Insights into polymer versus oligosaccharide synthesis:: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium
Insights into polymer versus oligosaccharide synthesis:: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium
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DOI：
10.1042/bj20070600
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发表时间：
2007-10-15
影响因子：
4.1
通讯作者：
Seibel, Juergen
中科院分区：
文献类型：
--
作者：
Homann, Arne;Biedendieck, Rebekka;Seibel, Juergen
在巨大芽孢杆菌DSM 319的细胞培养物的上清液中鉴定了一种新的果聚糖蔗糖酶。为了测试特定氨基酸残基对果聚糖蔗糖酶催化的贡献，在大肠杆菌中异源产生野生型酶沿着16种基于序列比对和结构信息的变体。纯化的酶进行了动力学表征，并确定了每个变体的产物谱。比较来自革兰氏阳性枯草芽孢杆菌和革兰氏阴性重氮营养葡萄糖酸醋杆菌的果聚糖蔗糖酶的X射线结构，结合相应的产物光谱，确定了负责产物特异性和催化的关键氨基酸残基。高度保守的区域如先前描述的RDP和DXXER基序被鉴定为重要的。与寡糖合成相比，位于氨基酸残基Arg（370）和Asn（252）的两个关键结构差异在聚合物中具有高度相关性。
A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of specific amino acid residues to levansucrase catalysis, the wild-type enzyme along with 16 variants based on sequence alignments and structural information were heterologously produced in Escherichia coli. The purified enzymes were characterized kinetically and the product spectrum of each variant was determined. Comparison of the X-ray structures of the levansucrases from Gram-positive Bacillus subtilis and Gram-negative Gluconacetobacter diazotrophicus in conjunction with the corresponding product spectra identified crucial amino acid residues responsible for product specificity and catalysis. Highly conserved regions such as the previously described RDP and DXXER motifs were identified as being important. Two crucial structural differences localized at amino acid residues Arg(370) and Asn(252) were of high relevance in polymer compared with oligosaccharide synthesis.