Formation of fibrillar multimers of oat beta-glucosidase isoenzymes is mediated by the As-Glu1 monomer.
Oat beta-glucosidase (EC 3.2.1.21) exists in two isomeric forms of homomultimer (type I) and heteromultimer (type II), which are comprised of two 60 kDa monomers of As-Glu1 and As-Glu2. The cDNA of As-Glu2 was cloned in this study, whereas As-Glu1 was previously cloned as As-P60. The As-Glu2 cDNA encodes a plastid-directing transit peptide of 57 amino acid residues and a mature protein of 521 amino acid residues. The amino acid sequence of As-Glu2 is highly homologous to that of As-Glu1, except for their C-terminal portions. When the two cDNAs of the mature proteins were expressed as T7.Tag-fused proteins in Escherichia coli, they produced soluble and enzymatically active T7.Tag-As-Glu1 and T7.Tag-As-Glu2 proteins. The T7.Tag-As-Glu1 was assembled into a donut-shaped hexamer ring which was in turn stacked in integer numbers to form long fibrillar homomultimers of different lengths with a molecular mass of up to several million daltons. On the other hand, the T7.Tag-As-Glu2 primarily formed a dimer rather than a multimer. When both cDNAs of As-Glu1 and As-Glu2 were co-expressed as T7.Tag-fused mature proteins, they were also assembled into a hexamer ring comprised of the two monomers in a 1:1 stoichiometry. The heteromeric hexamer was stacked in smaller numbers to form the heteromultimer of T7. Tag-As-Glu1 and -As-Glu2. The results indicate that the As-Glu1 monomer plays a crucial role in the formation of both the As-Glu1 homomultimer and the As-Glu1 and As-Glu2 heteromultimer. We describe here a unique structure for the oat beta-glucosidase fibrillar multimer that is formed by stacking the hexamer rings composed of As-Glu1 and/or As-Glu2.
Kim
Y W
YW
Department of Genetic Engineering, College of Natural Sciences, Kyungpook National University, Taegu, 702-701, South Korea.
Kang
K S
KS
Kim
S Y
SY
Kim
I S
IS
D000595:Amino Acid Sequence; D018554:Avena / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017343:Genes, Plant / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D016415:Sequence Alignment; D012995:Solubility; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D007527:Isoenzymes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D001617:beta-Glucosidase