Glycobiology, Vol 8, 625-632, Copyright © 1998 by Society for Glycobiology
AR Dal Nogare, N Dan and MA Lehrman
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.
ORIGINAL ARTICLES
Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase
Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center At Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9041, USA.
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