Bejger Magdalena, Imiolczyk Barbara, Clavel Damien, Gilski Miroslaw, Pajak Agnieszka, Marsolais Frédéric, Jaskolski Mariusz
Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.
Acta Crystallogr D Biol Crystallogr. 2014 Jul;70(Pt 7):1854-72. doi: 10.1107/S1399004714008700. Epub 2014 Jun 29.
Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type L-asparaginase from Phaseolus vulgaris (PvAspG1) differing in the type of associated alkali metal ions (K(+), Na(+) or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type L-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to α and β subunits, thus creating the mature heterotetramer or dimer of heterodimers (αβ)2. The αβ subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two β-sheets flanked on each side by a layer of helices. In addition to the sodium loop' (here referred to as the stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the `activation loop') in subunit α (residues Val111-Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type L-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K(+) is coordinated) or OFF (when Na(+) is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent L-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.
植物型L-天冬酰胺酶是Ntn-水解酶家族的一个亚类，可分为具有不同底物偏好的钾依赖性和非钾依赖性酶。虽然非钾依赖性酶已得到充分表征，但关于钾依赖性酶成员的结构数据尚未见报道，无法阐明其催化机制对碱金属阳离子的有趣依赖性。本文报道了来自菜豆的一种钾依赖性植物型L-天冬酰胺酶（PvAspG1）的三种晶体结构（其结合的碱金属离子类型不同，分别为K⁺、Na⁺或两者皆有），并将不同离子的结构后果与酶活性相关联。与所有植物型L-天冬酰胺酶一样，未成熟的PvAspG1是由两条蛋白质链组成的同型二聚体，两条链均经过自催化切割形成α和β亚基，从而形成成熟的异源四聚体或异源二聚体（αβ）₂二聚体。PvAspG1的αβ亚基折叠方式与非钾依赖性酶相似，由两层β折叠片层夹在中间，两侧各有一层螺旋。除了非钾依赖性植物型天冬酰胺酶中已知的“钠环”（此处称为“稳定环”）外，钾依赖性PvAspG1酶在亚基α（第111位缬氨酸至第118位丝氨酸）中还含有另一个碱金属结合环（“激活环”）。PvAspG1的活性位点位于这两个金属结合环之间，紧邻三个作为催化开关的残基，即组氨酸117、精氨酸224和谷氨酸250，这是在植物型L-天冬酰胺酶中首次发现的新特征。对三种PvAspG1结构的比较表明，结合在激活环中的金属离子如何影响其构象，将催化开关设置为开启（当K⁺配位时）或关闭（当Na⁺配位时），分别允许或阻止反应底物/产物在活性位点的锚定。此外，有人提出激活环的最后一个残基丝氨酸118参与了钾依赖性机制。本文将PvAspG1的结构与非钾依赖性L-天冬酰胺酶（LlA、EcAIII和hASNase3）以及其他Ntn-水解酶（AGA和Tas1）的结构进行了比较，并结合非晶体学研究进行了讨论。
