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

Abstract 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.