Crystal Structure of Hypothetical Fructose-Specific EIIB from Escherichia coli.

We have solved the crystal structure of a predicted fructose-specific enzyme IIBfruc from Escherichia coli (EcEIIBfruc) involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system transferring carbohydrates across the cytoplasmic membrane. EcEIIBfruc belongs to a sequence family with more than 5,000 sequence homologues with 25–99% amino-acid sequence identity. It reveals a conventional Rossmann-like α-β-α sandwich fold with a unique β-sheet topology. Its C-terminus is longer than its closest relatives and forms an additional β-strand whereas the shorter C-terminus is random coil in the relatives. Interestingly, its core structure is similar to that of enzyme IIBcellobiose from E. coli (EcIIBcel) transferring a phosphate moiety. In the active site of the closest EcEIIBfruc homologues, a unique motif CXXGXAHT comprising a P-loop like architecture including a histidine residue is found. The conserved cysteine on this loop may be deprotonated to act as a nucleophile similar to that of EcIIBcel. The conserved histidine residue is presumed to bind the negatively charged phosphate. Therefore, we propose that the catalytic mechanism of EcEIIBfruc is similar to that of EcIIBcel transferring phosphoryl moiety to a specific carbohydrate.