Crystal structure of hypothetical fructose-specific EIIB from Escherichia coli

We have solved the crystal structure of a predicted fructose- specific enzyme IIB^fruc from Escherichia coli (EcEIIB^fruc) involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system transferring carbohydrates across the cytoplasmic membrane. EcEIIB^fruc 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 IIB^cellobiose from E. coli (EcIIB^cel) transferring a phosphate moiety. In the active site of the closest EcEIIB^fruc 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 EcIIB^cel. The conserved histidine residue is presumed to bind the negatively charged phosphate. Therefore, we propose that the catalytic mechanism of EcEIIB^fruc is similar to that of EcIIB^cel transferring phosphoryl moiety to a specific carbohydrate.