AbstractPolyisoprenyl‐phosphate N‐acetylaminosugar‐1‐phosphate transferases (PNPTs) constitute a family of eukaryotic and prokaryotic membrane proteins that catalyze the transfer of a sugar‐1‐phosphate to a phosphoisoprenyl lipid carrier. All PNPT members share a highly conserved 213‐Valine‐Phenylalanine‐Methionine‐Glycine‐Aspartic acid‐217 (VFMGD) motif. Previous studies using the MraY protein suggested that the aspartic acid residue in this motif, D267, is a nucleophile for a proposed double‐displacement mechanism involving the cleavage of the phosphoanhydride bond of the nucleoside. Here, we demonstrate that the corresponding residue in the E. coli WecA, D217, is not directly involved in catalysis, as its replacement by asparagine results in a more active enzyme. Kinetic data indicate that the D217N replacement leads to more than twofold increase in Vmax without significant change in the Km for the nucleoside sugar substrate. Furthermore, no differences in the binding of the reaction intermediate analog tunicamycin were found in D217N as well as in other replacement mutants at the same position. We also found that alanine substitutions in various residues of the VFMGD motif affect to various degrees the enzymatic activity of WecA in vivo and in vitro. Together, our data suggest that the highly conserved VFMGD motif defines a common region in PNPT proteins that contributes to the active site and is likely involved in the release of the reaction product.