Biochemical and functional studies on the Burkholderia cepacia complex bceN gene, encoding a GDP-D-mannose 4,6-dehydratase

PLoS One. 2013;8(2):e56902. doi: 10.1371/journal.pone.0056902. Epub 2013 Feb 27.

Abstract

This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. Vmax and Km values of 1.5±0.2 µmol.min(-1).mg(-1) and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl2 per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated Ki of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biocatalysis
  • Burkholderia cepacia complex / enzymology*
  • Burkholderia cepacia complex / genetics*
  • Genes, Bacterial / genetics*
  • Guanosine Diphosphate Mannose / metabolism
  • Histidine / metabolism
  • Humans
  • Hydro-Lyases / chemistry
  • Hydro-Lyases / genetics*
  • Hydro-Lyases / metabolism*
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation / genetics
  • Oligopeptides / metabolism
  • Polysaccharides, Bacterial / biosynthesis
  • Protein Multimerization
  • Sequence Analysis, DNA
  • Substrate Specificity

Substances

  • His-His-His-His-His-His
  • Oligopeptides
  • Polysaccharides, Bacterial
  • cepacian
  • Guanosine Diphosphate Mannose
  • Histidine
  • Hydro-Lyases
  • GDPmannose 4,6-dehydratase

Associated data

  • GENBANK/JN987864

Grants and funding

This work was partially funded by FEDER and FCT, Portugal (contracts PTDC/EBB-BIO/098352/2008 and PTDC/BIA-MIC/119091/2010, and a doctoral and post-doctoral grant to J.R.F. and S.A.S., respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.