鼠李糖乳杆菌 2'-脱氧核糖基转移酶，一种用于核苷修饰的新型生物催化剂。
Lactobacillus reuteri 2'-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides.
机构信息
Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/ José Antonio Nováis 2, 28040 Madrid, Spain.
出版信息
Appl Environ Microbiol. 2010 Mar;76(5):1462-70. doi: 10.1128/AEM.01685-09. Epub 2010 Jan 4.
Abstract
A novel type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% alpha-helix, 10% beta-strand, 16% beta-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T(m)) of 64 degrees C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40 degrees C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2'-fluorodeoxyribonucleosides was carried out.
摘要
从乳杆菌（Lactobacillus reuteri）中克隆并在大肠杆菌中过表达了一种新型 II 型核苷 2'-脱氧核糖基转移酶（LrNDT）。对重组 LrNDT 进行了结构和功能表征。沉降平衡分析显示其为 114 kDa 的同六聚体分子。圆二色性研究表明，该酶二级结构中包含 55%的α-螺旋、10%的β-折叠、16%的β-片层和 19%的无规卷曲。荧光、圆二色性和差示扫描量热法研究表明，LrNDT 具有热稳定性，其熔点（T(m)）为 64°C。该酶在较宽的 pH 范围（4.6 至 7.9）内具有高活性，在 pH 4 至 7.9 之间也非常稳定。最适反应温度为 40°C。重组 LrNDT 能够合成天然和非天然核苷类似物，提高了文献中描述的活性，并且令人惊讶的是，还表现出了以前在这种酶中尚未描述的新的阿拉伯糖基转移酶活性。此外，还进行了新型阿拉伯核苷和 2'-氟脱氧核苷的合成。
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