Literature summary for 4.2.2.3 extracted from

Tondervik, A.; Klinkenberg, G.; Aarstad, O.; Drablos, F.; Ertesvag, H.; Ellingsen, T.; Skjak-Brak, G.; Valla, S.; Sletta, H.
Isolation of mutant alginate lyases with cleavage specificity for Di-guluronic acid linkages (2010), J. Biol. Chem., 285, 35284-35292.

Application

Application	Comment	Organism
analysis	the enzyme, wild-type and mutant variants, can be used for alginate fine structure elucidation	Klebsiella pneumoniae

Cloned(Commentary)

Cloned (Comment)	Organism
gene alyA, expression in Escherichia coli strain DH5alpha	Klebsiella pneumoniae

Protein Variants

Protein Variants	Comment	Organism
A78S	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 820 U/mg, against poly(alpha-L-guluronic acid) 938 U/mg. Ratio of activities 1.1	Klebsiella pneumoniae
A78S	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
A78S/T89I	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 228 U/mg, against poly(alpha-L-guluronic acid) 34.3 U/mg. Ratio of activities 0.2	Klebsiella pneumoniae
A78S/T89I	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
A78S/T89I/A217E	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 187 U/mg, against poly(alpha-L-guluronic acid) 29.8 U/mg. Ratio of activities 0.2	Klebsiella pneumoniae
A78S/T89I/A217E	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
G26E	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 692 U/mg, against poly(alpha-L-guluronic acid) 787 U/mg. Ratio of activities 1.1	Klebsiella pneumoniae
G26E	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
G26E/P39H	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 246 U/mg, against poly(alpha-L-guluronic acid) 19.5 U/mg. Ratio of activities 0.1. In the absence of Ca2+, no detectable activity against G-M linkages	Klebsiella pneumoniae
G26E/P39H	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
G304V	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 828 U/mg, against poly(alpha-L-guluronic acid) 878 U/mg. Ratio of activities 1.1	Klebsiella pneumoniae
G304V	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
I51M	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 731 U/mg, against poly(alpha-L-guluronic acid) 786 U/mg. Ratio of activities 1.1	Klebsiella pneumoniae
I51M	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
I51M/T89I	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 227 U/mg, against poly(alpha-L-guluronic acid) 18.8 U/mg. Ratio of activities 0.1	Klebsiella pneumoniae
I51M/T89I	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
I51M/T89I/G304V	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 153 U/mg, against poly(alpha-L-guluronic acid) 13.3 U/mg. Ratio of activities 0.1	Klebsiella pneumoniae
I51M/T89I/G304V	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
additional information	engineering of different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure	Klebsiella pneumoniae
additional information	modification of the specificity of the Klebsiella pneumoniae AlyA to obtain a lyase with preference for cleaving only G-G linkages, random mutagenesis and library screening, overview	Klebsiella pneumoniae
P39H	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 356 U/mg, against poly(alpha-L-guluronic acid) 37 U/mg. Ratio of activities 0.1	Klebsiella pneumoniae
P39H	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
P39T	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 216 U/mg, against poly(alpha-L-guluronic acid) 71 U/mg. Ratio of activities 0.3	Klebsiella pneumoniae
P39T	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
S35R	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 443 U/mg, against poly(alpha-L-guluronic acid) 448 U/mg. Ratio of activities 1.0	Klebsiella pneumoniae
S35R	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
S35R/P39T	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 32.6 U/mg, against poly(alpha-L-guluronic acid) 6.9 U/mg. Ratio of activities 0.2	Klebsiella pneumoniae
S35R/P39T	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
S35R/P39T/A224V	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 43 U/mg, against poly(alpha-L-guluronic acid) 3.4 U/mg. Ratio of activities 0.1	Klebsiella pneumoniae
S35R/P39T/A224V	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
S37I	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 53.8 U/mg, against poly(alpha-L-guluronic acid) 4.3 U/mg. Ratio of activities 0.1	Klebsiella pneumoniae
S37I	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
S86L	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 31 U/mg, against poly(alpha-L-guluronic acid) 9.1 U/mg. Ratio of activities 0.3	Klebsiella pneumoniae
S86L	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
T85A	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) 24.8 is U/mg, against poly(alpha-L-guluronic acid) 4.4 U/mg. Ratio of activities 0.32	Klebsiella pneumoniae
T85A	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
T89I	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 218 U/mg, against poly(alpha-L-guluronic acid) 31 U/mg. Ratio of activities 0.1	Klebsiella pneumoniae
T89I	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
V6I	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is858 U/mg, against poly(alpha-L-guluronic acid) 851 U/mg. Ratio of activities 1.0	Klebsiella pneumoniae
V6I	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae
V6I/T85A	activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) is 23.6 U/mg, against poly(alpha-L-guluronic acid) 2.2 U/mg. Ratio of activities 0.1	Klebsiella pneumoniae
V6I/T85A	random mutagenesis, mutant activities with and ratio of polyG (alpha-L-guluronic acid) to polyM (beta-D-mannuronic acid) compared to the wild-type enzyme	Klebsiella pneumoniae

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
extracellular	lyase AlyA is secreted by strain SM0524	Klebsiella pneumoniae	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	activates	Klebsiella pneumoniae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Klebsiella pneumoniae	alginate lyases degrade the polysaccharide by cleaving the glycosidic linkages through a beta-elimination reaction. Lyase AlyA is bifunctional and shows activities toward both polyG (alpha-L-guluronic acid), activity of EC 4.2.2.11, and polyM (beta-D-mannuronic acid). AlyA is endolytic, acting on G-blocks and MG-blocks where G-M linkages are cleaved in the latter substrate. Substrate specificities of diverse enzyme mutants, overview	?	-	?
additional information	Klebsiella pneumoniae SM0524	alginate lyases degrade the polysaccharide by cleaving the glycosidic linkages through a beta-elimination reaction. Lyase AlyA is bifunctional and shows activities toward both polyG (alpha-L-guluronic acid), activity of EC 4.2.2.11, and polyM (beta-D-mannuronic acid). AlyA is endolytic, acting on G-blocks and MG-blocks where G-M linkages are cleaved in the latter substrate. Substrate specificities of diverse enzyme mutants, overview	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Klebsiella pneumoniae	-	gene alyA	-
Klebsiella pneumoniae	-	polysaccharide lyase family 7 lyase	-
Klebsiella pneumoniae SM0524	-	gene alyA	-
Klebsiella pneumoniae SM0524	-	polysaccharide lyase family 7 lyase	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
937	-	substrate poly(beta-D-mannuronic acid/alpha-L-guluronic acid), 22°C, pH 7.5	Klebsiella pneumoniae

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	alginate lyases degrade the polysaccharide by cleaving the glycosidic linkages through a beta-elimination reaction. Lyase AlyA is bifunctional and shows activities toward both polyG (alpha-L-guluronic acid), activity of EC 4.2.2.11, and polyM (beta-D-mannuronic acid). AlyA is endolytic, acting on G-blocks and MG-blocks where G-M linkages are cleaved in the latter substrate. Substrate specificities of diverse enzyme mutants, overview	Klebsiella pneumoniae	?	-	?
additional information	alginate lyases degrade the polysaccharide by cleaving the glycosidic linkages through a beta-elimination reaction. Lyase AlyA is bifunctional and shows activities toward both polyG (alpha-L-guluronic acid), activity of EC 4.2.2.11, and polyM (beta-D-mannuronic acid). AlyA is endolytic, acting on G-blocks and MG-blocks where G-M linkages are cleaved in the latter substrate. Substrate specificities of diverse enzyme mutants, overview	Klebsiella pneumoniae SM0524	?	-	?
poly(beta-(1->4)-D-mannuronic acid/alpha-L-guluronic acid)	alternating structure of alpha-L-guluronic acid and beta-D-mannuronic acid. In wild-type, ratio of activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) to poly(alpha-L-guluronic acid) is 1.2	Klebsiella pneumoniae	?	-	?
poly(beta-(1->4)-D-mannuronic acid/alpha-L-guluronic acid)	alternating structure of alpha-L-guluronic acid and beta-D-mannuronic acid. In wild-type, ratio of activity against poly(beta-D-mannuronic acid/alpha-L-guluronic acid) to poly(alpha-L-guluronic acid) is 1.2	Klebsiella pneumoniae SM0524	?	-	?

Synonyms

Synonyms	Comment	Organism
alginate lyase	-	Klebsiella pneumoniae
AlyA	-	Klebsiella pneumoniae
lyase AlyA	-	Klebsiella pneumoniae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Klebsiella pneumoniae

General Information

General Information	Comment	Organism
evolution	AlyA belongs to the polysaccharide lyase family 7	Klebsiella pneumoniae