Generation of up-regulated allosteric variants of potato ADP-glucose pyrophosphorylase by reversion genetics.
Mutagenesis of the large subunit (LS) of the potato ADP-glucose pyrophosphorylase generated an enzyme, P52L, that was insensitive to 3-phosphoglycerate (3-PGA). To identify additional residues involved in 3-PGA interaction, we subjected P52L LS DNA to a second round of mutagenesis and identified second-site revertants by their ability to restore glycogen accumulation as assessed by iodine (I2) staining. Enzymes from class I revertants with normal I2-staining had an 11- to 49-fold greater affinity for the activator 3-PGA compared with the P52L mutant and a decreased sensitivity to the inhibitor orthophosphate. Sequence analysis of these class I revertants identified a P66L mutation in R4, an E38K mutation in R20, and a G101N mutation in R10 and R32. These mutations appear to restore 3-PGA binding by counteracting the effect of the P52L mutation because introducing E38K or G101N into the wild-type LS led to enzyme variants with higher affinity for the activator 3-PGA and increased resistance to the inhibitor orthophosphate. The generation of these revertant enzymes provides additional structure-function information on the allosteric regulation of higher plant ADP-glucose pyrophosphorylases and validates a strategy for developing novel variants of the enzyme that may be useful in manipulating starch biosynthesis in higher plants.
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
T W
TW
Greene
I H
IH
Kavakli
M L
ML
Kahn
T W
TW
Okita
D000495:Allosteric Site / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D017343:Genes, Plant; D014644:Genetic Variation; D051599:Glucose-1-Phosphate Adenylyltransferase; D005988:Glyceric Acids / Q000378:metabolism / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics*; D013213:Starch / Q000096:biosynthesis; D015854:Up-Regulation
D017931:DNA Primers; D005988:Glyceric Acids; C005156:3-phosphoglycerate; D013213:Starch; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase