Structural and functional characterization of the Clostridium perfringens N-acetylmannosamine-6-phosphate 2-epimerase essential for the sialic acid salvage pathway

J Biol Chem. 2014 Dec 19;289(51):35215-24. doi: 10.1074/jbc.M114.604272. Epub 2014 Oct 15.


Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys(66) as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. (1)H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys(66) for the epimerization reaction with participation of neighboring Arg(43), Asp(126), and Glu(180) residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.

Keywords: 1H NMR Spectroscopy; Carbohydrate; Enzyme Mechanism; Mutagenesis; Sialic Acid; Sugar 2-Epimerase; Sugar Phosphate; X-ray Crystallography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / analogs & derivatives
  • Acetylglucosamine / metabolism
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biocatalysis
  • Carbohydrate Epimerases / chemistry
  • Carbohydrate Epimerases / genetics
  • Carbohydrate Epimerases / metabolism*
  • Clostridium perfringens / enzymology*
  • Clostridium perfringens / genetics
  • Crystallography, X-Ray
  • Hexosamines / metabolism
  • Kinetics
  • Lysine / genetics
  • Lysine / metabolism
  • Magnetic Resonance Spectroscopy
  • Metabolic Networks and Pathways
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • N-Acetylneuraminic Acid / metabolism*
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protons
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Sugar Phosphates / metabolism


  • Bacterial Proteins
  • Hexosamines
  • N-acetylmannosamine 6-phosphate
  • Protons
  • Sugar Phosphates
  • N-acetylglucosamine-1-phosphate
  • Carbohydrate Epimerases
  • N-acetylmannosamine-6-phosphate epimerase
  • N-Acetylneuraminic Acid
  • Lysine
  • Acetylglucosamine

Associated data

  • PDB/1Y0E
  • PDB/1YXY
  • PDB/3IGS
  • PDB/3Q58
  • PDB/4UTT
  • PDB/4UTU
  • PDB/4UTW