Functional analyses of plasmodium ferredoxin Asp97Tyr mutant related to artemisinin resistance of human malaria parasites
Functional analyses of plasmodium ferredoxin Asp97Tyr mutant related to artemisinin resistance of human malaria parasites
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DOI：
10.1093/jb/mvab070
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发表时间：
2021-08-20
影响因子：
2.7
通讯作者：
Morihisa, Rena
中科院分区：
文献类型：
--
作者：
Kimata-Ariga, Yoko;Morihisa, Rena
疟疾疟原虫顶质体铁氧还蛋白（PfFd）C端Asp 97 Tyr突变与疟原虫对一线抗疟药青蒿素的耐药性密切相关。我们以前发现PfFd C端的芳香族氨基酸在与其电子传递伙伴Fd-NADP（+）还原酶（PfFNR）相互作用中起重要作用。在这里，PfFd和PfFNR之间的芳香族-芳香族相互作用的重要性被示出使用PfFNR的定点突变体与PfFd的电子转移反应的动力学分析。通过色氨酸荧光滴定法测定PfFd的解离常数（K-d）和PfFNR动力学分析中的米氏常数（K-m），进一步表明PfFd的Asp 97 Tyr突变增加了PfFNR的亲和力。与野生型PfFd相比，D97 Y PfFd在较低浓度下抑制PfFNR的心肌黄酶活性。抗坏血酸自由基清除活性的PfFd和电子转移活性的异质性FD依赖性酶与D97 Y PfFd低于野生型PfFd。这些结果表明PfFd的D97 Y突变体与PfFNR的结合比野生型PfFd更紧密，因此可能抑制PfFNR的功能，这可能与青蒿素的作用有关。
Mutation of Asp97Tyr in the C-terminal region of ferredoxin (PfFd) in the apicoplast of malaria parasites was recently reported to be strongly related to the parasite's resistance to the frontline antimalarial drug, artemisinin We previously showed that the aromatic amino acid in the C-terminal region of PfFd is important for the interaction with its electron transfer partner, Fd-NADP(+) reductase (PfFNR). Here, the importance of the aromatic-aromatic interaction between PfFd and PfFNR was shown using the kinetic analysis of the electron transfer reaction of site-directed mutants of PfFNR with PfFd. Mutation of Asp97Tyr of PfFd was further shown to increase the affinity with PfFNR by the measurements of the dissociation constant (K-d) using tryptophan fluorescence titration and the Michaelis constant (K-m) in the kinetic analysis with PfFNRs. Diaphorase activity of PfFNR was inhibited by D97Y PfFd at lower concentration as compared to wild-type PfFd. Ascorbate radical scavenging activity of PfFd and electron transfer activity to a heterogeneous Fd-dependent enzyme was lower with D97Y PfFd than that of wild-type PfFd. These results showed that D97Y mutant of PfFd binds to PfFNR tighter than wild-type PfFd, and thus may suppress the function of PfFNR which could be associated with the action of artemisinin.