Generation, characterization, and heterologous expression of wild-type and up-regulated forms of Arabidopsis thaliana leaf ADP-glucose pyrophosphorylase.
ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch biosynthesis of higher plants, consists of a pair of regulatory large (LS) and catalytically small (SS) subunits. In plants, these subunits are coded by multiple genes resulting in the formation of tissue-specific enzyme forms, which are differentially regulated during plant growth and development. Some AGPase isoforms differ in catalytic and regulatory properties as well as intracellular location. In an effort to gain a better understanding of the role of the leaf AGPase in carbon partitioning and its effect on plant productivity, the Arabidopsis leaf AGPase containing the mature forms of the SS and LS was expressed in a heterologous expression system and characterized enzymatically. The Arabidopsis recombinant AGPase had kinetic values for 3-phosphoglyceric acid, glucose-1-phosphate and Mg(2+) similar to those of the native enzyme. As the N-terminus of the LS has been suggested to be involved in enzyme function, the length of the N-terminal region was extended or shortened. Of the five modified LSs analyzed, only the T5 form lacking six residues of the mature N-terminus was able to form detectable levels of enzyme activity, indicating that the N-terminal region is critical for enzyme function. Two up-regulatory LS mutations that allosterically activate the potato enzyme, a stem isoform, were introduced into the corresponding Arabidopsis LS sequences and co-expressed with wild-type SS. Both modified enzymes showed up-regulatory properties, indicating that these specific residue changes were also operational in the leaf isoform.
Institute of Biological Chemistry and School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
I Halil
IH
Kavakli
Chikako
C
Kato
Sang-Bong
SB
Choi
Kyung-Hwan
KH
Kim
Peter R
PR
Salamone
Hiroyuki
H
Ito
Thomas W
TW
Okita
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D051599:Glucose-1-Phosphate Adenylyltransferase; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics*; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics; D013213:Starch / Q000096:biosynthesis
D029681:Arabidopsis Proteins; D017931:DNA Primers; D010940:Plant Proteins; D013213:Starch; D009713:Nucleotidyltransferases; C496715:ADP-glucose pyrophosphorylase, Arabidopsis; D051599:Glucose-1-Phosphate Adenylyltransferase