Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions.
Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42 degreesC. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.
Greene
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Horticultural Sciences Department, University of Florida, P.O. Box 110690, 2211 Fifield Hall, Gainesville, FL 32611-0690, USA.
Hannah
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D051599:Glucose-1-Phosphate Adenylyltransferase; D006003:Glycogen / Q000096:biosynthesis; D001467:Hordeum / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012275:Oryza / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology; D014908:Triticum / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006003:Glycogen; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase