Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein
Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein
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拟南芥谷氨酰-tRNA还原酶与其刺激蛋白复合物的晶体结构
DOI：
10.1073/pnas.1400166111
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发表时间：
2014-05-06
影响因子：
11.1
通讯作者：
Liu, Lin
中科院分区：
文献类型：
--
作者：
Zhao, Aiguo;Fang, Ying;Liu, Lin
意义谷氨酰-tRNA还原酶(GluTR)催化的谷氨酰-tRNA还原是四吡咯生物合成途径中的限速步骤和关键调控步骤。在含叶绿体的光合作用生物中，GlutR结合蛋白(GluBP)是一种新发现的空间调节蛋白，它分配Glutr来合成不同的四吡咯产物。我们发现GluBP提高了GluTR的催化效率。GluTR-GluBP复合体的结构表明，GluBP结合促进GluTR进入氢化物转移状态，这是谷氨酰-tRNA还原的第二步，揭示了催化过程的结构细节。这些发现澄清了关于Glutr激活和调节的一系列争论。GluBP的结构还表明，GluBP可能在血红素代谢中具有新的作用。在植物、藻类和大多数细菌中，四氢吡咯的生物合成始于依赖NADPH的谷氨酰tRNA还原酶(GluTR)对谷氨酰tRNA的还原。Glutr催化的反应是限速步骤，而Glutr是四吡咯生物合成途径的多种翻译后调节的靶点，如血红素反馈抑制。最近发现的一种GluTR调节蛋白，GluTR结合蛋白(GluBP)，通过将GluTR分布到不同的细胞器下位置，在空间上组织了四个吡咯的合成。在这里，我们报道了来自拟南芥的GluTR-GluBP的复杂结构。二聚体GluBP通过其C-末端结构域与V型GluTR二聚体的催化结构域对称结合。观察到GluTRNADPH结合域的实质性构象变化，证实了NADPH结合域的旋转是氢化物从NADPH转移到底物的假设。Arg146是新陈代谢通道的“守门人”，它采用不同的构象，这可能代表了底物识别和产物释放的步骤。偶联酶分析表明，GluBP促进了GluTR的催化效率，使5-氨基乙酰丙酸的形成速率提高了近三倍。此外，无论GluBP是否存在，血红素都能以浓度依赖的方式抑制GluTR的活性。结构比对表明GluBP属于参与血红素代谢的血红素结合家族。我们提出了一个Glutr的催化机制模型，通过该模型，光合作用生物可以实现对四吡咯生物合成的精确调控。
Significance The glutamyl-tRNA reductase (GluTR)-catalyzed reduction of glutamyl-tRNA is the rate-limiting and a pivotal regulation step in the tetrapyrrole biosynthetic pathway. In chloroplast-containing photosynthetic organisms, GluTR binding protein (GluBP) is a newly identified spatial regulator that allocates GluTR for synthesis of different tetrapyrrole products. We find that GluBP stimulates GluTR catalytic efficiency. The structure of the GluTR–GluBP complex shows that GluBP binding promotes GluTR to a hydride-transferring state, the second step of the glutamyl-tRNA reduction, revealing structural details for the catalytic process. These findings clarify a series of arguments regarding the activation and regulation of GluTR. The GluBP structure also suggests that GluBP may have a novel role in heme metabolism. Tetrapyrrole biosynthesis in plants, algae, and most bacteria starts from the NADPH-dependent reduction of glutamyl-tRNA by glutamyl-tRNA reductase (GluTR). The GluTR-catalyzed reaction is the rate-limiting step, and GluTR is the target of multiple posttranslational regulations, such as heme feedback inhibition, for the tetrapyrrole biosynthetic pathway. A recently identified GluTR regulator, GluTR binding protein (GluBP), has been shown to spatially organize tetrapyrrole synthesis by distributing GluTR into different suborganellar locations. Here we report the complex structure of GluTR–GluBP from Arabidopsis thaliana. The dimeric GluBP binds symmetrically to the catalytic domains of the V-shaped GluTR dimer via its C-terminal domain. A substantial conformational change of the GluTR NADPH-binding domain is observed, confirming the postulated rotation of the NADPH-binding domain for hydride transfer from NADPH to the substrate. Arg146, “guarding the door” for metabolic channeling, adopts alternative conformations, which may represent steps involved in substrate recognition and product release. A coupled enzyme assay shows that GluBP stimulates GluTR catalytic efficiency with an approximate threefold increase of the 5-aminolevulinic acid formation rate. In addition, the GluTR activity can be inhibited by heme in a concentration-dependent way regardless of the presence of GluBP. A structural alignment indicates that GluBP belongs to a heme-binding family involved in heme metabolism. We propose a catalytic mechanism model for GluTR, through which photosynthetic organisms can achieve precise regulation of tetrapyrrole biosynthesis.