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 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.
Petreikov
Marina
M
Department of Vegetable Research, Volcani Center-ARO, Bet Dagan 50250, Israel.
Eisenstein
Miriam
M
Yeselson
Yelena
Y
Preiss
Jack
J
Schaffer
Arthur A
AA
D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D010940:Plant Proteins; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins; D051599:Glucose-1-Phosphate Adenylyltransferase