Distinctive regulatory properties of pyruvate kinase 1 from Aedes aegypti mosquitoes.
Distinctive regulatory properties of pyruvate kinase 1 from Aedes aegypti mosquitoes.
复制标题
DOI：
10.1016/j.ibmb.2018.12.010
复制
发表时间：
2019-01
影响因子：
3.8
通讯作者：
N. Petchampai;Claribel Murillo-Solano;J. Isoe;J. Pizarro;P. Y. Scaraffia
中科院分区：
文献类型：
--
作者：
N. Petchampai;Claribel Murillo-Solano;J. Isoe;J. Pizarro;P. Y. Scaraffia
雌性埃及伊蚊是虫媒病毒的载体，可引起具有公共卫生意义的疾病。新的代谢靶点的发现对于改善蚊虫控制策略至关重要。我们最近证明了葡萄糖氧化支持氨解毒。蚊。丙酮酸激酶（Pyruvate kinase， PK, EC 2.7.1.40）催化糖酵解途径的最后一步。在大多数生物体中，一种或多种变构效应物控制PK活性。然而，蚊子体内PK的动力学性质和结构尚未见报道。在这项研究中，在theA中发现了两个编码PKs的可选剪接mRNA变体（AaPK1和AaPK2）。aegyptigenome。AaPK1 mRNA变体编码一个529个氨基酸的蛋白，估计分子量为~ 57 kDa。该蛋白在大肠杆菌中表达并纯化。鉴定了AaPK1的动力学性质。重组蛋白也被结晶并确定其三维结构。我们发现丙氨酸、谷氨酰胺、脯氨酸、丝氨酸和果糖-1-磷酸对AaPK1表现出典型的变构激活。核酮糖-5-磷酸作为AaPK1的变构抑制剂，但其抑制作用被丙氨酸、谷氨酰胺、脯氨酸和丝氨酸逆转。此外，果糖-1,6-二磷酸能减弱氨基酸对AaPK1的变构激活，而葡萄糖-6-磷酸能减弱丙氨酸和丝氨酸对AaPK1的变构激活。AaPK1结构显示在变构位点存在果糖-1,6-二磷酸。总之，我们的研究结果表明，特定的氨基酸和磷酸化糖密切调节着AaPK1的构象动力学和催化变化。独特的AaPK1变构特性支持该酶在蚊子代谢中的复杂作用。
FemaleAedes aegyptimosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification inA. aegypti.Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in theA. aegyptigenome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of ∼57 kDa, was cloned. The protein was expressed inEscherichia coliand purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism.