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Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates.

ABSTRACT: Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one-pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate-binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one-pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.

SUBMITTER: Barad BA

PROVIDER: S-EPMC7096708 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates.

Barad Benjamin A BA Liu Lin L Diaz Roberto E RE Basilio Ralp R Van Dyken Steven J SJ Locksley Richard M RM Fraser James S JS

Protein science : a publication of the Protein Society 20200121 4

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mo ...[more]

PMID: 31930591

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Project description:BackgroundThe biodegradation of chitin is an important part of the carbon and nitrogen cycles in nature. Speeding up the biotransformation of chitin substrates can not only reduce pollution, but also produce high value-added products. However, this process is strictly regulated by the catalytic efficiency of the chitinolytic machinery. Therefore, it is necessary to study the mode of action and compound mechanisms of different chitin-degrading enzymes in depth to improve the catalytic efficiency of the chitinolytic machinery.ResultsThe thermophilic bacterium Streptomyces sp. F-3 showed comparatively high chitin degradation activities. To elucidate the mechanism underlying chitin hydrolysis, six chitin degradation-related enzymes were identified in the extracellular proteome of Streptomyces sp. F-3, including three chitinases (SsChi18A, SsChi18B, and SsChi18C) from the GH18 family, one GH19 chitinase (SsChi19A), one GH20 β-N-acetylhexosaminidase (SsGH20A), and one lytic polysaccharide monooxygenase (SsLPMO10A) from the AA10 family. All were upregulated by chitin. The heterologously expressed hydrolases could withstand temperatures up to 70 °C and were stable at pH values of 4 to 11. Biochemical analyses displayed that these chitin degradation-related enzymes had different functions and thus showed synergistic effects during chitin degradation. Furthermore, based on structural bioinformatics data, we speculated that the different action modes among the three GH18 chitinases may be caused by loop differences in their active site architectures. Among them, SsChi18A is probably processive and mainly acts on polysaccharides, while SsChi18B and SsChi18C are likely endo-non-processive and displayed higher activity on the degradation of chitin oligosaccharides. In addition, proteomic data and synergy experiments also indicated the importance of SsLPMO10A, which could promote the activities of the hydrolases and increase the monosaccharide content in the reaction system, respectively.ConclusionsIn this article, the chitinolytic machinery of a thermophilic Streptomyces species was studied to explore the structural basis for the synergistic actions of chitinases from different GH18 subfamilies. The elucidation of the degradation mechanisms of these thermophilic chitinases will lay a theoretical foundation for the efficient industrialized transformation of natural chitin.

Dataset Information

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates.

Publications

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates.

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