Heat stability of maize endosperm ADP-glucose pyrophosphorylase is enhanced by insertion of a cysteine in the N terminus of the small subunit.
ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. However, plant AGPases differ in several parameters, including spatial and temporal expression, allosteric regulation, and heat stability. AGPases of cereal endosperms are heat labile, while those in other tissues, such as the potato (Solanum tuberosum) tuber, are heat stable. Sequence comparisons of heat-stable and heat-labile AGPases identified an N-terminal motif unique to heat-stable enzymes. Insertion of this motif into recombinant maize (Zea mays) endosperm AGPase increased the half-life at 58 degrees C more than 70-fold. Km values for physiological substrates were unaffected, although Kcat was doubled. A cysteine within the inserted motif gives rise to small subunit homodimers not found in the wild-type maize enzyme. Placement of this N-terminal motif into a mosaic small subunit containing the N terminus from maize endosperm and the C terminus from potato tuber AGPase increases heat stability more than 300-fold.
Linebarger
Carla R Lyerly
CR
Program in Plant Molecular and Cellular Biology and Horticultural Sciences, University of Florida, Gainesville, Florida 32610-0245, USA.
Boehlein
Susan K
SK
Sewell
Aileen K
AK
Shaw
Janine
J
Hannah
L Curtis
LC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004795:Enzyme Stability; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D021122:Protein Subunits; D011994:Recombinant Proteins; D051599:Glucose-1-Phosphate Adenylyltransferase; D003545:Cysteine