Exploring the effect of tandem non-native non-F-type lectin carbohydrate-binding domains on Streptosporangium roseum α-L-Fucosidase activity.

Microbial carbohydrate-active enzymes often contain non-catalytic carbohydrate-binding modules, which enhance the activity of the catalytic domain by proximity, targeting, or disruptive effects. We have previously biochemically characterized a GH29 α-L-fucosidase from Streptosporangium roseum, demonstrated that its C-terminal F-type lectin domain (FLD) improves the enzyme activity of its tandem N-terminal α-L-fucosidase domain towards fucooligosaccharides, and proposed a "bind and jump" model to explain this FLD-driven improvement of α-L-fucosidase activity. Here, we have explored the use of non-native, non-FLD carbohydrate-binding domains to similarly enhance the enzymatic activity of the α-L-fucosidase domain. We employed three carbohydrate-binding domains (MG1D1, MN3α, and MU1α) that we previously identified from the gut microbial metagenome by functional screening, and fused them to the N-terminus of the S. roseum α-L-fucosidase domain. The constructs showed activity on a synthetic substrate, 4-Methylumbelliferyl-α-L-fucopyranoside. However, when assaying these constructs on the natural fucosylated oligosaccharides, Lewis a tetrasaccharide and 2'-fucosyllactose, we did not observe a statistically significant improvement in the α-L-fucosidase activity of MN3α-SrFuc, MG1D1-SrFuc, or MU1α-SrFuc, compared to the SrFuc construct containing only the α-L-fucosidase domain. Our results indicate that the tandem positioning of these non-native, non-FLD carbohydrate-binding domains does not significantly improve the α-L-fucosidase activity of SrFuc towards these oligosaccharides. Additional chimeric constructs with domain organizations designed to closely mimic the wild-type protein SrFucNaFLD, and further studies focused on the mechanistic basis of the FLD-mediated improvement of α-L-fucosidase activity, might help translate the strategy and design chimeric α-L-fucosidases with non-native, non-FLD carbohydrate-binding domains that exhibit improved α-L-fucosidase activity towards these oligosaccharides.