Dataset Information

Functional characterization of Lpt3 and Lpt6, the inner-core lipooligosaccharide phosphoethanolamine transferases from Neisseria meningitidis.

ABSTRACT: The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His(6) and Lpt6-His(6) were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro.

SUBMITTER: Wenzel CQ

PROVIDER: S-EPMC2798236 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional characterization of Lpt3 and Lpt6, the inner-core lipooligosaccharide phosphoethanolamine transferases from Neisseria meningitidis.

Wenzel Cory Q CQ St Michael Frank F Stupak Jacek J Li Jianjun J Cox Andrew D AD Richards James C JC

Journal of bacteriology 20100101 1

The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucro ...[more]

PMID: 19854897

Similar Datasets

Project description:The first x-ray crystallographic structure of a CAZY family-52 glycosyltransferase, that of the membrane associated ?2,3/?2,6 lipooligosaccharide sialyltransferase from Neisseria meningitidis serotype L1 (NST), has been solved to 1.95 Å resolution. The structure of NST adopts a GT-B-fold common with other glycosyltransferase (GT) families but exhibits a novel domain swap of the N-terminal 130 residues to create a functional homodimeric form not observed in any other class to date. The domain swap is mediated at the structural level by a loop-helix-loop extension between residues Leu-108 and Met-130 (we term the swapping module) and a unique lipid-binding domain. NST catalyzes the creation of ?2,3- or 2,6-linked oligosaccharide products from a CMP-sialic acid (Neu5Ac) donor and galactosyl-containing acceptor sugars. Our structures of NST bound to the non-hydrolyzable substrate analog CMP-3F((axial))-Neu5Ac show that the swapping module from one monomer of NST mediates the binding of the donor sugar in a composite active site formed at the dimeric interface. Kinetic analysis of designed point mutations observed in the CMP-3F((axial))-Neu5Ac binding site suggests potential roles of a requisite general base (Asp-258) and general acid (His-280) in the NST catalytic mechanism. A long hydrophobic tunnel adjacent to the dimer interface in each of the two monomers contains electron density for two extended linear molecules that likely belong to either the two fatty acyl chains of a diglyceride lipid or the two polyethylene glycol groups of the detergent Triton X-100. In this work, Triton X-100 maintains the activity and increases the solubility of NST during purification and is critical to the formation of ordered crystals. Together, the mechanistic implications of the NST structure provide insight into lipooligosaccharide sialylation with respect to the association of substrates and the essential membrane-anchored nature of NST on the bacterial surface.

Project description:A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.

Project description:Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.

Dataset Information

Functional characterization of Lpt3 and Lpt6, the inner-core lipooligosaccharide phosphoethanolamine transferases from Neisseria meningitidis.

Publications

Functional characterization of Lpt3 and Lpt6, the inner-core lipooligosaccharide phosphoethanolamine transferases from Neisseria meningitidis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets