The structure of a prophenoloxidase (PPO) from Anopheles gambiae provides new insights into the mechanism of PPO activation.
The structure of a prophenoloxidase (PPO) from Anopheles gambiae provides new insights into the mechanism of PPO activation.
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DOI：
10.1186/s12915-015-0225-2
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发表时间：
2016-01-05
期刊：
影响因子：
5.4
通讯作者：
Jiang H
中科院分区：
文献类型：
--
作者：
Hu Y;Wang Y;Deng J;Jiang H
酚氧化酶（PO）催化的黑化是昆虫抵御病原和寄生虫感染的普遍防御机制。在冈比亚按蚊（Anopheles gambiae）等蚊子中，黑色素包膜是一种抵抗某些引起疟疾和丝虫病的寄生虫的机制。PO最初由血细胞合成，并作为无活性的酚氧化酶（PPO）释放到血淋巴中，在识别外来入侵者时被丝氨酸蛋白酶级联激活。PPO活化和催化的机制一直不清楚。本文报道了A. gambiae中PPO8在2.6 Å分辨率下的晶体结构。PPO8形成同二聚体，每个亚基显示经典的III型二铜活性中心。我们的分子对接和诱变研究发现了一个新的底物结合位点，Glu364作为催化残基负责单酚和二酚底物的去质子化。Glu364突变严重破坏了AgPPO8的单酚羟化酶和二酚氧化酶活性。我们的数据表明，新发现的底物结合口袋是催化的实际位点，PPO激活可以在不提取之前被认为是底物“占位符”的保守苯丙氨酸残基的情况下实现。我们介绍了蚊子PPO的结构和功能数据。我们的研究结果揭示了一个新的底物结合位点，Glu364被鉴定为PO酶活性的关键催化残基。我们的数据为分子水平上的PPO激活提供了一个新的模型，它不同于规范的机制，需要从先前认为的底物结合位点提取阻断苯丙氨酸残基。该研究为PPO的活化机制和酶催化提供了新的见解。本文的在线版本（doi:10.1186/s12915-015-0225-2）包含补充材料，可供授权用户使用。
Phenoloxidase (PO)-catalyzed melanization is a universal defense mechanism of insects against pathogenic and parasitic infections. In mosquitos such as Anopheles gambiae, melanotic encapsulation is a resistance mechanism against certain parasites that cause malaria and filariasis. PO is initially synthesized by hemocytes and released into hemolymph as inactive prophenoloxidase (PPO), which is activated by a serine protease cascade upon recognition of foreign invaders. The mechanisms of PPO activation and PO catalysis have been elusive. Herein, we report the crystal structure of PPO8 from A. gambiae at 2.6 Å resolution. PPO8 forms a homodimer with each subunit displaying a classical type III di-copper active center. Our molecular docking and mutagenesis studies revealed a new substrate-binding site with Glu364 as the catalytic residue responsible for the deprotonation of mono- and di-phenolic substrates. Mutation of Glu364 severely impaired both the monophenol hydroxylase and diphenoloxidase activities of AgPPO8. Our data suggested that the newly identified substrate-binding pocket is the actual site for catalysis, and PPO activation could be achieved without withdrawing the conserved phenylalanine residue that was previously deemed as the substrate ‘placeholder’. We present the structural and functional data from a mosquito PPO. Our results revealed a novel substrate-binding site with Glu364 identified as the key catalytic residue for PO enzymatic activities. Our data offered a new model for PPO activation at the molecular level, which differs from the canonical mechanism that demands withdrawing a blocking phenylalanine residue from the previously deemed substrate-binding site. This study provides new insights into the mechanisms of PPO activation and enzymatic catalysis of PO. The online version of this article (doi:10.1186/s12915-015-0225-2) contains supplementary material, which is available to authorized users.