Abstact

The subunit interface plays a substantial role in the structures and functions of oligomeric enzymes, yet targeted mutations remain difficult to predict. Here, we targeted 2-keto-3-deoxy-d-xylonate dehydratase (CcXylX), the rate-limiting catalyst in the Weimberg pathway for d-xylose catabolism and a member of the fumarylacetoacetate hydrolase (FAH) family, which forms a compact homodimer. Guided by its crystal structure, we engineered the dimer interface and obtained triple mutant L210A/P181Q/Q308A, which showed a 6.04-fold increase in catalytic efficiency. Molecular dynamics simulations revealed that moderate enhancement of intersubunit flexibility accelerates substrate binding. When the mutant was coupled with other Weimberg enzymes in a one-pot process, 77% of d-xylose was converted to 56.05 +/- 0.39 g/L alpha-ketoglutaric acid within 6 h. Moreover, this strategy is also applicable to other dimeric enzymes within the FAH family. This study highlights a promising strategy for engineering dimeric enzymes with a higher catalytic efficiency for producing valuable chemicals.