Crystal structure of the branching enzyme I (BEI) from Oryza sativa L with implications for catalysis and substrate binding
Crystal structure of the branching enzyme I (BEI) from Oryza sativa L with implications for catalysis and substrate binding
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DOI：
10.1093/glycob/cwr049
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发表时间：
2011-08-01
期刊：
影响因子：
4.3
通讯作者：
Kimura, Makoto
中科院分区：
文献类型：
--
作者：
Noguchi, Junji;Chaen, Kimiko;Kimura, Makoto
淀粉分支酶催化α -1,4键的断裂和α -1,4葡聚糖的转移，在支链淀粉中形成α -1,6分支点。水稻分枝酶I （BEI）的序列分析表明其具有模块化结构，其中心-淀粉酶结构域两侧分别是n端碳水化合物结合模块48和-淀粉酶c结构域。我们以大肠杆菌糖原BE为搜索模型，采用分子置换法在1.9 a分辨率下确定了BEI的晶体结构。尽管BEI有三个模块结构，但其形状大致为椭球状，具有两个球状结构域，形成一个突出的凹槽，该凹槽被认为是α -葡聚糖结合位点。氨基酸残基Asp344和Glu399分别作为亲核试剂和一般酸/碱在催化过程中发挥重要作用，它们位于凹槽的中心裂缝处。此外，结构比较显示，在BEI中，延长的环结构导致底物结合位点变窄，而在肺炎克雷伯菌普鲁兰酶中，缩短的环结构使相应亚位点的空间变大。这种结构差异可能归因于不同的催化反应，分别是BEI的转糖基化和普鲁兰酶的水解。
Starch-branching enzyme catalyzes the cleavage of alpha-1, 4-linkages and the subsequent transfer of alpha-1,4 glucan to form an alpha-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central alpha-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the alpha-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 A by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the alpha-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase.