Lysine-81 and Threonine-82 on Maize β-Glucosidase Isozyme Glu1 Are the Key Amino Acids Involved in β-Glucosidase Aggregating Factor Binding
| Autor: | Asim Esen, Hyun Young Yu, Farooqahmed S. Kittur, David R. Bevan |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Threonine Molecular Sequence Data Electrophoretic Mobility Shift Assay Peptide Plasma protein binding Biology Zea mays Biochemistry Isozyme Chromatography Affinity Protein Structure Secondary Structure-Activity Relationship Protein structure Affinity chromatography Sequence Analysis Protein Amino Acid Sequence Cloning Molecular Peptide sequence Plant Proteins chemistry.chemical_classification Lysine beta-Glucosidase Molecular biology Amino acid Isoenzymes chemistry Mutant Proteins Carrier Proteins Sequence Alignment Protein Binding |
| Zdroj: | Biochemistry. 48:2924-2932 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | In certain maize genotypes (nulls), beta-glucosidase specifically interacts with a chimeric lectin called beta-glucosidase aggregating factor (BGAF), resulting in high molecular weight complexes. Previously, we showed that three regions (S1-T29, E50-N127, and F466-A512) on the maize beta-glucosidase isozyme Glu1 are involved in interaction and aggregation with BGAF. Recently, we found that the peptide span I72-T82 within E50-N127 is essential and sufficient for BGAF binding, whereas the S1-T29 and F466-A512 regions are required for formation of large complexes. To define the contribution of individual amino acids in the above three regions to BGAF binding, we constructed mutant beta-glucosidases based on sequence differences between maize beta-glucosidase and sorghum beta-glucosidase (dhurrinase 2, Dhr2), which does not bind BGAF. Binding was evaluated by gel-shift assay and affinity by frontal affinity chromatography (FAC). In the gel-shift assay, Glu1 mutants K81E and T82Y failed to bind BGAF, and their FAC profiles were essentially similar to that of Dhr2, indicating that these two amino acids within the I72-T82 region are important for BGAF binding. Substitution of N481 with E (as in Dhr2) lowered affinity for BGAF, whereas none of the mutations in the S1-T29 region showed any effect on BGAF binding. To further confirm the importance of K81 and T82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four amino acids (I72, N75, K81, and T82) that differ between Glu1 and Dhr2 in the peptide span I72-T82 are required to impart BGAF-binding ability to Dhr2. |
| Databáze: | OpenAIRE |
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