Publications

• Purification and characterization of the lipid A disaccharide synthase (LpxB) from Escherichia coli, a peripheral membrane protein.

  Metzger L.E., Raetz C.R.

  Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member of CAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine + 2,3-diacylglucosamine 1-phosphate --> 2',3'-diacylglucosamine-(beta,1'-6)-2,3-diacylglucosamine 1- ... >> More

  Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member of CAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine + 2,3-diacylglucosamine 1-phosphate --> 2',3'-diacylglucosamine-(beta,1'-6)-2,3-diacylglucosamine 1-phosphate + UDP. LpxB is a target for the development of new antibiotics, but no member of family 19, which consists entirely of LpxB orthologues, has been characterized mechanistically or structurally. Here, we have purified E. coli and Haemophilus influenzae LpxB to near homogeneity on a 10-100 mg scale using protease-cleavable His(10)-tagged constructs. E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micelle assay system, suggesting that catalysis occurs at the membrane interface. E. coli LpxB (M(r) approximately 43 kDa) sediments with membranes at low salt concentrations but is largely solubilized with buffers of high ionic strength. It purifies with 1.6-3.5 mol of phospholipid/mol of LpxB polypeptide. Transmission electron microscopy reveals the accumulation of aberrant intracellular membranes when LpxB is overexpressed. Mutagenesis of LpxB identified two conserved residues, D89A and R201A, for which no residual catalytic activity was detected. Our results provide a rational starting point for structural studies. << Less

  Biochemistry 48:11559-11571(2009) [PubMed] [EuropePMC]

• Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase.

  Crowell D.N., Reznikoff W.S., Raetz C.R.H.

  The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence sugg ... >> More

  The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D. N. Crowell, M. S. Anderson, and C. R. H. Raetz, J. Bacteriol. 168:152-159, 1986). We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene. This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB. There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA. The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled. A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction. These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E. coli chromosome. The accompanying paper by Tomasiewicz and McHenry (J. Bacteriol. 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon. << Less

  J. Bacteriol. 169:5727-5734(1987) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The biosynthesis of gram-negative endotoxin. Formation of lipid A disaccharides from monosaccharide precursors in extracts of Escherichia coli.

  Ray B.L., Painter G., Raetz C.R.

  We have discovered an enzyme in the cytosol of Escherichia coli that generates lipid A disaccharides from monosaccharide precursors by the following route: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2,3-diacyl-GlcN (beta, 1----6) 2,3-diacyl-GlcN-1-P + UDP. Previous studies from our laboratory ha ... >> More

  We have discovered an enzyme in the cytosol of Escherichia coli that generates lipid A disaccharides from monosaccharide precursors by the following route: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2,3-diacyl-GlcN (beta, 1----6) 2,3-diacyl-GlcN-1-P + UDP. Previous studies from our laboratory have documented the presence in vivo of the precursors 2,3-diacylglucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) (lipid X of E. coli) and UDP-2,3-diacylglucosamine (UDP-2,3-diacyl-GlcN) (Bulawa, C.E., and Raetz, C.R.H.J. Biol. Chem. 259, 4846-4851). Both substrates are novel glucosamine-derived phospholipids, acylated with beta-hydroxymyristoyl moieties, and they accumulate in E. coli mutants defective in the pgsB gene. Synthetic ADP-, GDP-, and CDP-2,3-diacylglucosamines are inefficient substrates compared to the naturally occurring UDP derivative. The free-acid form of the tetraacyldisaccharide 1-phosphate product (C68H129N2O20P) that is generated in vitro has Mr = 1325.74 as judged by fast atom bombardment mass spectrometry. Mild acid hydrolysis (0.1 M HCl for 30 min at 100 degrees C) liberates greater than 95% of the phosphate moiety as Pi. Detailed analysis by 1H and 13C NMR spectroscopy confirms the presence of a phosphate residue at position 1 of the disaccharide, an alpha-anomeric configuration at the reducing end, and a beta, 1----6 linkage between the two glucosamines. Importantly the disaccharide 1-phosphate synthase is missing in extracts of E. coli strains harboring the pgsB1 mutation, consistent with the massive accumulation of 2,3-diacyl-GlcN-1-P and UDP-2,3-diacyl-GlcN in vivo. The enzymatic reaction reported here represents a major biosynthetic route for the formation of lipid A disaccharides in E. coli and other Gram-negative bacteria. An in vitro system for the biosynthesis of lipid A disaccharides has not been described previously. << Less

  J. Biol. Chem. 259:4852-4859(1984) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structure of lipid A disaccharide synthase LpxB from Escherichia coli.

  Bohl H.O., Shi K., Lee J.K., Aihara H.

  Most Gram-negative bacteria are surrounded by a glycolipid called lipopolysaccharide (LPS), which forms a barrier to hydrophobic toxins and, in pathogenic bacteria, is a virulence factor. During LPS biosynthesis, a membrane-associated glycosyltransferase (LpxB) forms a tetra-acylated disaccharide ... >> More

  Most Gram-negative bacteria are surrounded by a glycolipid called lipopolysaccharide (LPS), which forms a barrier to hydrophobic toxins and, in pathogenic bacteria, is a virulence factor. During LPS biosynthesis, a membrane-associated glycosyltransferase (LpxB) forms a tetra-acylated disaccharide that is further acylated to form the membrane anchor moiety of LPS. Here we solve the structure of a soluble and catalytically competent LpxB by X-ray crystallography. The structure reveals that LpxB has a glycosyltransferase-B family fold but with a highly intertwined, C-terminally swapped dimer comprising four domains. We identify key catalytic residues with a product, UDP, bound in the active site, as well as clusters of hydrophobic residues that likely mediate productive membrane association or capture of lipidic substrates. These studies provide the basis for rational design of antibiotics targeting a crucial step in LPS biosynthesis. << Less

  Nat Commun 9:377-377(2018) [PubMed] [EuropePMC]