Plasmodium ookinete-secreted chitinase and parasite penetration of the mosquito peritrophic matrix

Langer, Rebecca C.; Vinetz, Joseph M.

doi:10.1016/s1471-4922(01)01918-3

Cited by 60 publications

(48 citation statements)

References 19 publications

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“…Studies with soluble oligosaccharide substrates showed that the W97A mutant hydrolyzes (GlcNAc) 4 (data not shown) and (GlcNAc) 6 (Figs. 3 C and D) Ϸ4-fold faster than the wild-type enzyme, the initial specific activities for (GlcNAc) 6 degradation being 33 s Ϫ1 and 131 s Ϫ1 for ChiB wild-type and W97A, respectively.…”

Section: Resultsmentioning

confidence: 95%

Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides

Horn

Sikorski²,

Cederkvist

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

253

306

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Many enzymes that hydrolyze insoluble crystalline polysaccharides such as cellulose and chitin guide detached single-polymer chains through long and deep active-site clefts, leading to processive (stepwise) degradation of the polysaccharide. We have studied the links between enzyme efficiency and processivity by analyzing the effects of mutating aromatic residues in the substrate-binding groove of a processive chitobiohydrolase, chitinase B from Serratia marcescens. Mutation of two tryptophan residues (Trp-97 and Trp-220) close to the catalytic center (subsites ؉1 and ؉2) led to reduced processivity and a reduced ability to degrade crystalline chitin, suggesting that these two properties are linked. Most remarkably, the loss of processivity in the W97A mutant was accompanied by a 29-fold increase in the degradation rate for single-polymer chains as present in the soluble chitin-derivative chitosan. The properties of the W220A mutant showed a similar trend, although mutational effects were less dramatic. Processivity is thought to contribute to the degradation of crystalline polysaccharides because detached single-polymer chains are kept from reassociating with the solid material. The present results show that this processivity comes at a large cost in terms of enzyme speed. Thus, in some cases, it might be better to focus strategies for enzymatic depolymerization of polysaccharide biomass on improving substrate accessibility for nonprocessive enzymes rather than on improving the properties of processive enzymes.cellulose ͉ chitin ͉ chitinase ͉ chitosan ͉ processivity

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Section: Resultsmentioning

confidence: 95%

Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides

Horn

Sikorski²,

Cederkvist

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

253

306

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“…Screening of the Sigma Library of Pharmacologically Active Compounds identified nine hits of 1,280. These were subjected to further screening using the DMAB assay, which measures the amount of amino sugars (here only N-acetylhexosamines) liberated from the substrate GlcNAc 6 . Two of the initial hits were not confirmed, because their IC 50 values were above 1 mM.…”

Section: Resultsmentioning

confidence: 99%

“…The interactions of all inhibitors with the VhChiA are unique and entirely different from the interactions of the enzyme with the GlcNAc 6 substrate; the inhibitors are accommodated in the active site of the enzyme largely through hydrophobic interactions, and only very few potential hydrogen bonds were observed. The detailed interactions between the enzyme, the substrate GlcNAc 6 , and the inhibitor DEQ as representative example are depicted in the supplemental Fig. S1.…”

Section: Resultsmentioning

confidence: 99%

“…S1. This "Ligplot" representation illustrates the extended structure of GlcNAc 6 , exhibiting a number of hydrogen bonds and hydrophobic interactions with various side chains that surround the multiple binding sites. In marked contrast, the contacts with the inhibitors, such as DEQ, are made up almost exclusively of hydrophobic interactions.…”

Section: Resultsmentioning

confidence: 99%

“…The cause of malaria is the Plasmodium parasite. During the reproductive stage of their complex life cycles, the zygotes (so-called ookinetes) produce significant levels of chitinases to penetrate the chitin-containing wall of the mosquito midgut (6). This stage of development is crucial because it allows the parasites to be repetitively transmitted from mosquitoes to humans.…”

mentioning

confidence: 99%

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Potent Family-18 Chitinase Inhibitors

Pantoom

Vetter

Prinz

et al. 2011

Journal of Biological Chemistry

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Structure–function analysis of cysteine residues in the plasmodium falciparum chitinase, PfCHT1

et al. 2022

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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 chitinbinding 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 humaninfecting 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.

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Plasmodium ookinete-secreted chitinase and parasite penetration of the mosquito peritrophic matrix

Cited by 60 publications

References 19 publications

Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides

Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides

Potent Family-18 Chitinase Inhibitors

Structure–function analysis of cysteine residues in the plasmodium falciparum chitinase, PfCHT1

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