Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds

cDNA clones encoding an isoform of starch branching enzyme, RBE4, have been identified from a developing rice seed cDNA library, using a synthetic oligonucleotide probe corresponding to the N-terminal amino acid sequence of RBE4. The cDNA-derived amino acid sequence indicated that RBE4 is initially produced as a precursor protein of 841 amino acids, including a 53-residue transit peptide at the N-terminus. The mature form of RBE4 shared a high degree of sequence identity (80%) with mature RBE3, and possessed an N-terminal extra sequence, as found in RBE3. Northern blot analysis demonstrated that the RBE4 gene is expressed in both leaves and developing seeds. The RBE4 gene was distinguished from the RBE1 and RBE3 genes by expression at the earlier stages of seed development. To examine enzymatic functions of RBE4, recombinant proteins were produced in Escherichia coli cells, and purified by two chromatographic separations. The branched alpha-glucans produced by the recombinant enzymes from potato amylose revealed the different patterns of oligosaccharide chain transfer. The peak of major branches of the products by RBE3 or RBE4 was 6 glucose units, whereas the peaks of major branches of the products by RBE1 were 6 and 11 glucose units. The similar property between RBE3 and RBE4 is supported by high similarity of the amino acid sequences between them.