Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4
Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4
复制标题
DOI：
10.1002/pro.432
复制
发表时间：
2010-08-01
期刊：
影响因子：
8
通讯作者：
Wimmer, Reinhard
中科院分区：
文献类型：
--
作者：
Buchinger, Edith;Aachmann, Finn L.;Wimmer, Reinhard
藻酸盐差向异构酶是大型多结构域蛋白，能够将 β-D-甘露糖醛酸 (M) 上的 C5 差向异构化，将其转化为聚合藻酸盐中的 α-L-古洛糖醛酸 (G)。维氏固氮菌分泌七种差向异构酶家族，每种差向异构酶都能产生具有特征性 G 分布模式的藻酸盐。所有七种差向异构酶均由两种类型的模块组成，分别表示为 A 和 R，数量不同。利用溶液态 NMR 研究这些酶的尝试受到其尺寸的阻碍 - 最小的差向异构酶 AlgE4（由一个 A 模块和一个 R 模块组成）为 58 kDa，导致大量信号重叠，损害了 NMR 谱的解释。因此，我们使用发菜天然分裂的内含肽通过蛋白质转拼获得了分段H-2、N-15标记的AlgE4同位素（A-[H-2，N-15]-R和[H-2，N-15]-A-R）。天然 AlgE4 和连接版本的 NMR 光谱非常吻合，证明了蛋白质结构的保守性。通过两种不同的酶活性测定验证了连接的AlgE4的活性，证明连接的AlgE4表现出与野生型AlgE4相同的催化活性。
Alginate epimerases are large multidomain proteins capable of epimerising C5 on beta-D-mannuronic acid (M) turning it into alpha-L-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution-state NMR are hampered by their size-the smallest epimerase, AlgE4, consisting of one A- and one R-module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally H-2, N-15 labeled AlgE4 isotopomeres (A-[H-2, N-15]-R and [H-2, N-15]-A-R) by protein trans-splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4.