Cyclization Decoded: Engineering Amylomaltase for Efficient α-Glucan Transformations

α-Glucan, broadly distributed in nature or produced artificially by biotransformation, is fundamental to life. Amylomaltases (AMs) can modulate the biochemical properties of α-glucan through a distinctive cyclization feature to form α-1,4-glucoside-linked large-ring polysaccharides (cycloamyloses; CAs), endowing α-glucan with significant health benefits and medical value. While the industrial application of CAs continues to progress, the mechanistic intricacies of the cyclization process─and its nuanced interplay with hydrolysis─remain only partially understood. Here, we harness large-scale computations in synergy with biochemical experiments to unravel, at atomic resolution, the full covalent and noncovalent catalytic mechanism of AMs, revealing that cyclization (12.8 kcal/mol) outcompetes hydrolysis (17.5 kcal/mol) as the dominant pathway. Furthermore, we identify that postglycosylation noncovalent polysaccharide chain transfer emerges as the decisive factor in cyclization, particularly for AM-degree of polymerization (DP) greater than 30 substrate complexes, where this noncovalent step (≥13.6 kcal/mol) dictates the rate of CA production. 57 variants with enhanced activity (up to a 2.3-fold increase) were engineered in our biochemical experiments by strategically modulating the chain transfer step. Enzyme kinetics results suggest that the improvement in enzyme performance largely stems from the decrease in enzyme-substrate affinity along the substrate transfer pathway. Additionally, through mass spectrometry, CAs with DP ranging from 22 to 61 were detected, confirming our theoretical results. We also quantify the kinetic competitive balance between cyclization and hydrolysis, providing clear engineering blueprints for the AM family. This work delivers a systematic, molecular-level understanding of CA biosynthesis from an industrial-performance perspective, paving the way for next-generation CA innovations.