Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure
Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure
复制标题
DOI：
10.1038/s42003-019-0505-4
复制
发表时间：
2019-07-17
影响因子：
5.9
通讯作者：
Woodward, Jeremy D.
中科院分区：
文献类型：
--
作者：
Mulelu, Andani E.;Kirykowicz, Angela M.;Woodward, Jeremy D.
腈酶是将腈转化为酸和/或酰胺的螺旋酶。所有的植物都有特异的-氰丙氨酸的腈酶4同源物，而在一些植物中，新功能化产生了特异性改变的腈酶。植物硝化酶底物大小和特异性与螺旋扭转相关，但缺乏这种关系的分子细节。在这里，我们确定，据我们所知，第一个接近原子分辨率（3.4埃）的低温电镜结构的活性螺旋硝化酶，硝化酶4从拟南芥。我们对三个残基（R95， S224和L169）进行了位点饱和诱变，并产生了一个螺旋扭曲改变的突变体，该突变体接受不被已知植物腈酶催化的底物。我们揭示了α 2和α 3之间的一个环限制了结合袋的长度，并提出它作为螺旋扭转的函数而改变位置。这些见解将使我们能够开始设计用于化学酶合成的腈酶。
Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for beta-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 angstrom) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between alpha 2 and alpha 3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.