C, screening and method optimization, X-ray diffraction structure determination and analysis at 1.75 A resolution Zobellia galactanivorans
4.2.2.11 purified recombinant catalytic domain of AlyA1PL7, hanging drop vapor diffusion method, 0.002 ml of 11.3 mg/ml protein solution with 0.1 mg/ml oligoguluronate, are mixed with 0.001 ml of reservoir solution containing 0.2 M KSCN and 28% PEG-MME 2000, and equilibration against 0.5 ml reservoir solution, 21°C, screening and method optimization, X-ray diffraction structure determination and analysis at 1.43 A resolution Zobellia galactanivorans
4.2.2.11 purified recombinant full-length dimeric AlyA5, hanging drop vapor diffusion method, 0.002 ml of 7.7 mg/ml protein solution with 0.1 mg/ml oligoglucuronate, are mixed with 0.001 ml of reservoir solution containing PEG 3350 and 0.2 M sodium/potassium tartrate, and equilibration against 02 ml reservoir solution, 21°C, screening and method optimization, X-ray diffraction structure determination and analysis at 1.75 A resolution Zobellia galactanivorans

Inhibitors

EC Number Inhibitors Comment Organism Structure
4.2.2.11 EDTA 90% inhibition Zobellia galactanivorans
4.2.2.11 EGTA 95% inhibition Zobellia galactanivorans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.2.3 additional information
-
additional information dimeric AlyA5 shows two-phase kinetics with alginate, overview Zobellia galactanivorans
4.2.2.11 additional information
-
additional information dimeric AlyA5 shows two-phase kinetics with alginate, overview Zobellia galactanivorans
4.2.2.11 1.753
-
sodium alginate with L-guluronate content of 66.7%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans
4.2.2.11 3.087
-
sodium alginate with L-guluronate content of 52.6%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans
4.2.2.11 6.18
-
sodium alginate with L-guluronate content of 33.3%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.2.3 Ca2+ at least 2 mM Ca2+ in the reaction mixture is essential for the activity Zobellia galactanivorans
4.2.2.11 Ca2+ at least 2 mM Ca2+ in the reaction mixture is essential for the activity Zobellia galactanivorans
4.2.2.11 Ca2+ requires a divalent cation for activity, interaction between the enzyme and divalent cations takes place during substrate fixation and not before Zobellia galactanivorans
4.2.2.11 NaCl activates, best at 150-200 mM Zobellia galactanivorans

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.2.2.3 38300
-
monomeric recombinant His-tagged enzyme, gel filtration Zobellia galactanivorans
4.2.2.3 42000
-
x * 42000, recombinant His-tagged enzyme, SDS-PAGE Zobellia galactanivorans
4.2.2.3 69500
-
dimeric recombinant His-tagged enzyme, gel filtration Zobellia galactanivorans
4.2.2.11 38300
-
monomeric recombinant His-tagged enzyme, gel filtration Zobellia galactanivorans
4.2.2.11 42000
-
x * 42000, recombinant His-tagged enzyme, SDS-PAGE Zobellia galactanivorans
4.2.2.11 69500
-
dimeric recombinant His-tagged enzyme, gel filtration Zobellia galactanivorans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.2.3 sodium alginate Zobellia galactanivorans AlyA5 cleaves unsaturated units, alpha-L-guluronate or beta-D-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion, cf. EC 4.2.2.11 ?
-
?
4.2.2.3 sodium alginate Zobellia galactanivorans DSM 12802 AlyA5 cleaves unsaturated units, alpha-L-guluronate or beta-D-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion, cf. EC 4.2.2.11 ?
-
?
4.2.2.11 sodium alginate Zobellia galactanivorans AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches ?
-
?
4.2.2.11 sodium alginate Zobellia galactanivorans AlyA5 cleaves unsaturated units, alpha-L-guluronate or beta-D-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion, cf. EC 4.2.2.3 ?
-
?
4.2.2.11 sodium alginate Zobellia galactanivorans DSM 12802 AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches ?
-
?

Organism

EC Number Organism UniProt Comment Textmining
4.2.2.3 Zobellia galactanivorans G0L2Y1
-
-
4.2.2.3 Zobellia galactanivorans DSM 12802 G0L2Y1
-
-
4.2.2.11 Zobellia galactanivorans G0KZG8
-
-
4.2.2.11 Zobellia galactanivorans G0L2Y1
-
-
4.2.2.11 Zobellia galactanivorans DSM 12802 G0KZG8
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.2.3 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration to homogeneity Zobellia galactanivorans
4.2.2.11 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration to homogeneity Zobellia galactanivorans

Reaction

EC Number Reaction Comment Organism Reaction ID
4.2.2.11 [alpha-1,4-L-guluronate]n = alpha-L-guluronate + 4-deoxy-alpha-L-erythro-hex-4-enuronosyl-[alpha-1,4-L-guluronate]n-2 AlyA1PL7 alginate lyase acts via the beta-elimination mechanism, producing the 4,5-unsaturated 4-deoxy-L-erythro-hex-4-enepyranosyluronate (DELTA) on the nonreducing end, catalytic mechanism of G-specific alginate lyases, overview Zobellia galactanivorans
4.2.2.11 [alpha-1,4-L-guluronate]n = alpha-L-guluronate + 4-deoxy-alpha-L-erythro-hex-4-enuronosyl-[alpha-1,4-L-guluronate]n-2 AlyA1PL7 alginate lyase acts via the beta-elimination mechanism, producing the 4,5-unsaturated 4-deoxy-L-erythro-hex-4-enepyranosyluronate (DELTA) on the nonreducing end, catalytic mechanism of guluronate-specific alginate lyases, overview Zobellia galactanivorans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.2.3 additional information the enzyme has a broad substrate tolerance and can cleave mannuronate-mannuronate, mannuronate-guluronate, and guluronate-guluronate linkages at the nonreducing end. The activity is depending on the block structure Zobellia galactanivorans ?
-
?
4.2.2.3 additional information the enzyme has a broad substrate tolerance and can cleave mannuronate-mannuronate, mannuronate-guluronate, and guluronate-guluronate linkages at the nonreducing end. The activity is depending on the block structure Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.3 poly(beta-D-manuronate)
-
Zobellia galactanivorans ?
-
?
4.2.2.3 poly(beta-D-manuronate)
-
Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.3 sodium alginate AlyA5 cleaves unsaturated units, alpha-L-guluronate or beta-D-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion, cf. EC 4.2.2.11 Zobellia galactanivorans ?
-
?
4.2.2.3 sodium alginate LC mass and NMR spectrometris product analysis Zobellia galactanivorans ?
-
?
4.2.2.3 sodium alginate AlyA5 cleaves unsaturated units, alpha-L-guluronate or beta-D-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion, cf. EC 4.2.2.11 Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.3 sodium alginate LC mass and NMR spectrometris product analysis Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.11 additional information alginate pentasaccharides that can be cleaved by AlyA1PL7 are GGGGG, GGMGG, GGGMG, and GGMMG Zobellia galactanivorans ?
-
?
4.2.2.11 additional information the enzyme has a broad substrate tolerance and can cleave M-M, M-G, and G-G linkages at the nonreducing end. The activity is depending on the block structure Zobellia galactanivorans ?
-
?
4.2.2.11 additional information alginate pentasaccharides that can be cleaved by AlyA1PL7 are GGGGG, GGMGG, GGGMG, and GGMMG Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.11 additional information the enzyme has a broad substrate tolerance and can cleave M-M, M-G, and G-G linkages at the nonreducing end. The activity is depending on the block structure Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.11 poly(alpha-L-guluronate)
-
Zobellia galactanivorans ?
-
?
4.2.2.11 poly(alpha-L-guluronate) unmodified substrate poly-G blocks and substrate that with its reducing end being reduced using sodium borohydride prior to the digestion Zobellia galactanivorans ?
-
?
4.2.2.11 poly(alpha-L-guluronate)
-
Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.11 sodium alginate AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches Zobellia galactanivorans ?
-
?
4.2.2.11 sodium alginate AlyA5 cleaves unsaturated units, alpha-L-guluronate or beta-D-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion, cf. EC 4.2.2.3 Zobellia galactanivorans ?
-
?
4.2.2.11 sodium alginate AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches, no activity with poly-(mannuronate-guluronate) and poly-(mannuronate-mannuronate), minimal recognition pattern of AlyA1PL7, overview Zobellia galactanivorans ? mainly trisaccharide and tetrasaccharide oligomers of alginate with a total content of 41% and 36%, respectively, around 19% disaccharide and only a small amount of pentamers and hexamers, LC mass and NMR spectrometric product analysis ?
4.2.2.11 sodium alginate LC mass and NMR spectrometris product analysis Zobellia galactanivorans ?
-
?
4.2.2.11 sodium alginate AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches Zobellia galactanivorans DSM 12802 ?
-
?
4.2.2.11 sodium alginate AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches, no activity with poly-(mannuronate-guluronate) and poly-(mannuronate-mannuronate), minimal recognition pattern of AlyA1PL7, overview Zobellia galactanivorans DSM 12802 ? mainly trisaccharide and tetrasaccharide oligomers of alginate with a total content of 41% and 36%, respectively, around 19% disaccharide and only a small amount of pentamers and hexamers, LC mass and NMR spectrometric product analysis ?

Subunits

EC Number Subunits Comment Organism
4.2.2.3 monomer or dimer x * 42000, recombinant His-tagged enzyme, SDS-PAGE Zobellia galactanivorans
4.2.2.11 ? 2 * 265000-29000, recombinant His-tagged enzyme, SDS-PAGE Zobellia galactanivorans
4.2.2.11 monomer or dimer x * 42000, recombinant His-tagged enzyme, SDS-PAGE Zobellia galactanivorans
4.2.2.11 More overall structure of AlyA1PL7 is a PL7 beta-sandwich jelly roll-fold, formed by two anti-parallel beta-sheets stacked against each other. The outer convex sheet is composed of five beta-strands, and the inner concave sheet is composed of seven beta-strands, forming a groove that harbors the catalytic active site Zobellia galactanivorans

Synonyms

EC Number Synonyms Comment Organism
4.2.2.3 AlyA5
-
Zobellia galactanivorans
4.2.2.11 AlyA1PL7
-
Zobellia galactanivorans
4.2.2.11 AlyA5
-
Zobellia galactanivorans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.2.3 30
-
assay at Zobellia galactanivorans
4.2.2.11 30
-
-
Zobellia galactanivorans
4.2.2.11 30
-
assay at Zobellia galactanivorans

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
4.2.2.3 10 45 temperature profile, overview Zobellia galactanivorans

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.2.11 12.66
-
sodium alginate with L-guluronate content of 66.7%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans
4.2.2.11 17.89
-
sodium alginate with L-guluronate content of 52.6%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans
4.2.2.11 19.51
-
sodium alginate with L-guluronate content of 33.3%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.2.3 7
-
-
Zobellia galactanivorans
4.2.2.11 7
-
-
Zobellia galactanivorans
4.2.2.11 7.5
-
assay at Zobellia galactanivorans

pH Range

EC Number pH Minimum pH Maximum Comment Organism
4.2.2.3 additional information
-
pH profile, narrow activity range, overview Zobellia galactanivorans
4.2.2.11 additional information
-
pH profile, narrow activity range, overview Zobellia galactanivorans
4.2.2.11 4 8.5 no activity outside this range Zobellia galactanivorans

General Information

EC Number General Information Comment Organism
4.2.2.3 evolution AlyA5 belongs to the PL7 family, subfamily 5, the genome of Zobellia galactanivorans contains seven putative alginate lyase genes, five of which are localized within two clusters comprising additional carbohydrate-related genes, phylogenetic analysis. Despite a common jelly roll-fold, these striking differences of the mode of action are explained by a distinct active site topology, an open cleft in AlyA1PL7, whereas AlyA5 displays a pocket topology due to the presence of additional loops partially obstructing the catalytic groove. In contrast to PL7 alginate lyases from terrestrial bacteria, both enzymes proceed according to a calcium-dependent mechanism Zobellia galactanivorans
4.2.2.3 additional information secondary structure comparison of AlyA5 and AlyA1PL7 Zobellia galactanivorans
4.2.2.11 evolution AlyA1PL7 is an endolytic guluronate lyase and belongs to the PL7 family, subfamily 1, the genome of Zobellia galactanivorans contains seven putative alginate lyase genes, five of which are localized within two clusters comprising additional carbohydrate-related genes, phylogenetic analysis. Despite a common jelly roll-fold, these striking differences of the mode of action are explained by a distinct active site topology, an open cleft in AlyA1PL7, whereas AlyA5 displays a pocket topology due to the presence of additional loops partially obstructing the catalytic groove. In contrast to PL7 alginate lyases from terrestrial bacteria, both enzymes proceed according to a calcium-dependent mechanism Zobellia galactanivorans
4.2.2.11 evolution AlyA5 belongs to the PL7 family, subfamily 5, the genome of Zobellia galactanivorans contains seven putative alginate lyase genes, five of which are localized within two clusters comprising additional carbohydrate-related genes, phylogenetic analysis. Despite a common jelly roll-fold, these striking differences of the mode of action are explained by a distinct active site topology, an open cleft in AlyA1PL7, whereas AlyA5 displays a pocket topology due to the presence of additional loops partially obstructing the catalytic groove. In contrast to PL7 alginate lyases from terrestrial bacteria, both enzymes proceed according to a calcium-dependent mechanism Zobellia galactanivorans
4.2.2.11 additional information secondary structure comparison of AlyA5 and AlyA1PL7 Zobellia galactanivorans

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.2.2.11 3.16
-
sodium alginate with L-guluronate content of 33.3%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans
4.2.2.11 5.79
-
sodium alginate with L-guluronate content of 52.6%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans
4.2.2.11 7.22
-
sodium alginate with L-guluronate content of 66.7%, pH 7.5, 30°C, recombinant enzyme Zobellia galactanivorans