Dataset Information

Structural characterization of a nonhydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A.

ABSTRACT: Bacterial nonhydrolyzing UDP-N-acetylglucosamine 2-epimerases catalyze the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). UDP-ManNAc is an important intermediate in the biosynthesis of certain cell-surface polysaccharides, including those in some pathogenic bacteria, such as Neisseria meningitidis and Streptococcus pneumoniae. Many of these epimerases are allosterically regulated by UDP-GlcNAc, which binds adjacent to the active site and is required to initiate UDP-ManNAc epimerization. Here, two crystal structures of UDP-N-acetylglucosamine 2-epimerase from Neisseria meningitidis serogroup A (NmSacA) are presented. One crystal structure is of the substrate-free enzyme, while the other structure contains UDP-GlcNAc substrate bound to the active site. Both structures form dimers as seen in similar epimerases, and substrate binding to the active site induces a large conformational change in which two Rossmann-like domains clamp down on the substrate. Unlike other epimerases, NmSacA does not require UDP-GlcNAc to instigate the epimerization of UDP-ManNAc, although UDP-GlcNAc was found to enhance the rate of epimerization. In spite of the conservation of residues involved in binding the allosteric UDP-GlcNAc observed in similar UDP-GlcNAc 2-epimerases, the structures presented here do not contain UDP-GlcNAc bound in the allosteric site. These structural results provide additional insight into the mechanism and regulation of this critical enzyme and improve the structural understanding of the ability of NmSacA to epimerize modified substrates.

SUBMITTER: Hurlburt NK

PROVIDER: S-EPMC7605110 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

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

Structural characterization of a nonhydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets