title: Biochemical and crystallographic characterization of the starch branching enzyme I (BEI) from Oryza sativa L.
abstract: Starch branching enzyme (SBE) catalyzes the cleavage of alpha-1.4-linkages and the subsequent transfer of alpha-1.4 glucan to form an alpha-1.6 branch point in amylopectin. We overproduced rice branching enzyme I (BEI) in Escherichia coli cells, and the resulting enzyme (rBEI) was characterized with respect to biochemical and crystallographic properties. Specific activities were calculated to be 20.8 units/mg and 2.5 units/mg respectively when amylose and amylopectin were used as substrates. Site-directed mutations of Tyr235, Asp270, His275, Arg342, Asp344, Glu399, and His467 conserved in the alpha-amylase family enzymes drastically reduced catalytic activity of rBEI. This result suggests that the structures of BEI and the other alpha-amylase family enzymes are similar and that they share common catalytic mechanisms. Crystals of rBEI were grown under appropriate conditions and the crystals diffracted to a resolution of 3.0 A on a synchrotron X-ray source.
authors.affiliation: Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors.forename: Nhuan Thi
authors.initials: NT
authors.lastname: Vu
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shimada
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Kakuta
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakashima
authors.forename: Hiroko
authors.initials: H
authors.lastname: Ida
authors.forename: Toshiro
authors.initials: T
authors.lastname: Omori
authors.forename: Aiko
authors.initials: A
authors.lastname: Nishi
authors.forename: Hikaru
authors.initials: H
authors.lastname: Satoh
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D015061:1,4-alpha-Glucan Branching Enzyme