Abstract
Carbohydrate-binding modules (CBMs) function independently to assist carbohydrate-active enzymes. Family 21 CBMs contain ~100 amino acid residues, and some members have starch-binding functions or glycogen-binding activities. We report here the first structure of a family 21 CBM from the starch binding domain (SBD) of Rhizopus oryzae glucoamylase (RoCBM21) determined by nuclear magnetic resonance spectroscopy. This CBM has a □-sandwich fold with an immunoglobulin-like structure. Ligand-binding properties of RoCBM21 were analyzed by chemical shift perturbations and automated docking. Structural comparisons with previously reported SBDs revealed two types of topologies, type I and type II, with CBM20, CBM25, CBM26, and CBM41 belonging to the type I topology group, whereas CBM21 and CBM34 belonging type II topology. According to the chemical shift perturbations, RoCBM21 contains two ligand-binding sites—as do most SBDs. Residues in site II are similar to those found in the family 20 CBM from Aspergillus niger glucoamylase (AnCBM20). Site I, however, is enclosed by two flexible asparagine-rich loops that are unique and only found in some members of CBM21s. Additionally, docking of a large amylose molecule (comprising 26 glucose residues) into RoCBM21 highlights possible mechanism that SBDs may interact with the structured portion of starch. The structure of RoCBM21 helps us characterize the structural features of SBDs and to understand the basis of protein-carbohydrate recognitions.