Yu Hyun Young, Kittur Farooqahmed S, Bevan David R, Esen Asim
Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406, USA.
Biochemistry. 2009 Apr 7;48(13):2924-32. doi: 10.1021/bi900012h.
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.
在某些玉米基因型（无效基因型）中，β-葡萄糖苷酶与一种名为β-葡萄糖苷酶聚集因子（BGAF）的嵌合凝集素特异性相互作用，形成高分子量复合物。此前，我们发现玉米β-葡萄糖苷酶同工酶Glu1上的三个区域（S1-T29、E50-N127和F466-A512）参与了与BGAF的相互作用和聚集。最近，我们发现E50-N127区域内的肽段I72-T82对于BGAF结合至关重要且足够，而S1-T29和F466-A512区域对于大复合物的形成是必需的。为了确定上述三个区域中单个氨基酸对BGAF结合的贡献，我们基于玉米β-葡萄糖苷酶和高粱β-葡萄糖苷酶（苦杏仁苷酶2，Dhr2）之间的序列差异构建了突变型β-葡萄糖苷酶，后者不与BGAF结合。通过凝胶迁移试验评估结合情况，并通过前沿亲和色谱法（FAC）评估亲和力。在凝胶迁移试验中，Glu1突变体K81E和T82Y未能与BGAF结合，它们的FAC图谱与Dhr2的基本相似，表明I72-T82区域内的这两个氨基酸对于BGAF结合很重要。用E取代N481（如在Dhr2中）降低了对BGAF的亲和力，而S1-T29区域的所有突变对BGAF结合均无任何影响。为了进一步证实K81和T82对BGAF结合的重要性，我们构建了一些Dhr2突变体，结果表明，在肽段I72-T82中，Glu1和Dhr2之间不同的所有四个氨基酸（I72、N75、K81和T82）都是赋予Dhr2与BGAF结合能力所必需的。
