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)

-Glucosidase zymograms of certain maize genotypes (nulls) do not show any activity bands after electrophoresis. We have shown that a chimeric lectin called -glucosidase aggregating factor (BGAF) is responsible for the absence of -glucosidase activity bands on zymograms. BGAF specifically binds to maize -glucosidase isozymes Glu1 and Glu2 and forms large, insoluble complexes. Furthermore, we have previously shown that the N-terminal (Glu 50 -Val 145 ) and the C-terminal (Phe 466 -Ala 512 ) regions contain residues that make up the BGAF binding site on maize Glu1. However, sequence comparison between sorghum glucosidases (dhurrinases, Dhr1 and Dhr2), to which BGAF does not bind, and maize βglucosidases, 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 -glucosidases. The results showed that a region spanning 11 amino acids (Ile 72 -Thr 82 ) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser 1 -Thr 29 , together with C-terminal region Phe 466 -Ala 512 , affects the size of Glu1-BGAF complexes. To determine the importance of each region for binding, we determined the dissociation constants (Kd) of chimeric -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 Ile 72 -Thr 82 on Glu1 for BGAF binding, we constructed chimeric Dhr2 (C-11, Dhr2 whose Val 72 -Glu 82 region was replaced with the Ile 72 -Thr 82 region of Glu1). C-11 binds to BGAF, indicating that the Ile 72 -Thr 82 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

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