Structure-Guided Engineering of Xylitol 4-dehydrogenase Unlocks Efficient L-Xylulose Biosynthesis: Mechanistic Insights into Catalytic Enhancement.

L-Xylulose, a potent α-glucosidase inhibitor and indispensable chiral precursor for L-nucleoside drugs, is highly valuable. However, chemical synthesis often suffers from poor atom economy and tedious protection and deprotection steps of functional groups. Here, a green biocatalytic route was explored by retrieving five XDH4 genes and selecting AYXDH4 as the best candidate. Through homology modeling and site-directed mutagenesis targeting potential synergy sites in the α-helix, the double mutant T171A/S172A was generated, which exhibited 43.97% higher catalytic efficiency (kcat/Km) than the wild type, alongside an increase in optimal temperature from 35 °C to 40 °C. Structural analysis revealed that flexibility modulation of an adjacent α-helix triggers hydrogen bond rearrangements, thereby enhancing substrate binding and hydride transfer. Coupling with NADH oxidase regenerates NAD+ in situ, eliminating cofactor addition and producing only water. The system produced 6.61 ± 0.52 g/L L-xylulose, 1.9-fold higher than the wild type, demonstrating a green, atom-economic platform for L-xylulose production.