Effect of domain architecture and non-native F-type lectin domains on a <i>Streptosporangium roseum</i> α-L-fucosidase

Microbial carbohydrate-active enzymes frequently have tandem carbohydrate-binding modules that enable enhanced enzyme activity on carbohydrates via targeting and proximity effects. CBM47 is a family of L-fucose-binding F-type Lectin Domains (FLDs) found in proteins with diverse domain architectures and possible roles in directing biological functions to fucosylated niches. In one such FLD-containing protein, Streptosporangium roseum α-L-fucosidase (SrFucNaFLD), the FLD enhances the enzyme activity of the tandem-positioned α-L-fucosidase domain for small, aqueous, freely diffusible, fucosylated oligosaccharides. Here, we performed domain engineering experiments on SrFucNaFLD to dissect and understand the spatial role of the domains on α-L-fucosidase activity. We found that the central NPCBM-associated (Na) domain was dispensable for optimal enhancement of α-L-fucosidase activity; however, the N-terminal to C-terminal domain order was critical, perhaps because it altered the relative spatial orientation of the α-L-fucosidase domain and the FLD, thereby affecting substrate access. We also explored the effect of replacing the native FLD on this protein with non-native FLDs (with different α-L-fucoside binding profiles) from different organisms. We found no enhancement of α-L-fucosidase activity towards the natural oligosaccharide substrates, Lewis a tetraose, H type-2 triaose, and H type-2 tetraose, by non-native FLDs other than Actinomyces turicensis FLD, which incidentally also exists in tandem with an α-L-fucosidase domain in the native context. Our results suggest that the S. roseum α-L-fucosidase is a well-optimized system with fine-tuned substrate channelling from the FLD to the tandem α-L-fucosidase domain. Our study has implications for future engineering studies of carbohydrate-active enzymes.