Characterization of the AGPase large subunit isoforms from tomato indicates that the recombinant L3 subunit is active as a monomer

The enzyme AGPase [ADP-Glc (glucose) pyrophosphorylase] catalyses a rate-limiting step in starch synthesis in tomato (Solanum lycopersicon) fruit, which undergoes a transient period of starch accumulation. It has been a generally accepted paradigm in starch metabolism that the enzyme naturally funct...

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Veröffentlicht in:Biochemical journal 2010-06, Vol.428 (2), p.201-212
Hauptverfasser: Petreikov, Marina, Eisenstein, Miriam, Yeselson, Yelena, Preiss, Jack, Schaffer, Arthur A
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container_end_page 212
container_issue 2
container_start_page 201
container_title Biochemical journal
container_volume 428
creator Petreikov, Marina
Eisenstein, Miriam
Yeselson, Yelena
Preiss, Jack
Schaffer, Arthur A
description The enzyme AGPase [ADP-Glc (glucose) pyrophosphorylase] catalyses a rate-limiting step in starch synthesis in tomato (Solanum lycopersicon) fruit, which undergoes a transient period of starch accumulation. It has been a generally accepted paradigm in starch metabolism that the enzyme naturally functions primarily as a heterotetramer comprised of two large subunits (L) and two small subunits (S). The tomato genome harbours a single gene encoding S and three genes for L proteins, which are expressed in both a tissue- and time-specific manner. In the present study the allosteric contributions of the different L subunits were compared by expressing each one in Escherichia coli, in conjunction with S and individually, and characterizing the resulting enzyme activity. Our results indicate different kinetic characteristics of the tomato L1/S and L3/S heterotetramers. Surprisingly, the recombinant L3 protein was also active when expressed alone and size-exclusion and immunoblotting showed that it functioned as a monomer. Subunit interaction modelling pointed to two amino acids potentially affecting subunit interactions. However, directed mutations did not have an impact on subunit tetramerization. These results indicate a hitherto unknown active role for the L subunit in the synthesis of ADP-Glc.
doi_str_mv 10.1042/BJ20091777
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subjects Blotting, Western
Escherichia coli - genetics
Escherichia coli - metabolism
Glucose-1-Phosphate Adenylyltransferase - chemistry
Glucose-1-Phosphate Adenylyltransferase - genetics
Glucose-1-Phosphate Adenylyltransferase - metabolism
Kinetics
Lycopersicon esculentum - enzymology
Lycopersicon esculentum - genetics
Mutagenesis, Site-Directed
Plant Proteins - chemistry
Plant Proteins - genetics
Plant Proteins - metabolism
Protein Isoforms - chemistry
Protein Isoforms - genetics
Protein Isoforms - metabolism
Protein Subunits - chemistry
Protein Subunits - genetics
Protein Subunits - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Tandem Mass Spectrometry
title Characterization of the AGPase large subunit isoforms from tomato indicates that the recombinant L3 subunit is active as a monomer
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