Lysine-81 and Threonine-82 on Maize β-Glucosidase Isozyme Glu1 Are the Key Amino Acids Involved in β-Glucosidase Aggregating Factor Binding

Abstract

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.

Caterpillar attack triggers accumulation of the toxic maize protein RIP2

“…Some of the maize proteins identified in FAW frass in this study have been implicated in plant defense against herbivores and pathogens. Beta‐D‐glucosidase has been shown to activate DIMBOA, a secondary metabolite toxic to the European corn borer ( Ostrinia nubilalis ; Yu et al ., ). Lipoxygenase (LOX) and allene oxide synthase (AOS) catalyze steps in the JA biosynthetic pathway and the production of JA in response to herbivory triggers many plant defenses against insects (Howe & Jander, ).…”

Genome-wide analysis of the beta-glucosidase gene family in maize (Zea mays L. var B73)

“…3; see also Fig. S6) are involved in binding to the protein beta-glucosidase aggregating factor (BGAF) (Yu et al 2009b). The key amino acid pair KT is only conserved in BGLU1 (GRMZM2G016890) and BGLU2 (GRMZM2G008247) but not in all other BGLU isoforms that rather contain YHRYKEDV as consensus (Fig.…”