Isolation and characterization of a higher plant ADP-glucose pyrophosphorylase small subunit homotetramer.
ADP-glucose pyrophosphorylase (AGPase) is the allosterically regulated gateway for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays a key role in the modulation of photosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield potential. AGPase is a tetrameric enzyme; in higher plants it consists of two regulatory large subunits (LS) and two catalytic small subunits (SS), while in cyanobacteria and prokaryotes the enzyme is homotetrameric. The potato SS gene in pML10 was mutated by hydroxylamine and mutants were screened for elevated homotetrameric activity by iodine vapor staining. This search strategy led to the isolation of SS mutants (SUP-1, TG-15) that had pyrophosphorylase activity in the absence of the LS. TG-15 has a leucine to phenylalanine change at position 48 (L(48)F) that corresponds to a phenylalanine residue at the analogous position in the Escherichia coli homotetrameric AGPase as well as a valine to isoleucine change at position 59 (V(59)I). TG-15 was partially purified and kinetic analysis revealed substrate and effector affinities equal to wild type heterotetrameric enzyme with the exception of ATP binding.
Salamone
P R
PR
Institute for Biological Chemistry, Washington State University, Pullman, 99164-6340, USA.
Greene
T W
TW
Kavakli
I H
IH
Okita
T W
TW
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D021122:Protein Subunits; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase