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- W4224283325 abstract "The Plasmodium ookinete uses chitinase activity to penetrate the acellular, chitin-containing peritrophic matrix to invade the mosquito vector. Plasmodium ookinetes from different parasite clades secrete two structurally distinct forms of chitinase, one, a short form lacking a C-terminal putative chitin-binding domain (CBD), the other, a long form with both proenzyme and C-terminal putative chitin-binding domains. Here, we structurally and functionally characterize the three cysteines in the short chitinase of the human-infecting malaria parasite, P. falciparum testing the hypothesis that one unpaired cysteine would not contribute to chitinase-specific enzymatic activity which would identify this residue as potentially involved in intermolecular disulfide bonding and heteromultimeric invasion complex formation as previously described. To test this hypothesis, we produced and characterized recombinant wild-type and cysteine-mutation PfCHT1 proteins in E. coli and used biophysical and enzymatic approaches to examine their enzymatic activities and chitin-binding affinities. The cysteine-203 PfCHT1 mutation had no effect on chitinolytic and chitin-binding functions. The cysteine-220 and cysteine-230 mutants were enzymatically inactive and did not bind to chitin. The artificial intelligence-based protein prediction algorithm, AlphaFold, correctly identified the involvement of cys-220 and cys-230 in the intramolecular disulfide linkages key to maintaining properly folded chitinase structural integrity. AlphaFold predicted that cys-203 cysteine is surface exposed and thus involved in intermolecular protein-protein interaction. Production of the cys-to-ser 203 PfCHT1 mutant facilitated recombinant protein production. Future cellular and biochemical studies are needed to further understand details of Plasmodium ookinete mosquito midgut invasion." @default.
- W4224283325 created "2022-04-26" @default.
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- W4224283325 date "2022-04-09" @default.
- W4224283325 modified "2023-10-15" @default.
- W4224283325 title "Structure–function analysis of cysteine residues in the <i>plasmodium falciparum</i> chitinase, <scp>PfCHT1</scp>" @default.
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- W4224283325 doi "https://doi.org/10.1002/pro.4289" @default.
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