Structural analysis of Bacillus pumilus xylanase

X-ray crystalline analysis of Bacillus pumilus was done at 2.2 A^^resolution。Xylanase分子有一个大小为30 x40 x35 A^，并且在分子中观察到了一个精细的分子被建议成为催化反应站点。自从酶和磷酸酶催化-1，4-glycosidic Bond的羟基，一种催化机制可能与酶中的两种相似。作为催化站点的两种酸性氨基酸溶液的氧化物和重金属修饰。在另一只手上，在xylanase中，功能上重要的居住地将被保留在那些与不同起源相同的地方。B.从Bacillus和fungi、Asp^<21>、Glu^<93>、Asp^<121>和Glu^<182>衍生出的5个xylanase之间的序列排列。pumilus xylanase是一种由酸性化合物组成的。Asp^<21>没有被放置在分子的裂缝中。如果计算两种氨基酸之间的距离，最可能的对催化站点是Glu^<93>和Glu^<182>。在两种驻地的站点指示突变之前，木聚糖酶结构基因的区域编码信号序列被使用合成寡核苷酸替换的启动代码。结构基因中的限制性站点也已被修改为在E中创建独特的站点和在高表达载体中的下游战术推进器。coli，pKP 1500。然后， Glu^<93>和Glu^<182>改变为Asp或Ser. The mutated enzymes were purified to homogeniety from E. coli cell extract。突变酶的活动没有被检测到除了Glu^<93>到ASP的突变。在Asp^ enzyme中观察到的一小部分活动<93>是Vm值的标记递减的结果。

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.