Functional characteristics of C-terminal regions of starch-branching enzymes from developing seeds of kidney bean (Phaseolus vulgaris L.)

Plant starch-branching enzyme (SBE) catalyzes the formation of alpha-1,6-branch points in amylopectin and therefore plays a pivotal role in determining the fine structure of starch. We have previously shown that developing kidney bean (Phaseolus vulgaris L.) seeds contain two SBE isozymes (designated PvSBE1 and PvSBE2), which display distinct enzymatic properties. To understand the biochemical basis for the differences in kinetic properties of these SBE isozymes, a series of chimeric enzymes were constructed to determine whether the differences in enzymatic properties were attributable to unique peptide sequences located at the C-terminal ends of these SBE isozymes. Of the six chimeric enzymes constructed, three showed significant branching enzyme activity. These chimeric enzymes were purified to apparent homogeneity and their kinetic properties compared to recombinant PvSBE1 and PvSBE2. The first chimeric enzyme, I-(1Ca/2Cb), in which a portion of the C-terminal region of PvSBE1 was substituted by the corresponding region of PvSBE2, showed nearly the same K value for amylose as recombinant PvSBE2 (rPvSBE2) instead of as for rPvSBE1. The second enzyme, II-(1Ca/2Cb), where part of the C-terminal region of PvSBE2 was replaced with the corresponding region of PvSBE1, showed no apparent changes in amylose specificity as the parental rPvSBE2. The third enzyme, II-(1Ca/1Cb), where the C-terminal region of PvSBE2 was replaced with the corresponding regions of PvSBE1, had a K-m value for amylose much like rPvSBE1 rather than rPvSBE2. Chain transfer experiments by chimeric enzymes revealed that the proportion of dp 6 chains transferred by I-(1Ca/2Cb) enzyme was much higher than that by rPvSBE1, whereas II-(1Ca/1Cb) showed reduced capacity than rPvSBE2 in transferring linear glucans of this chain length. These results suggest that the C-terminal regions of the two PvSBEs have different roles in branching enzyme activity; the C-terminal region of PvSBE1 confers specificity to amylose while that of PvSBE2 confers specificity to the transfer of short chains. (C) 2004 Elsevier Ireland Ltd. All rights reserved.