Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii
Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii
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DOI：
10.1074/jbc.m114.567008
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发表时间：
2014-11-07
影响因子：
4.8
通讯作者：
Aachmann, Finn L.
中科院分区：
文献类型：
--
作者：
Buchinger, Edith;Knudsen, Daniel H.;Aachmann, Finn L.
棕色固氮细菌产生一个由七个分泌型和钙依赖型甘露聚糖酶C-5下异构酶(算法E1-7)组成的家族。这些异构体参与了海藻酸聚合物中β-D-甘露糖酸(M)向α-L古鲁糖酸(G)的异构化反应。同分异构体显示由一个或两个催化A-模块和一个至七个对A-模块具有激活作用的R-模块组成的模块化结构。在本研究中，我们用小角X射线散射方法确定了算法E6(AR1R2R3)中三个独立R-模块的核磁共振结构以及算法E4(AR)和算法E6的整体结构。此外，还用核磁共振和等温滴定量热法研究了海藻酸根与海藻酸盐的结合能力。该算法E6的R模块折叠成一个拉长的平行测试卷与一个浅的，带正电的凹槽横跨模块。小角X射线散射分析显示，两种酶的模块之间都有一定程度的柔韧性，整体呈拉长状。用所定义的海藻酸低聚物滴定R-模块，结果表明，这些低聚物与寡聚-M和MG之间都有很强的相互作用，而这些低聚物与海藻酸低聚物中单独的R-模块之间没有相互作用。算法E6中的所有三个R-模块的组合显示出与长M-齐聚物的弱相互作用。在算法E4和算法E6之间交换R-模块，产生了一种新的同向异构酶，称为算法E64，与算法E6相比，它具有更强的G-块形成能力。
The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for the epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel beta-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R- modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.