Relief of Xylose Binding to Cellobiose Phosphorylase by a Single Distal Mutation

Cellobiose phosphorylase (CBP) cleaves cellobiose-abundant in plant biomass-to glucose and glucose 1-phosphate. However, the pentose sugar xylose, also abundant in plant biomass, acts as a mixed-inhibitor and a substrate for the reverse reaction, limiting the industrial potential of CBP. Preventing xylose, which lacks only a single hydroxymethyl group relative to glucose, from binding to the CBP active site poses a spatial challenge for protein engineering, since simple steric occlusion cannot be used to block xylose binding without also preventing glucose binding. Using CRISPR-based chromosomal library selection, we identified a distal mutation in CBP, Y47H, responsible for improved cellobiose consumption in the presence of xylose. In silico analysis suggests this mutation may alter the conformation of the cellobiose phosphorylase dimer complex to reduce xylose binding to the active site. These results may aid in engineering carbohydrate phosphorylases for improved specificity in biofuel production, and also in the production of industrially important oligosaccharides.