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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)

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. / Ph. D.

Identiferoai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/27135
Date22 May 2009
CreatorsYu, Hyun Young
ContributorsBiology, Esen, Asim, Walker, Richard A., Yang, Zhaomin, Kittur, Farooqahmed S., Bevan, David R.
PublisherVirginia Tech
Source SetsVirginia Tech Theses and Dissertation
Detected LanguageEnglish
TypeDissertation
Formatapplication/pdf
RightsIn Copyright, http://rightsstatements.org/vocab/InC/1.0/
RelationDissertation_Hyun_Young_Yu.pdf

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