Identification of residues involved in catalytic activity of the invertingglycosyl transferase WbbE from Salmonella enterica serovar borreze

Synthesis of the O:54 O antigen of Salmonella enterica is initiated by the nonprocessive glycosyl transferase WbbE, assigned to family 2 of the glycos yl transferase enzymes (GT2). GT2 enzymes possess a characteristic N-termin al domain, domain A. Based on structural data from the GT2 representative S psA (S. J. Charnock and G. J. Davies, Biochemistry 38:6380-6385, 1999), thi s domain is responsible for nucleotide binding. It possesses two invariant Asp residues, the first forming a hydrogen bond to uracil and the second co ordinating a Mn2+ ion. Site-directed replacement of Asp41 (D41A) of WbbE, t he analogue of the first Asp residue of SpsA, revealed that this is not req uired for activity. WbbE possesses three Asp residues near the position ana logous to the second conserved residue. Whereas D95A reduced WbbE activity, activity in D93A and D96A mutants was abrogated, suggesting that either D9 3 or D96 may coordinate the Mn2+ ion. Our studies also identified a C-termi nal region of sequence conservation in 22 GT2 members, including WbbE. SpsA was not among these. This region is characterized by an EDGY) motif. The G lu and Asp residues of this motif were individually replaced in WbbE. E180D in WbbE had greatly reduced activity, and an E180Q replacement completely abrogated activity; however, D181E had no effect. E180 is predicted to resi de on a turn. Combined with the alignment of the motif with potential catal ytic residues in the GT2 enzymes ExoM and SpsA, we speculate that E180 is t he catalytic residue of WbbE. Sequence and predicted structural divergence in the catalytic region of GT2 members suggests that this is not a homogene ous family.