Structure-Guided Tunnel Engineering of Rhamnosyltransferase Cm1,2RhaT: Mechanistic Insights and Enhanced Catalytic...

Neohesperidin, the key precursor of the high-intensity sweetener neohesperidin dihydrochalcone, is severely limited in biosynthesis by the need for costly sugar donors and high-efficiency rhamnosyltransferases. A deep mechanistic understanding is crucial for boosting rhamnosyltransferase catalytic efficiency, but the absence of structural information has long been a bottleneck. Herein, the access transport tunnels for uridine diphosphate (UDP) rhamnose in Cm1,2RhaT were unveiled using dynamics simulations, and a highly active mutant was obtained via substrate tunnel engineering. We identified the critical residues in the UDP-rhamnose access tunnel and engineered the S50A mutant, which exhibits a 1.68-fold higher catalytic efficiency than the wild type toward UDP-rhamnose. Structural analyses showed that increased loop flexibility shortened the tunnel length, favoring substrate entry. This study provided insights into UDP-sugar binding mechanisms and offered a general strategy for engineering UDP-dependent glycosyltransferases to enhance catalytic performance.