Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes useUDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-Pas acceptors, and generate sugar-P-P-polyisoprenols. A series of sixconserved sequences, designated A through F and ranging from 5 to 13 aminoacid residues, has been identified in this family. To determine whetherthese conserved sequences are required for enzyme function, variousmutations were examined in hamster UDP- GlcNAc:dolichol-P GlcNAc-1-Ptransferase (GPT). Scramble mutations of sequences B-F, generated byscrambling the residues within each sequence, demonstrated that each isimportant in GPT. While E and F scrambles appeared to prevent stableexpression of GPT, scrambling of B- D resulted in GPT mutants that could bestably expressed and bound tunicamycin, but lacked enzymatic activity.Further, the C and D scramble mutants had an unexpected sorting defect.Replacement of sequences B-F with prokaryotic counterparts from either theB.subtilis mraY or E.coli rfe genes also affected GPT by preventingexpression of the mutant protein (B, F) or inhibiting its enzymaticactivity (C-E). For the C-E replacements, no acquisition of acceptoractivity for polyprenol-P, the fully unsaturated natural bacterialacceptor, was detected. These studies show that the conserved sequences ofthe UDP- GlcNAc/MurNAc family are important, and that the eukaryotic andprokaryotic counterparts are not freely interchangeable. Since severalmutants were efficiently expressed and bound tunicamycin, yet lackedenzymatic activity, the data are consistent with these sequences having adirect role in product formation.