Flexible Loop in Carbohydrate-Binding Module 48 Allosterically Modulates Substrate Binding of the 1,4-α-Glucan Branching Enzyme

The 1,4-alpha-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of alpha-1,6 branching points in starch and plays a key role in synthesis. To obtain mechanistic insights into the catalytic action of the enzyme, we first determined the crystal structure of GBE from Rhodothermus obamensis STB05 (RoGBE) to a resolution of 2.39 angstrom (PDB ID: 6JOY). The structure consists of three domains: domain A, domain C, and the carbohydrate-binding module 48 (CBM48). An engineered truncated mutant lacking the CBM48 domain (Delta CBM48) showed significantly reduced ligand binding affinity and enzyme activity. Comparison of the structures of RoGBE with other GBEs showed that CBM48 of RoGBE had a longer flexible loop. Truncation of the flexible loops resulted in reduced binding affinity and activity, thereby substantiating the importance of the optimum loop structure for catalysis. In essence, our study shows that CBM48, especially the flexible loop, plays an important role in substrate binding and enzymatic activity of RoGBE. Further, based on the structural analysis, kinetics, and activity assays on wild type and mutants, as well as homology modeling, we proposed a mechanistic model (called the lid model) to illustrate how the flexible loop triggers substrate binding, ultimately leading to catalysis.

Automated summary

The study focused on the crystal structure analysis of the GBE enzyme, and found that the CBM48 domain plays a crucial role in substrate binding and enzymatic activity of RoGBE. Comparison of RoGBE with other GBEs revealed that CBM48 of RoGBE has a longer flexible loop, and truncation of this loop resulted in reduced enzyme activity.