Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions
| Autor: | Thomas W. Greene, L C Hannah |
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| Rok vydání: | 1998 |
| Předmět: |
Macromolecular Substances
Protein subunit Molecular Sequence Data Mutant Glucose-1-Phosphate Adenylyltransferase Biology Zea mays Endosperm chemistry.chemical_compound Consensus Sequence Escherichia coli Amino Acid Sequence Cloning Molecular Triticum Solanum tuberosum chemistry.chemical_classification Multidisciplinary Sequence Homology Amino Acid Glycogen Wild type Hordeum Oryza Biological Sciences Nucleotidyltransferases Heterotetramer Recombinant Proteins Amino acid Biochemistry chemistry Seeds Mutagenesis Site-Directed Sequence Alignment |
| Zdroj: | Proceedings of the National Academy of Sciences. 95:13342-13347 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α- 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°C. 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, S h2hs33, 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. |
| Databáze: | OpenAIRE |
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