Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs
Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs
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亮氨酸拉链基序异二聚化促进的亚基剪接在 ω-转氨酶、丙氨酸脱氢酶和甲酸脱氢酶异亚基之间形成肽键
DOI：
10.3389/fbioe.2020.00686
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发表时间：
2020
影响因子：
5.7
通讯作者：
Wei Feng
中科院分区：
文献类型：
--
作者：
Rong Li;Yao Chen;K. Du;Wei Feng
对于多聚体酶R-ω-转氨酶（RTA）、丙氨酸脱氢酶（AlaDH）和甲酸脱氢酶（FDH），杂亚基之间的肽键形成已经通过内含肽介导的体内亚基剪接实现。亚基连接由富含精氨酸的亮氨酸拉链基序与富含谷氨酸的亮氨酸拉链基序的异二聚化触发。异亚基的一一连接构建配对酶RTA&AlaDH和AlaDH&FDH。基于蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）分析连接模式。圆二色谱（CD），荧光光谱，和二维FTIR光谱提供的信息的二级结构和稳定性的配对酶。基于微尺度热泳分析，分析了酶-底物相互作用。与混合的三种酶RTA + AlaDH + FDH相比，连接的酶RTA&AlaDH + AlaDH&FDH表现出更大的底物亲和力、更高的稳定性和显著增强的活性。
For the multimeric enzymes R-ω-transaminase (RTA), alanine dehydrogenase (AlaDH), and formate dehydrogenase (FDH), peptide bond formation between the hetrosubunits has been achieved by the intein-mediated in vivo subunit splicing. The subunit ligation is triggered by the heterodimerization of an arginine rich leucine zipper motif with a glutamic acid rich leucine zipper motif. The one-by-one ligation of hetrosubunits constructs the pairing enzymes RTA&AlaDH and AlaDH&FDH. The ligation modes were analyzed based on blue native polyacrylamide gel electrophoresis (BN-PAGE). The spectra of circular dichroism (CD), fluorescence, and two-dimensional FTIR provide information on the secondary structures and stability of the pairing enzymes. The enzyme-substrate interaction was analyzed based on microscale thermophoresis analysis. In contrast to the mixed three enzymes RTA + AlaDH + FDH, the ligated enzymes RTA&AlaDH + AlaDH&FDH exhibited a much larger substrate affinity, higher stability, and significantly enhanced activity.
影响因子：
4.8
作者：
Seidel, Susanne A. I.;Dijkman, Patricia M.;Lea, Wendy A.;van den Bogaart, Geert;Jerabek-Willemsen, Moran;Lazic, Ana;Joseph, Jeremiah S.;Srinivasan, Prakash;Baaske, Philipp;Simeonov, Anton;Katritch, Ilia;Melo, Fernando A.;Ladbury, John E.;Schreiber, Gideon;Watts, Anthony;Braun, Dieter;Duhr, Stefan
通讯作者：
Duhr, Stefan
影响因子：
16.6
作者：
Shah, Neel H.;Vila-Perello, Miquel;Muir, Tom W.
通讯作者：
Muir, Tom W.