Domain swapping between a cyanobacterial and a plant subunit ADP-glucose pyrophosphorylase.
ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo- and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N- and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3-phosphoglycerate and inorganic orthophosphate.
Iglesias
Alberto A
AA
Laboratorio de Enzimología Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral. Paraje El Pozo, CC 242, S3000ZAA, Santa Fe, Argentina.
Ballicora
Miguel A
MA
Sesma
Juliana I
JI
Preiss
Jack
J
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005988:Glyceric Acids / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010710:Phosphates / Q000494:pharmacology; D035281:Plant Tubers / Q000201:enzymology; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology*
D005988:Glyceric Acids; D010710:Phosphates; D011993:Recombinant Fusion Proteins; C005156:3-phosphoglycerate; D051599:Glucose-1-Phosphate Adenylyltransferase