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 use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably 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 the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.
Dal Nogare
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AR
Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center At Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9041, USA.
Dan
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Lehrman
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MA
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D016466:CHO Cells / Q000737:chemistry / Q000201:enzymology; D017124:Conserved Sequence / Q000235:genetics*; D006224:Cricetinae; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D018390:Gene Targeting; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C106504:UDP-GlcNAc-undecaprenyl phosphate N-acetylglucosaminyl 1-phosphate transferase; C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase