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.

Keywords: chitin; directed evolution; enzymes; glycobiology.

Figure 1

Engineering of hyperactive AMCase mutants. (a) Workflow for directed evolution of AMCase. Mutants of AMCase were generated via error‐prone PCR, then transformed and grown out from individual colonies in 96‐well blocks. After expression, activity was measured using the 4MU‐chitobioside substrate incubated with the expression media. (b) Distribution of activity for mutants with 1–3 mutations per construct. Vertical lines at 0 and 1 represent a catalytically dead negative control and a wild type positive control, respectively. The best two results are highlighted in purple and orange. (c) k _cat/K _m of purified hyperactive mutants using the 4MU‐chitobioside assay (d) Structure of AMCase catalytic domain (PDB 3RM9) highlighting A239T/L364Q (pink) and V246A (orange). The active site catalytic network is highlighted in teal, and an inhibitor (5‐(4‐(2‐[4‐bromophenoxy]ethyl)piperazine‐1‐yl)‐1H‐1,2,4‐triazol‐3‐amine)21 that binds to the active site cleft is shown in red

Figure 2

Activity comparisons of AMCase catalytic domain and full length enzyme. Difference in k _cat, K _m, and k _cat/K _m of AMCase catalytic domain and full length enzyme generated via (a) 4MU‐chitobioside assay, (b) colloidal chitin clearance assay, (c) reducing sugar generation assay quantified with potassium ferricyanide, (d) chitooligosaccharide oxidase coupled peroxidase assay. Error bars denote propagated SD of fit (accounting for covariance)

Figure 3

Engineered mutant activity with the novel chitooligosaccharide oxidase assay. Difference in k _cat/K _m of purified hyperactive mutants using the 4MU‐chitobioside assay. k _cat values are reported in units of 1/s. K _m values are reported in units % w/v. Error bars denote propagated SD of fit (accounting for covariance). The A239T/L364Q mutant had too little total activity to measure k _cat or K _m

Figure 4

Comparison of activity of AMCase asthma‐associated mutants. Measurement of k _cat/K _m for reversed asthma‐associated mutants in the mouse background using the (a) 4MU‐chitobioside and (b) chitO assays. Error bars denote propagated SD of fit (accounting for covariance)

Figure 5

Comparison of AMCase and Chitotriosidase. Differences in k _cat/K _m between AMCase (blue) and chitotriosidase (magenta) using (a) 4MU‐chitobioside, (b) 4MU‐chitotrioside, (c) chitooligosaccharide oxidase coupled peroxidase assay. Error bars denote propagated SD of fit (accounting for covariance)