Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, M5S 3B, Canada.
Center for the Analysis of Genome Evolution and Function, University of Toronto, 25 Willcocks Street, Toronto, Ontario, M5S 3B2, Canada.
Plant J. 2022 Feb;109(4):789-803. doi: 10.1111/tpj.15590. Epub 2021 Dec 5.
The shikimate pathway plays a central role in the biosynthesis of aromatic amino acids and specialized metabolites in plants. The first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) serves as a key regulatory point for the pathway in various organisms. These enzymes are important in regulating the shikimate pathway in multiple microbial systems. The mechanism of regulation of DAHPS is poorly understood in plants, and the role of tyrosine (Tyr) with respect to the three DAHPS isozymes from Arabidopsis thaliana was investigated. In vitro enzymatic analyses established that Tyr does not function as an allosteric regulator for the A. thaliana DAHPS isozymes. In contrast, Arabidopsis T-DNA insertional mutants for the DAHPS1 locus, dahps1, are hypersensitive to elevated Tyr. Tyr hypersensitivity can be reversed with tryptophan and phenylalanine supplementation, indicating that Tyr is affecting the shikimate pathway flux in the dahps1 mutant. Tyr treatment of Arabidopsis seedlings showed reduced accumulation of overexpressed DAHPS2 in the chloroplast. Further, bimolecular fluorescence complementation studies revealed that DAHPS2 interacts with a 14-3-3 protein in the cytosol, and this interaction is enhanced with Tyr treatment. This interaction with 14-3-3 may retain DAHPS2 in the cytosol, which prevents its ability to function in the chloroplast with elevated Tyr.
莽草酸途径在植物芳香族氨基酸和特殊代谢物的生物合成中起着核心作用。第一个酶，3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶（DAHPS）作为各种生物体中途径的关键调节点。这些酶在多种微生物系统中调节莽草酸途径的重要性。植物中 DAHPS 的调节机制知之甚少，本研究调查了色氨酸（Tyr）与拟南芥三种 DAHPS 同工酶的关系。体外酶分析确立了 Tyr 不作为拟南芥 DAHPS 同工酶的变构调节剂。相反，拟南芥 DAHPS1 基因座的 T-DNA 插入突变体 dahps1 对升高的 Tyr 表现出超敏反应。Tyr 超敏反应可以通过色氨酸和苯丙氨酸的补充来逆转，表明 Tyr 正在影响 dahps1 突变体中的莽草酸途径通量。Tyr 处理拟南芥幼苗显示叶绿体中过量表达的 DAHPS2 积累减少。此外，双分子荧光互补研究表明 DAHPS2 在细胞质中与一种 14-3-3 蛋白相互作用，而 Tyr 处理增强了这种相互作用。这种与 14-3-3 的相互作用可能将 DAHPS2 保留在细胞质中，从而阻止其在叶绿体中与升高的 Tyr 一起发挥作用。
