Discovery and Structural Analysis of a Novel Pectin-Specific Carbohydrate-Binding Module: The First Member of a New CBM Family

Pectin is a biologically and commercially valuable polysaccharide with broad applications. Carbohydrate-binding modules (CBMs) are independent domains associated with carbohydrate-active enzymes, serving as versatile tools for specific carbohydrate recognition. In this study, a CBM was identified within a putative pectin lyase sequence. This CBM specifically binds pectin, with its association constants (K_a) for homogalacturonan and rhamnogalacturonan-I measured using affinity electrophoresis as 1.7 ± 0.5 × 10⁶ M^-1 and 6.3 ± 0.2 × 10³ M^-1, respectively. The 2.00 Å resolution crystal structure of this CBM revealed a β-sandwich fold of two antiparallel β-sheets comprising 13 β-strands. The binding site was identified in the loop region, and molecular docking suggested that basic residues play a key role in binding. Its recombinant protein with Emerald Green Fluorescent Protein (EmGFP) enabled fluorescent staining and microscopic observation of pectin in diverse plants including carrot, citrus peel, okra, and pea, demonstrating its ability to bind pectin in the food matrix and its usefulness for in situ visualization. This CBM shows no homology to characterized proteins, proposing the existence of a new CBM family. This study enriches the specific recognition elements for pectin and would contribute to the structural annotation and functional understanding of multidomain pectin lyases.