Purification and characterization of GlcNAc-6-P 2-epimerase from Escherichia coli K92.
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 degrees C. Under these conditions, the K(m) 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.
Ferrero
Miguel Angel
MA
Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, León, Spain. ma.ferrero@unileon.es
Martínez-Blanco
Honorina
H
Lopez-Velasco
Federico Felino
FF
Ezquerro-Sáenz
Carlos
C
Navasa
Nicolas
N
Lozano
Sofia
S
Rodríguez-Aparicio
Leandro B
LB
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006595:Hexosamines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002412:Cations; D004269:DNA, Bacterial; D006595:Hexosamines; D010446:Peptide Fragments; D002238:Carbohydrate Epimerases; C121340:N-acylglucosamine-6-phosphate 2-epimerase; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine