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Literature summary for 3.2.1.68 extracted from

  • Facon, M.; Lin, Q.; Azzaz, A.M.; Hennen-Bierwagen, T.A.; Myers, A.M.; Putaux, J.L.; Roussel, X.; DHulst, C.; Wattebled, F.
    Distinct functional properties of isoamylase-type starch debranching enzymes in monocot and dicot leaves (2013), Plant Physiol., 163, 1363-1375.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene isa1, single expression of C-terminally His8-tagged subunit in Escherichia coli strain BL21(DE3), co-expression of untagged ISA1 subunit with C-terminally His8-tagged subunit ISA2 in Escherichia coli Arabidopsis thaliana
gene isa2, single expression of C-terminally His8-tagged subunit in Escherichia coli strain BL21(DE3) or co-expression with untagged ISA1 subunit in Escherichia coli Zea mays
gene isa2, single expression of C-terminally His8-tagged subunit in Escherichia coli strain BL21(DE3) or co-expression with untagged ISA1 subunit in Escherichia coli Arabidopsis thaliana
gene su-1, single expression of C-terminally His8-tagged subunit ISA1 in Escherichia coli strain BL21(DE3), co-expression of untagged ISA1 subunit with C-terminally His8-tagged subunit ISA2 in Escherichia coli. Zea mays ISA1 subunit is heterologously expressed in Arabidopsis thaliana lacking endogenous ISA1 or lacking both native ISA1 and ISA2 via transfection with Agrobacterium tumefaciens strain GV3101. The maize protein functions in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2 Zea mays

Protein Variants

Protein Variants Comment Organism
additional information construction of a single mutant line isa2-2 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity Arabidopsis thaliana
additional information construction of single mutant line isa1-1 by T-DNA insertion mutagenesis. The isa1-1 isa2-2 double mutant line is obtained by a cross between the two corresponding single mutants and screening of the progeny by PCR amplification of genomic DNA using a T-DNA end primer and gene-specific primers. The phenotype of the isa1-1 isa2-2 double mutant is complemented but the ZmISA1 activity, Zea mays ISA1 subunit is expressed via transfection with Agrobacterium tumefaciens strain GV3101 in Arabidopsis thaliana lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functions in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2 Arabidopsis thaliana

Localization

Localization Comment Organism GeneOntology No. Textmining
chloroplast
-
Zea mays 9507
-
chloroplast
-
Arabidopsis thaliana 9507
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
85000
-
1 * 85000, ISA1 monomer, SDS-PAGE Zea mays
85000
-
2 * 85000, ISA1 dimer, SDS-PAGE Zea mays
158000
-
ISA2 dimer, gel filtration and analytical ultracentrifugation Zea mays

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
amylopectin + H2O Arabidopsis thaliana
-
?
-
?
amylopectin + H2O Zea mays
-
?
-
?
amylopectin + H2O Arabidopsis thaliana Col-0
-
?
-
?
Arabidopsis starch + H2O Arabidopsis thaliana
-
?
-
?
Arabidopsis starch + H2O Arabidopsis thaliana Col-0
-
?
-
?
beta-limit dextrin + H2O Arabidopsis thaliana
-
?
-
?
beta-limit dextrin + H2O Arabidopsis thaliana Col-0
-
?
-
?
maize starch + H2O Zea mays
-
?
-
?
additional information Zea mays Zea mays subunit ISA 2 alone is catalytically inactive ?
-
?

Organism

Organism UniProt Comment Textmining
Arabidopsis thaliana O04196 gene isa1
-
Arabidopsis thaliana Q8L735 gene isa2
-
Arabidopsis thaliana Col-0 O04196 gene isa1
-
Arabidopsis thaliana Col-0 Q8L735 gene isa2
-
Zea mays
-
gene isa2
-
Zea mays B6U0X5 gene su-1
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His8-tagged single subunit ISA1 or ISA1 in complex with His8-tagged ISA2 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Arabidopsis thaliana
recombinant C-terminally His8-tagged single subunit ISA1 or ISA1 in complex with His8-tagged ISA2 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Zea mays
recombinant C-terminally His8-tagged single subunit ISA2 or His8-tagged ISA2 in complex with ISA1 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Zea mays
recombinant C-terminally His8-tagged single subunit ISA2 or His8-tagged ISA2 in complex with ISA1 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Arabidopsis thaliana

Source Tissue

Source Tissue Comment Organism Textmining
endosperm only ISA1, no ISA2 Zea mays
-
leaf
-
Arabidopsis thaliana
-
leaf ISA1 and ISA2 Zea mays
-
additional information not in endosperm Zea mays
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
amylopectin + H2O
-
Arabidopsis thaliana ?
-
?
amylopectin + H2O
-
Zea mays ?
-
?
amylopectin + H2O
-
Arabidopsis thaliana Col-0 ?
-
?
Arabidopsis starch + H2O
-
Arabidopsis thaliana ?
-
?
Arabidopsis starch + H2O
-
Arabidopsis thaliana Col-0 ?
-
?
beta-limit dextrin + H2O
-
Arabidopsis thaliana ?
-
?
beta-limit dextrin + H2O
-
Zea mays ?
-
?
beta-limit dextrin + H2O
-
Arabidopsis thaliana Col-0 ?
-
?
maize starch + H2O
-
Zea mays ?
-
?
additional information Zea mays subunit ISA 2 alone is catalytically inactive Zea mays ?
-
?

Subunits

Subunits Comment Organism
dimer 2 * 85000, ISA1 dimer, SDS-PAGE Zea mays
monomer 1 * 85000, ISA1 monomer, SDS-PAGE Zea mays

Synonyms

Synonyms Comment Organism
AtISA1
-
Arabidopsis thaliana
AtISA2
-
Arabidopsis thaliana
ISA1
-
Arabidopsis thaliana
ISA1
-
Zea mays
ISA2
-
Zea mays
ISA2
-
Arabidopsis thaliana
isoamylase-type starch debranching enzyme 1
-
Arabidopsis thaliana
isoamylase-type starch debranching enzyme 1
-
Zea mays
isoamylase-type starch debranching enzyme 2
-
Zea mays
isoamylase-type starch debranching enzyme 2
-
Arabidopsis thaliana
ZmISA1
-
Zea mays
ZmISA2
-
Zea mays

General Information

General Information Comment Organism
evolution the enzyme belongs to the glycosyl hydrolase family 13 GH13, and harbors a carbohydrate-binding module family 48 (CBM48) domain Zea mays
additional information Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview Arabidopsis thaliana
additional information Arabidopsis thaliana subunit ISA1 requires subunit ISA2 as a partner for enzymatic function. Images of purified starch granules of wild-type and mutant lines, overview. Recombinant AtISA1 is capable of enzymatic activity if mixed with recombinant AtISA2 but does not display such function on its own Arabidopsis thaliana
additional information Zea mays subunit ISA1 is active by itself and does not require subunit ISA2 for activity, subunit ISA 2 alone is catalytically inactive Zea mays
additional information Zea mays subunit ISA1 is active by itself, either in vitro or in transgenic Arabidopsis leaves, and does not require subunit ISA2 for activity Zea mays