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Structural analysis of Bacillus pumilus xylanase

短小芽孢杆菌木聚糖酶的结构分析

基本信息

批准号：
62560105
负责人：
ATSUHIKO SHINMYO
金额：
$ 1.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62560105/
关键词：
Bacillus pumilus xylanase X-ray crystalline analysis active site 部位特異的変異 X線解析

项目摘要

X-ray crystalline analysis of xylanase of Bacillus pumilus was done at 2.2 A^^゜ resolution. Xylanase molecule has a size of 30 x 40 x 35 A^^゜ and a cleft observed in the molecule was suggested to be the site of catalytic reaction. Since xylanase and lysozyme catalyze hydrolysis of -1,4-linkage of glycosidic bond, a catalytic mechanism might be similar in both enzymes. Chemical and heavy metal modification of xylanase suggested two acidic amino acid residues as catalytic site. On the other hand, functionally important residues in xylanase will be conserved in those from different origins. Among sequence alignments of 5 xylanases derived from Bacillus and fungi, Asp^<21>, Glu^<93>, Asp^<121> and Glu^<182> in B. pumilus xylanase were conserved acidic residues. Asp^<21> was not located in the cleft of the molecule. By taking account the distance between two amino acids, most possible pair as catalytic site was Glu^<93> and Glu^<182>.Before site-directed mutation of the two residues, the region coding signal sequence of the structural gene of xylanase was replaced by the initiation codon using synthetic oligonucleotides. Restriction sites in the structural gene were also modified to create unique sites and ligated downstream of tac promoter in high expression vector in E. coli, pKP1500. Then, Glu^<93> and Glu^<182> were changed to Asp or Ser. The mutated enzymes were purified to homogeniety from E. coli cell extract. Activity of the mutated enzymes was not detected except the mutation of Glu^<93> to ASP. A little activity observed in Asp^<93> enzyme was the result of marked decrease of Vm value.

用X射线衍射法对短小芽孢杆菌木聚糖酶的晶体结构进行了分析。木聚糖酶分子的大小为30 × 40 × 35埃，在分子中观察到的裂缝被认为是催化反应的位点。由于木聚糖酶和溶菌酶催化糖苷键的-1，4-键的水解，因此两种酶的催化机理可能相似。木聚糖酶的化学修饰和重金属修饰均提示两个酸性氨基酸残基为催化位点。另一方面，木聚糖酶中重要的功能残基在不同来源的木聚糖酶中是保守的。对芽孢杆菌和真菌的5种木聚糖酶进行了序列比对，发现B中有Asp^<21>、Glu^<93>、Asp^<121>和Glu^<182>。pumilus木聚糖酶是保守的酸性残基。Asp^<21>不位于分子的裂隙中。考虑到两个氨基酸之间的距离，最可能作为催化位点的对是Glu^<93>和Glu^<182>。在对这两个残基进行定点突变之前，使用人工合成的寡核苷酸将木聚糖酶结构基因的编码信号区序列替换为起始密码子。对结构基因的限制性酶切位点进行了改造，使之成为一个独特的酶切位点，并将其连接到大肠杆菌高效表达载体中的tac启动子下游。coli，pKP1500。然后将Glu^<93>和Glu^<182>分别突变为Asp或Ser，并从大肠杆菌中纯化得到纯的突变酶。大肠杆菌细胞提取物。除Glu_（1-x）突变为ASP外，未检测到突变酶的活性<93>。Asp^酶中观察到的少量活性<93>是Vm值显着降低的结果。