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2007 | 54 | 2 | 387-399
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Purification and characterization of GlcNAc-6-P 2-epimerase from Escherichia coli K92

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N-Acetylmannosamine (ManNAc) is the first committed intermediate in sialic acid metabolism. Thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. In prokaryotic organisms, UDP-N-acetylglucosamine (GlcNAc) 2-epimerase and GlcNAc-6-P 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc-6-P, respectively. We have purified for the first time native GlcNAc-6-P 2-epimerase from bacterial source to apparent homogeneity (1 200 fold) using Butyl-agarose, DEAE-FPLC and Mannose-6-P-agarose chromatography. By SDS/PAGE the pure enzyme showed a molecular mass of 38.4 ± 0.2 kDa. The maximum activity was achieved at pH 7.8 and 37°C. Under these conditions, the Km calculated for GlcNAc-6-P was 1.5 mM. The 2-epimerase activity was activated by Na+ and inhibited by mannose-6-P but not mannose-1-P. Genetic analysis revealed high homology with bacterial isomerases. GlcNAc-6-P 2-epimerase from E. coli K92 is a ManNAc-inducible protein and is detected from the early logarithmic phase of growth. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes the biosynthesis of sialic acid, GlcNAc-6-P 2-epimerase plays a catabolic role. When E. coli grows using ManNAc as a carbon source, this enzyme converts the intracellular ManNAc-6-P generated into GlcNAc-6-P, diverting the metabolic flux of ManNAc to GlcNAc.
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  • Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain
  • Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain
  • Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain
  • Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain
  • Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain
  • Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain
  • Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain
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