Literature summary for 4.2.2.11 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.

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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
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

Crystallization (Comment)

Organism

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

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

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

KM Value [mM]

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

Metals/Ions

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

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
sodium alginate	Zobellia galactanivorans	AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches	?	-	?
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	?	-	?
sodium alginate	Zobellia galactanivorans DSM 12802	AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Zobellia galactanivorans	G0KZG8	-	-
Zobellia galactanivorans	G0L2Y1	-	-
Zobellia galactanivorans DSM 12802	G0KZG8	-	-

Purification (Commentary)

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

Reaction

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	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	a
[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	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	alginate pentasaccharides that can be cleaved by AlyA1PL7 are GGGGG, GGMGG, GGGMG, and GGMMG	Zobellia galactanivorans	?	-	?
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	?	-	?
additional information	alginate pentasaccharides that can be cleaved by AlyA1PL7 are GGGGG, GGMGG, GGGMG, and GGMMG	Zobellia galactanivorans DSM 12802	?	-	?
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	?	-	?
poly(alpha-L-guluronate)	-	Zobellia galactanivorans	?	-	?
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	?	-	?
poly(alpha-L-guluronate)	-	Zobellia galactanivorans DSM 12802	?	-	?
sodium alginate	AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches	Zobellia galactanivorans	?	-	?
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	?	-	?
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	?
sodium alginate	LC mass and NMR spectrometris product analysis	Zobellia galactanivorans	?	-	?
sodium alginate	AlyA1PL7 is an endolytic guluronate lyase that preferentially cleaves guluronate stretches	Zobellia galactanivorans DSM 12802	?	-	?
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

Subunits	Comment	Organism
?	2 * 265000-29000, recombinant His-tagged enzyme, SDS-PAGE	Zobellia galactanivorans
monomer or dimer	x * 42000, recombinant His-tagged enzyme, SDS-PAGE	Zobellia galactanivorans
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

Synonyms	Comment	Organism
AlyA1PL7	-	Zobellia galactanivorans
AlyA5	-	Zobellia galactanivorans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	-	Zobellia galactanivorans
30	-	assay at	Zobellia galactanivorans

Turnover Number [1/s]

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

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	-	Zobellia galactanivorans
7.5	-	assay at	Zobellia galactanivorans

pH Range

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

General Information

General Information	Comment	Organism
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
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
additional information	secondary structure comparison of AlyA5 and AlyA1PL7	Zobellia galactanivorans

kcat/KM [mM/s]

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