Purification and characterization of maize starch synthase I and its truncated forms

Arch Biochem Biophys. 1998 May 1;353(1):64-72. doi: 10.1006/abbi.1998.0613.

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.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Chromatography, Affinity
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Cytoplasmic Granules / enzymology
  • Escherichia coli
  • Genes, Plant
  • Kinetics
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Starch Synthase / chemistry
  • Starch Synthase / isolation & purification*
  • Starch Synthase / metabolism*
  • Substrate Specificity
  • Zea mays / enzymology*

Substances

  • Recombinant Proteins
  • Starch Synthase

Associated data

  • GENBANK/AF036891