Lysine-81 and threonine-82 on maize beta-glucosidase isozyme Glu1 are the key amino acids involved in beta-glucosidase aggregating factor binding.
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.
Yu
Hyun Young
HY
Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406, USA.
Kittur
Farooqahmed S
FS
Bevan
David R
DR
Esen
Asim
A
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
C426244:BGAF protein, Zea mays; D002352:Carrier Proteins; D007527:Isoenzymes; D050505:Mutant Proteins; D010940:Plant Proteins; D013912:Threonine; D001617:beta-Glucosidase; D008239:Lysine