|Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
The cDNA clones coding for RBE1, RBE3, and RBE4 have been identified from a developing rice seed cDNA library in lambdagt11. The mature proteins of RBE1, RBE3, and RBE4 contain 756,760, and 788 amino acids with calculated molecular masses of 86,734,86,376, and 89,745 Da, respectively. RBE1 shares a noticeable degree of sequence identity (49 and 47%) with RBE3 and RBE4, respectively, whereas the RBE3 sequence is 80% identical to that of RBE4. These isoforms of braching enzyme all share a high degree of sequence identity in the middle regions of the protein molecules. Interestingly, the consensus sequences of the four regions, which form the catalytic sites of amylolytic enzymes, are conserved in the regions. Thus, plant branching enzyme as well as the bacterial and mammalian enzymes belongs to a family of amylolytic enzymes. This fact implies that branching enzyme possesses two enzymatic functions such as cleavage of alpha-1,4-glucosidic linkages and transfer of the newly formed reducin
g ends to other alpha-1,4-linked chains. There are two structural differences among three isoforms of rice branching enzyme ; RBE1 possesses an approximately 50-residue extra sequence at the carboxyl terminus, and lacks an amino-terminal sequence of almost 70 residues (90 residues for RBE4), as compared with RBE3 and RBE4. Another difference is that the RBE1 sequence contains an 8-residue sequence specifically present in this isoform at the carboxyl-terminal half. On the contrary, RBE3 and RBE4 have an 11-residue specific sequence that is deficient in RBE1.
To examine enzymatic functions of three isoforms of rice branching enzyme, we have established an expression system of the cDNA fragments in Escherichia coli. We have also prepared a lot of mutant proteins, including chimeric enzymes between RBE1 and RBE3, "deletion proteins" without the amino-or carboxyl-terminal sequences of the mature enzymes, and "point-mutated proteins" with the replacement of cysteine residue (s) into serine. Our data demonstrate that RBE1 has the much higher specific activity in branching amylose and amylopectin than RBE3 and RBE4. An intriguing observation is that RBE4 is capable of branching amylose almost 3-fold more rapidly than RBE3 in spite of the considerably high degree of sequence similarity between these two isoforms. However, the unit-chain profiles of branched glucans produced from amylose by these three isoforms are relatively similar. Analysis of the mutant proteins indicates that third, sixth, and seventh cysteine residues from the amino terminus are important for the activity of RBE1. It is also concluded that the carboxyl-terminal and amino-terminal sequences of approximately 50 and 60 residues in RBE1 and RBE4, respectively, are not essential for the enzyme activity. Despite this fact, the difference of the amino acid sequences near both termini still seems to explain the distinct enzymatic functions among three isoforms. Less