Mizuno Masahiro, Koide Atsushi, Yamamura Akihiro, Akeboshi Hiromi, Yoshida Hiromi, Kamitori Shigehiro, Sakano Yoshiyuki, Nishikawa Atsushi, Tonozuka Takashi
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-Cho, Fuchu, Tokyo 183-8509, Japan.
J Mol Biol. 2008 Feb 8;376(1):210-20. doi: 10.1016/j.jmb.2007.11.098. Epub 2007 Dec 5.
An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H(f) to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-A resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 beta-strands and forms a beta-sandwich. Domain C, where the active site is located, forms a right-handed beta-helix, and the lengths of the pitches of each coil of the beta-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites +2 and +3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the beta-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.
黑曲霉ATCC9642产生的异普鲁兰酶（IPU）可水解普鲁兰的α-1,4-糖苷键生成异麦芽糖。尽管IPU不能水解葡聚糖，但它被归类于糖苷水解酶家族49（GH49），该家族的主要成员是水解葡聚糖的酶。IPU高度糖基化，难以获得其晶体。我们使用内切糖苷酶H(f)切割IPU的N-连接寡糖，并在此确定了IPU的无配体形式和异麦芽糖复合形式，二者均在1.7 Å分辨率下解析。IPU由结构域N和C通过短连接子连接而成，每个分子有11或12个N-乙酰葡糖胺残基的电子密度图。结构域N由13条β链组成，形成一个β折叠片层。活性位点所在的结构域C形成一个右手β螺旋，β螺旋每个螺旋圈的螺距长度与GH49葡聚糖酶和GH28聚半乳糖醛酸酶相似。尽管底物特异性不同，但IPU的整体结构类似于GH49酶微小青霉葡聚糖酶（Dex49A）。与IPU和Dex49A的活性位点相比，参与亚位点+2和+3的氨基酸残基不保守，与IPU结合的异麦芽糖的葡萄糖残基的取向与与Dex49A结合的异麦芽糖的相应葡萄糖残基完全不同。由β螺旋的第七个螺旋圈和结构域N的一个环所表征的催化裂隙的形状似乎在决定IPU对普鲁兰的特异性方面至关重要。
