Crystal structure of pullulanase: Evidence for parallel binding of oligosaccharides in the active site
Crystal structure of pullulanase: Evidence for parallel binding of oligosaccharides in the active site
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DOI：
10.1016/j.jmb.2006.03.058
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发表时间：
2006-06-09
影响因子：
5.6
通讯作者：
Katsuya, Yoshio
中科院分区：
文献类型：
--
作者：
Mikami, Bunzo;Iwamoto, Hiroyuki;Katsuya, Yoshio
肺炎克雷伯氏菌普鲁兰酶及其与葡萄糖（G1）、麦芽糖（G2）、异麦芽糖（isoG 2）、麦芽三糖（G3）或麦芽四糖（G4）的复合物的晶体结构已经通过使用SPring-8的同步辐射源在约1.7-1.9埃分辨率下被精修。改进的模型包含920-1052个氨基酸残基，942-1212个水分子，4或5个钙离子和结合糖部分。该酶由五个结构域（N1，N2，N3，A和C）组成。NI结构域仅在具有G3或G4的复合物的结构中清晰可见。N1和N2结构域是普鲁兰酶的特征结构域，而N3、A和C结构域与假单胞菌异淀粉酶的结构域具有弱的相似性。N1结构域是一种新型的糖结合结构域，具有一个钙离子位点（CBM 41）。一个G1结合在亚位点-2，而两个G2结合在类似于-2的-1和类似于+1的+2，两个G3，-1类似于-3和类似于0 '的+2，和两个G4，-1类似于-4和类似于-1'的+2。两个结合的G3和G4分子在活性裂缝中几乎平行，并相互作用。亚位点-1类似于-4和+1类似于+2，包括催化残基Glu 706和Asp 677，在普鲁兰酶和α-淀粉酶之间是保守的，表明普鲁兰酶强烈识别分支点和分支糖残基，而亚位点0'和-1'识别主链α-1，4葡聚糖的非还原末端，对普鲁兰酶和异淀粉酶是特异性的。比较表明，周围的活性裂缝的构象差异，连同结构域的组织，决定了不同的底物特异性之间的普鲁兰酶和异淀粉酶。(c)2006爱思唯尔有限公司保留所有权利。
The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9 angstrom resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The NI domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 similar to-2 and +2 similar to+1, two G3, -1 similar to-3 and +2 similar to 0', and two G4, -1 similar to-4 and +2 similar to-1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 similar to-4 and +1 similar to+2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and a-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase. (c) 2006 Elsevier Ltd. All rights reserved.