Determination of the Binding Site and the Key Amino Acids on Maize β-Glucosidase Isozyme Glu1 Involved in Binding to β-Glucosidase Aggregating Factor (BGAF)
dc.contributor.author | Yu, Hyun Young | en |
dc.contributor.committeechair | Esen, Asim | en |
dc.contributor.committeecochair | Bevan, David R. | en |
dc.contributor.committeemember | Walker, Richard A. | en |
dc.contributor.committeemember | Yang, Zhaomin | en |
dc.contributor.committeemember | Kittur, Farooqahmed S. | en |
dc.contributor.department | Biology | en |
dc.date.accessioned | 2014-03-14T20:10:28Z | en |
dc.date.adate | 2009-05-22 | en |
dc.date.available | 2014-03-14T20:10:28Z | en |
dc.date.issued | 2009-04-10 | en |
dc.date.rdate | 2009-05-22 | en |
dc.date.sdate | 2009-04-22 | en |
dc.description.abstract | β-Glucosidase zymograms of certain maize genotypes (nulls) do not show any activity bands after electrophoresis. We have shown that a chimeric lectin called β-glucosidase aggregating factor (BGAF) is responsible for the absence of β-glucosidase activity bands on zymograms. BGAF specifically binds to maize β-glucosidase isozymes Glu1 and Glu2 and forms large, insoluble complexes. Furthermore, we have previously shown that the N-terminal (Glu⁵⁰-Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions contain residues that make up the BGAF binding site on maize Glu1. However, sequence comparison between sorghum β-glucosidases (dhurrinases, Dhr1 and Dhr2), to which BGAF does not bind, and maize β-glucosidases, and an examination of the 3-D structure of Glu1 suggested that the BGAF binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²-Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹-Thr²⁹, together with C-terminal region Phe⁴⁶⁶-Ala⁵¹², affects the size of Glu1-BGAF complexes. To determine the importance of each region for binding, we determined the dissociation constants (K<sub>d</sub>) of chimeric β-glucosidase-BGAF interactions. The results demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²-Thr⁸² on Glu1 for BGAF binding, we constructed chimeric Dhr2 (C-11, Dhr2 whose Val⁷²-Glu⁸² region was replaced with the Ile⁷²-Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²-Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding. We also constructed mutant β-glucosidases to identify and define the contribution of individual amino acids in the above three regions to BGAF binding. In the N-terminal region (Ile⁷²-Thr⁸²), critical region for BGAF binding, Glu1 mutants K81E and T82Y failed to bind BGAF in the gel-shift assay and their frontal affinity chromatography (FAC) profiles were essentially similar to that of sorghum β-glucosidase (dhurrinase 2, Dhr2), a non-binder, indicating that these two amino acids within Ile⁷²-Thr⁸² region are essential for BGAF binding. In the extreme N-terminal (Ser¹-Thr²⁹) and C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions, N481E [substitution of asparagine-481 with glutamic acid (as in Dhr)] showed lower affinity for BGAF, whereas none of the single amino acid substitutions in the Ser¹-Thr²⁹ region showed any effect on BGAF binding indicating that these regions play a minor role. To further confirm the importance of lysine-81 and threonine-82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four unique amino acids (isoleucine-72, asparagine-75, lysine-81, and threonine-82) of Glu1 in the peptide span Ile⁷²-Thr⁸² are required to impart BGAF binding ability to Dhr2. The sequence comparison among plant β-glucosidases supports the hypothesis that BGAF binding is specific to maize β-glucosidases because only maize β-glucosidases have threonine at position 82. | en |
dc.description.degree | Ph. D. | en |
dc.identifier.other | etd-04222009-131105 | en |
dc.identifier.sourceurl | http://scholar.lib.vt.edu/theses/available/etd-04222009-131105/ | en |
dc.identifier.uri | http://hdl.handle.net/10919/27135 | en |
dc.publisher | Virginia Tech | en |
dc.relation.haspart | Dissertation_Hyun_Young_Yu.pdf | en |
dc.rights | In Copyright | en |
dc.rights.uri | http://rightsstatements.org/vocab/InC/1.0/ | en |
dc.subject | β-glucosidase aggregating factor (BGAF) | en |
dc.subject | β-glucosidase | en |
dc.subject | protein-protein interaction | en |
dc.title | Determination of the Binding Site and the Key Amino Acids on Maize β-Glucosidase Isozyme Glu1 Involved in Binding to β-Glucosidase Aggregating Factor (BGAF) | en |
dc.type | Dissertation | en |
thesis.degree.discipline | Biology | en |
thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
thesis.degree.level | doctoral | en |
thesis.degree.name | Ph. D. | en |
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