Semi-rational Engineering of Xylulokinase from Lactococcus lactis N8 for Enhanced Catalytic Efficiency via Synergistic Hotspot Mutations.

Efficient xylose bioconversion is limited by the catalytic performance of xylulokinase (XylB), which phosphorylates d-xylulose to xylulose-5-phosphate. This study aimed to improve XylB from Lactococcus lactis N8 using a multi-dimensional semi-rational design strategy integrating sequence conservation, structure-based screening, mutagenesis, molecular docking, and molecular dynamics simulations. Asn301 was identified as a key functional hotspot, and Met259 was found to modulate the active-site hydrophobic environment. Combinatorial mutagenesis generated two superior variants, XylBM259H/N301G and XylBM259H/N301H, with more than 200% higher specific activity than the wild-type XylB (XylBWT) while retaining stable expression and environmental adaptability. Mechanistic analyses showed strengthened ATP binding, improved d-xylulose positioning, enhanced ligand affinity, and balanced local flexibility with structural compactness. These findings provide efficient XylB variants and a transferable strategy for engineering lignocellulose-related enzymes.