The N-terminal region of the starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and structural stability
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概要
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Starch-branching enzymes (SBEs) play a pivotal role in determining the fine structure of starch by catalyzing the syntheses of α-1,6-branch points. They are the members of the α-amylase family and have four conserved regions in a central (β/α)8 barrel, including the catalytic sites. Although the role of the catalytic barrel domain of an SBE is known, that of its N- and C-terminal regions remain unclear. We have previously shown that the C-terminal regions of the two SBE isozymes (designated as PvSBE1 and PvSBE2) from kidney bean (Phaseolus vulgaris L.) have different roles in branching enzyme activity. To understand the contribution of the N-terminal region to catalysis, six chimeric enzymes were constructed between PvSBE1 and PvSBE2. Only one enzyme (1Na/2Nb)-II, in which a portion of the N-terminal region of PvSBE2 was substituted by the corresponding region of PvSBE1, retained 6% of the PvSBE2 activity. The N-terminal truncated form (ΔN46-PvSBE2), lacking 46 N-terminal residues of PvSBE2, lost enzyme activity and stability to proteolysis. To investigate the possible function of this region, three residues (Asp-15, His-24, and Arg-28) among these 46 residues were subjected to site-directed mutagenesis. The purified mutant enzymes showed nearly the same Km values as PvSBE2 but had lower Vmax values and heat stabilities than PvSBE2. These results suggest that the N-terminal region of the kidney bean SBE is essential for maximum enzyme activity and thermostability.
- Elsevier Ltd.の論文
著者
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森 春英
北海道大学大学院農学研究院応用生命科学部門
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Matsui H
Division Of Applied Bioscience Graduate School Of Agriculture Hokkaido University
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Ito Hiroyuki
Division Of Applied Bioscience Graduate School Of Agriculture Hokkaido University
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Matsui Hirokazu
Dept. Biosci. Chem. Fac. Agric. Hokkaido Univ.
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Matsui Hirokazu
Hokkaido Univ. Sapporo Jpn
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Takeda Yasuhiro
Department Of Animal Science Utsunomiya University
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Hiraga Susumu
Department Of Applied Biosciences Graduate School Of Agriculture Hokkaido University
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Ito H
Division Of Applied Bioscience Graduate School Of Agriculture Hokkaido University
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Hamada Shigeki
Laboratory Of Biochemistry Research Faculty Of Agriculture Hokkaido University
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Matsui Hirokazu
Laboratory Of Biochemistry Research Faculty Of Agriculture Hokkaido University
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