Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.
Polyisoprenyl-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 V(max)  without significant change in the K(m)  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.
Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Sarah E
SE
Furlong
Miguel A
MA
Valvano
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C083617:wecA protein, E coli