Structural and biochemical investigation of two Arabidopsis shikimate kinases: The heat-inducible isoform is thermostable
Structural and biochemical investigation of two Arabidopsis shikimate kinases: The heat-inducible isoform is thermostable
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DOI：
10.1002/pro.640
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发表时间：
2011-07-01
期刊：
影响因子：
8
通讯作者：
Christendat, Dinesh
中科院分区：
文献类型：
--
作者：
Fucile, Geoffrey;Garcia, Christel;Christendat, Dinesh
植物莽草酸激酶（SK; EC 2.7.1.71）的表达是芳香族氨基酸生物合成莽草酸途径中的中间步骤，在环境应激和发育要求的特定条件下以亚型特异性方式诱导。尽管其重要的生理作用，植物SK的实验结构尚未确定和植物SK调节的生化性质是未知的。拟南芥基因组编码两个SK，AtSK 1和AtSK 2。我们证明了AtSK 2是高度不稳定的，在37摄氏度时会失活，而热诱导的同种型AtSK 1是热稳定的，在相同的条件下在这个温度下完全活跃。我们确定了AtSK 2的晶体结构，第一个SK结构从植物王国，并进行生物物理表征AtSK 1和AtSK 2了解这种机制的热调节。AtSK 2的晶体结构通常与细菌SK保守，其中添加了形成三磷酸腺苷结合位点的一部分的推定的调节磷酸化基序。热诱导亚型AtSK 1在溶液中形成同源二聚体，与AtSK 2相比，其形成促进其相对热稳定性。计算机模拟分析确定了可能有助于AtSK 1稳定性的AtSK 1位点变体。我们的研究结果表明，AtSK 1执行一个独特的功能，在热应激条件下，AtSK 2可以成为失活。我们讨论了这些发现的背景下，通过SK介导的控制稳态浓度的莽草酸调节代谢通量竞争下游途径。
The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform-specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37 degrees C whereas the heat-induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. We determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat-induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK-mediated control of steady state concentrations of shikimate.