Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized Limosilactobacillus reuteri N1 GtfB Enzyme

alpha-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important alpha-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-alpha-glucanotransferase of high enzyme activity (60.8 U mg(-1)) from Limosilactobacillus reuteri N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like alpha-glucan with (alpha 1 -> 6) linkages in linear chains and branch points. Protein structural analysis of LrN1 GtfB revealed the potential crucial residues at subsites -2 similar to+2, particularly H265, Y214, and R302, in the active center as well as previously unidentified surface binding sites. Furthermore, molecular dynamic simulations have provided unprecedented insights into linkage specificity hallmarks of the enzyme. Therefore, LrN1 GtfB represents a potent enzymatic tool for starch conversion, and this study promotes our knowledge on the structure-function relationship of GH70 GtfB alpha-glucanotransferases, which might facilitate the production of tailored alpha-glucans by enzyme engineering in future.