title: Purification and characterization of maize starch synthase I and its truncated forms.
abstract: Comparison of the protein sequences deduced from the cDNAs of maize granule-bound starch synthase, Escherichia coli glycogen synthase, and maize starch synthase I (SSI) reveals that maize SSI contains an N-terminal extension of 93 amino acids. In order to study the properties of maize SSI and to understand the functions of the maize SSI N-terminal extension, the gene coding for full-length SSI (SSI-1) and genes coding for N-terminally truncated SSI (SSI-2 and SSI-3) were individually expressed in E. coli. Here we describe for the first time the purification of a higher plant starch synthase to apparent homogeneity. Its kinetic properties were therefore studied in the absence of interfering amylolytic enzymes. The specific activities of the purified SSI-1, SSI-2, and SSI-3 were 22.5, 33.4, and 26.3 micromol Glc/min/mg of protein, respectively, which are eight times higher than those of partially purified SSI from developing maize endosperm. The full-length recombinant enzyme SSI-1 exhibited properties similar to those of the enzyme from maize endosperm. As observed for native maize enzyme, recombinant SSI-1 exhibited "unprimed" activity without added primer in the presence of 0.5 M citrate. Our results have clearly indicated that the catalytic center of SSI is not located in its N-terminal extension. However, N-terminal truncation decreased the enzyme affinity for amylopectin, with the Km for amylopectin of the truncated SSI-3 being about 60-90% higher than that of the full-length SSI-1. These results suggest that the N-terminal extension in SSI may not be directly involved in enzyme catalysis, but may instead regulate the enzyme binding of alpha-glucans. Additionally, the N-terminal extension may play a role in determining the localization of SSI to specific portions of the starch granule or it may regulate its interactions with other enzymes involved in starch synthesis.
authors.affiliation: ExSeed Genetics, L.L.C., Iowa State University, Food Science Building, Ames, Iowa 50011, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Imparl-Radosevich
authors.forename: P
authors.initials: P
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: A L
authors.initials: AL
authors.lastname: McKean
authors.forename: P L
authors.initials: PL
authors.lastname: Keeling
authors.forename: H
authors.initials: H
authors.lastname: Guan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000201:enzymology; D004926:Escherichia coli; D017343:Genes, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013214:Starch Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D013214:Starch Synthase