%0 Journal Article
%A Shivangi, s
%A Sandhu, Padmani
%A Kumar, Paras
%A Mahajan, Sonal
%A Subramanian, Srikrishna
%A Ramya, T. N. C.
%C JOURNALS DEPT, 2001 EVANS RD, CARY, NC 27513 USA
%D 2026
%F open:3454
%I OXFORD UNIV PRESS INC
%J GLYCOBIOLOGY
%K carbohydrate binding module; domain engineering; F-type lectin; Fucolectin; Fucosidase
%N 3
%T Effect of domain architecture and non-native F-type lectin domains on a Streptosporangium roseum α-L-fucosidase
%U http://crdd.osdd.net/open/3454/
%V 36
%X 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 alpha-L-fucosidase (SrFucNaFLD), the FLD enhances the enzyme activity of the tandem-positioned alpha-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 alpha-L-fucosidase activity. We found that the central NPCBM-associated (Na) domain was dispensable for optimal enhancement of alpha-L-fucosidase activity; however, the N-terminal to C-terminal domain order was critical, perhaps because it altered the relative spatial orientation of the alpha-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 alpha-L-fucoside binding profiles) from different organisms. We found no enhancement of alpha-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 alpha-L-fucosidase domain in the native context. Our results suggest that the S. roseum alpha-L-fucosidase is a well-optimized system with fine-tuned substrate channelling from the FLD to the tandem alpha-L-fucosidase domain. Our study has implications for future engineering studies of carbohydrate-active enzymes.
%Z Copyright of this Article belongs to OXFORD UNIV PRESS INC