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PMID:15456406

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Citation

Jam, M, Flament, D, Allouch, J, Potin, P, Thion, L, Kloareg, B, Czjzek, M, Helbert, W, Michel, G and Barbeyron, T (2005) The endo-beta-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours. Biochem. J. 385:703-13

Abstract

Two beta-agarase genes, agaA and agaB, were functionally cloned from the marine bacterium Zobellia galactanivorans. The agaA and agaB genes encode proteins of 539 and 353 amino acids respectively, with theoretical masses of 60 and 40 kDa. These two beta-agarases feature homologous catalytic domains belonging to family GH-16. However, AgaA displays a modular architecture, consisting of the catalytic domain (AgaAc) and two C-terminal domains of unknown function which are processed during secretion of the enzyme. In contrast, AgaB is composed of the catalytic module and a signal peptide similar to the N-terminal signature of prokaryotic lipoproteins, suggesting that this protein is anchored in the cytoplasmic membrane. Gel filtration and electrospray MS experiments demonstrate that AgaB is a dimer in solution, while AgaAc is a monomeric protein. AgaAc and AgaB were overexpressed in Escherichia coli and purified to homogeneity. Both enzymes cleave the beta-(1-->4) linkages of agarose in a random manner and with retention of the anomeric configuration. Although they behave similarly towards liquid agarose, AgaAc is more efficient than AgaB in the degradation of agarose gels. Given these organizational and catalytic differences, we propose that, reminiscent of the agarolytic system of Pseudoalteromonas atlantica, AgaA is specialized in the initial attack on solid-phase agarose, while AgaB is involved with the degradation of agarose fragments.

Links

PubMed PMC1134745 Online version:10.1042/BJ20041044

Keywords

Amino Acid Sequence; Base Sequence; Catalysis; Catalytic Domain; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Flavobacteriaceae/enzymology; Flavobacteriaceae/genetics; Glycoside Hydrolases/chemistry; Glycoside Hydrolases/genetics; Glycoside Hydrolases/isolation & purification; Glycoside Hydrolases/metabolism; Hydrolysis; Magnetic Resonance Spectroscopy; Marine Biology; Models, Molecular; Molecular Sequence Data; Molecular Weight; Phase Transition; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sepharose/chemistry; Sepharose/metabolism; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ZOBGA:AGAA

located_in

GO:0005576: extracellular region

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ZOBGA:AGAB

located_in

GO:0009279: cell outer membrane

ECO:0000304: author statement supported by traceable reference used in manual assertion

C

Seeded From UniProt

complete

ZOBGA:Q9RGX9

Contributes to

GO:0033916: beta-agarase activity

ECO:0000314:

F

Assists in cleaving the β-(1→4) linkages of agarose.

Figure 3 shows the hydrolysis of agarose by AgaA. After 24 hours,neoagarotetraose and neoagarohexaose were produced.

complete
CACAO 4696

ZOBGA:Q9RGX9

enables

GO:0033916: beta-agarase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ZOBGA:Q9RGX9

part_of

GO:0005576: extracellular region

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ZOBGA:Q9RGX9

involved_in

GO:0005975: carbohydrate metabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ZOBGA:AGAA

part_of

GO:0005576: extracellular region

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ZOBGA:AGAA

involved_in

GO:0005975: carbohydrate metabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ZOBGA:AGAA

enables

GO:0033916: beta-agarase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ZOBGA:AGAB

part_of

GO:0009279: cell outer membrane

ECO:0000304: author statement supported by traceable reference used in manual assertion

C

Seeded From UniProt

complete

ZOBGA:AGAB

involved_in

GO:0005975: carbohydrate metabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ZOBGA:AGAB

enables

GO:0033916: beta-agarase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ZOBGA:AGAB

enables

GO:0042803: protein homodimerization activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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