Structure of a prophenoloxidase (PPO) from Anopheles gambiae provides new insights into the mechanism of PPO activation
Date
2016-01-05Author
Hu, Yingxia
Wang, Yang
Deng, Junpeng
Jiang, Haobo
Metadata
Show full item recordAbstract
Background: 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. Results: Herein, we report the crystal structure of PPO8 from A. gambiae at 2.6 A 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'. Conclusions: 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.
Citation
Hu, Y., Wang, Y., Deng, J., & Jiang, H. (2016). The structure of a prophenoloxidase (PPO) from Anopheles gambiae provides new insights into the mechanism of PPO activation. BMC Biology, 14, Article 2. https://doi.org/10.1186/s12915-015-0225-2