Structural Determination and Functional Residues Analysis of a CBM99 Family Carbohydrate-Binding Module Targeting Porphyran

Porphyran is a bioactive polysaccharide extensively distributed in algae of the genus <i>Porphyra</i>. Carbohydrate-binding modules (CBMs) are independent domains often found in carbohydrate-active enzymes that function to bind carbohydrates and have various applications. Only one porphyran-binding CBM has been hitherto structurally characterized. The founding member (FvCBM99) of the CBM99 family was previously shown to exhibit a specific binding capacity to the primary constituent units of porphyran. In this study, the structure of FvCBM99 was determined at 1.75 Å resolution by X-ray crystallography. The protein adopts an overall β-sandwich fold with two antiparallel β-sheets comprising 7 β-strands. Site-directed mutagenesis analysis confirmed that residues W44, W49, K83, R87, and W93 are indispensable for the interaction of FvCBM99 with porphyran. The work delivers the first structural insights into the CBM99 family, which can guide the practical applications of FvCBM99 and promote the future discovery and characterization of porphyran-binding proteins.