Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase)
Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase)
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DOI：
10.1016/j.carres.2006.07.001
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发表时间：
2006-10-16
影响因子：
3.1
通讯作者：
Hecht, Hans-Juergen
中科院分区：
文献类型：
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作者：
Seibel, Juergen;Moraru, Roxana;Hecht, Hans-Juergen
在本研究中，我们对枯草芽孢杆菌NCIMB 11871中的果糖基转移酶（FTF，左旋蔗糖酶）（EC 2.4.1.162）进行了详细的受体和供体底物研究，并建立了底物酶复合物的结构模型，以详细研究活性位点氨基酸在酶的催化作用中的作用以及底物的范围和局限性。因此，我们分离了ftf基因，在大肠杆菌中表达，产生了左旋蔗糖酶。因此，详细的受体性质的影响，在糖苷受体相对于羟基的位置（2、3、4和6）从赤道到轴向的系统变化的果糖基化已经研究了新的低聚糖制备规模生产。这些研究提供了FTF反应的机理见解。作为底物或受体的葡萄糖苷衍生物的C-2和C-3羟基的构型和存在已被确定为反式果糖基化过程的速率限制。通过Arg360、Tyr411、Glu342、Trp85、Asp247和Arg246对受体和底物的稳定作用，C-4(1)构象中的d -甘黄苷残基与氨基酸网络配位，使反应速率合理。此外，我们还描述了第一个FTF反应，该反应催化L-糖（L-葡萄糖、L-鼠李糖、L-半乳糖、L-焦糖、L-木糖）以C-1(4)构象的β -(1 - bbbb2)-果糖转移到2- oh。在这些构象中，l -甘黄苷被相同的氢网络稳定。通过核磁共振和质谱分析确定了受体产物的结构。(c) 2006 Elsevier Ltd.版权所有。
In the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination Of D-glycopyranoside residues in C-4(1) conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the beta-(1 -> 2)-fructosyl transfer to 2-OH of L-sugars (L-glucose, L-rhamnose, L-galactose, L-fucose, L-Xylose) presumably in a C-1(4) conformation. In those conformations, the L-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis. (c) 2006 Elsevier Ltd. All rights reserved.