Structural Insights into the Substrate Recognition\nand Catalytic Mechanism of a GH16 βκ-Carrageenase from <i>Wenyingzhuangia fucanilytica</i>

Understanding\nthe substrate specificity of carrageenases has long\nbeen of interest in biotechnology applications. So far, the structural\nbasis of the βκ-carrageenase that hydrolyzes furcellaran,\na major hybrid carrageenan, remains unclear. Here, the crystal structure\nof Cgbk16A_Wf, as a representative of the βκ-carrageenase\nfrom GH16_13, was determined, and the structural characteristics of\nthis subfamily were elucidated for the first time. The substrate binding\nmode was clarified through a structure analysis of the hexasaccharide-bound\ncomplex and molecular docking. The binding pocket involves a conserved\ncatalytic motif and several specific residues associated with substrate\nrecognition. Functions of residues R88, E290, and E184 were validated\nthrough site-directed mutagenesis. Comparing βκ-carrageenase\nwith κ-carrageenase, we proposed that their different substrate\nspecificities are partly due to the distinct conformations of subsite\n−1. This research offers a comprehensive understanding of the\nrecognition mechanism of carrageenases and provides valuable theoretical\nsupport for enzyme modification and carrageenan oligosaccharide preparation.