Structural investigation of Fun168A unraveling the recognition mechanism of endo-1,3-fucanase towards sulfated fucan.

ABSTRACT

BACKGROUND: Sulfated fucan has gained interest due to its various physiological activities. Endo-1,3-fucanases are valuable tools for investigating the structure and establishing structure-activity relationships of sulfated fucan. However, the substrate recognition mechanism of endo-1,3-fucanases towards sulfated fucan remains unclear, limiting the application of endo-1,3-fucanases in sulfated fucan research. SCOPE AND APPROACH: This study presented the first crystal structure of endo-1,3-fucanase (Fun168A) and its complex with the tetrasaccharide product, utilizing X-ray diffraction techniques. The novel subsite specificity of Fun168A was identified through glycomics and nuclear magnetic resonance (NMR). KEY FINDINGS AND CONCLUSIONS: The structure of Fun168A was determined at 1.92 Å. Residues D206 and E264 acted as the nucleophile and general acid/base, respectively. Notably, Fun168A strategically positioned a series of polar residues at the subsites ranging from -2 to +3, enabling interactions with the sulfate groups of sulfated fucan through salt bridges or hydrogen bonds. Based on the structure of Fun168A and its substrate recognition mechanisms, the novel subsite specificities at the -2 and +2 subsites of Fun168A were identified. Overall, this study provided insight into the structure and substrate recognition mechanism of endo-1,3-fucanase for the first time and offered a valuable tool for further research and development of sulfated fucan.