Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.).
In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule-bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N-terminal noncatalytic region (N-domain), and a C-terminal catalytic region (C-domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N-domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N-domain, and recombinant kidney bean SSIII C-domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high k(cat) values, and that the K(m) values of recombinant kidney bean SSIII C-domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C-domain had similar chain-length specificities for the extension of glucan chains, indicating that the N-domain of kidney bean SSIII does not affect the chain-length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N-domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N-domain of kidney bean SSIII as a carbohydrate-binding module.
Senoura
Takeshi
T
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
Asao
Ayako
A
Takashima
Yoshinori
Y
Isono
Naoto
N
Hamada
Shigeki
S
Ito
Hiroyuki
H
Matsui
Hirokazu
H
D020134:Catalytic Domain; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000201:enzymology*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013214:Starch Synthase / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D013214:Starch Synthase