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

Abstract

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.

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

“…The topology of two members of this family, the bacterial protein MraY that is involved in peptidoglycan synthesis (Bouhss et al, 1999) and the eukaryotic GPT that is involved in protein glycosylation (Dan et al, 1996), has been characterized experimentally. Bacterial and eukaryotic proteins share discrete regions of conserved amino acid sequence, especially those located in hydrophilic segments of the protein that are exposed to the cytosolic face of the plasma membrane or the membrane of the endoplasmic reticulum (Bouhss et al, 1999 ;Dal Nogare & Lehrman, 1988).…”

“…Bacterial homologues also carry a large cytosolic loop that shares very little similarity with the eukaryotic members of the family (Bouhss et al, 1999 ;Dal Nogare & Lehrman, 1988). It has recently been proposed that this large cytosolic region, ranging from approximately 32 to 43 aa in length, may be important for the recognition of the nucleotide-sugar substrates and may also determine the specificity of such an interaction (Anderson et al, 2000).…”

“…The previous experiments did not distinguish whether the mutations have an effect on the ability of WecA to bind the nucleotide-sugar substrate or on the mechanism of transfer of GlcNAc 1-phosphate to Und-P. To differentiate between these possibilities, we used a binding assay involving the use of the UDP-GlcNAc analogue tunicamycin, which inhibits the activity of the enzyme (Dal Nogare & Lehrman, 1988). We reasoned that mutations in a putative binding region that would prevent the interaction between WecA and UDPGlcNAc would also prevent the binding of tunicamycin.…”

“…WecA is an UDP-N-acetylglucosamine (GlcNAc) : undecaprenyl-phosphate (Und-P) GlcNAc-1-phosphate transferase that belongs to a large family of eukaryotic and prokaryotic polyisoprenyl-phosphate Nacetylhexosamine-1-phosphate transferases (Anderson et al, 2000 ;Dal Nogare & Lehrman, 1988 ;Lehrman, 1994 ;Rick & Silver, 1996). The members of this family …”

Two Proteins Homologous to the N- and C-terminal Domains of the Bacterial Glycosyltransferase Murg Are Required for the Second Step of Dolichyl-linked Oligosaccharide Synthesis in Saccharomyces cerevisiae

“…Sequence data show that this enzyme belongs to a family of sugar phosphate transferases that act on phospolipid acceptors to initiate glycolipid synthesis in bacterial peptidoglycan, liposaccharide, exosaccharide, and capsular polysaccharide pathways (4,5). A practical consequence of these facts is that the potent antibiotic tunicamycin is not clinically useful because it blocks the first step of both the eukaryotic LLO (6) and bacterial peptidoglycan pathways (7).…”