Fig. 3.

Identification of positions for chemical modification. The crystal structure of the Y137A mutant of E. coli NAL in complex with Neu5Ac (PDB ID code 4BWL) (35) was used to select sites at which to incorporate Ncas. Residues were selected based on proximity (<8 Å) to the position of the Neu5Ac substrate (dark purple) and were separately mutated to cysteine residues. Residues at these positions are conserved between the E. coli and S. aureus enzymes, except for the following conservative changes (E. coli residue first): D170N, Y172F, Y190F, I247L, V251I, and F252Y.