文章基本信息

标题：Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)
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作者：Martín Hugo ; Alejandra Martínez ; Madia Trujillo 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：8
页码：E1326-E1335
DOI：10.1073/pnas.1618611114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [ Tc APx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H₂O₂ ( k = 2.9 × 10⁷ M⁻¹⋅s⁻¹) and catalytically decomposes H₂O₂ using Cc as the reducing substrate with higher efficiency than Asc ( k _cat/ K _m = 2.1 × 10⁵ versus 3.5 × 10⁴ M⁻¹⋅s⁻¹, respectively). Visible-absorption spectra of purified recombinant Tc APx-CcP after H₂O₂ reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp^233•+). Mutation of Trp²³³ to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp^233•+, a Cys²²²-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H₂O₂ addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp²³³ and Cys²²² is possible and likely to participate in the catalytic cycle. Recognizing the ability of Tc APx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). Tc APx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host–parasite interface. Tc APx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of Tc APx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.
关键词：Trypanosoma cruzi ; heme peroxidase ; oxidants ; virulence ; kinetics