The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of α-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 Å (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 (ΔCBM48) 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.

As the second largest production material, starch has important value in textile, food, chemical and other fields. The shortcomings of natural starch can be solved, and its properties can be improved by modifying its structure, developing original properties, or introducing new functions, making it more suitable for certain application requirements. At present, the methods of starch modification mainly include chemical, physical, and enzymatic modification. In comparison with the two traditional modification methods (chemical and physical modification) mentioned above, enzymatic modification has the advantages of mild conditions, high substrate selectivity, and high product safety, and it is the most ideal green modification method. In this paper, we present an overview of the modified starch by enzymatic structure design. The modification process and mechanism for granule starch and gelatinized starch are summarized. Further, the difficulties encountered in starch modification by enzymatic modification were also analyzed. These analyses could pave a way for understanding and broadening the preparation and applications of modified starch, and provide theoretical references for the utilization of amylase in starch modification.

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