Colominic acid is a capsular homopolymer from Escherichia coli K1 composed of α(2-8)-linked N-acetyl-d-neuraminic acid (NeuAc) residues. Recently, we have described that NeuAc synthesis in this bacterium occurs through the action of NeuAc lyase (EC 4.1.3.3) [Rodríguez-Aparicio, Ferrero and Reglero (1995) Biochem. J. 308, 501–505]. In the present work we analysed and characterized this enzyme. E. coli K1 NeuAc lyase is detected from the early logarithmic phase of growth, is induced by NeuAc and is not repressed by glucose. The enzyme was purified to apparent homogeneity (312-fold) using two types of hydrophobic chromatographies (butyl-agarose and phenyl-Sepharose CL-4B), gel filtration on Sephacryl S-200, and anion-exchange chromatography on DEAE-FPLC. The pure enzyme, whose amino acid composition and N-terminal amino acid sequence are also established, has a native molecular mass, estimated by gel filtration, of 135±3 kDa, whereas its molecular mass in SDS/PAGE was 33±1 kDa. The enzyme was able to synthesize and cleave NeuAc in a reversible reaction. The maximal rate of catalysis was achieved in 125 mM Tris/HCl buffer, pH 7.8, at 37 °C. Under these conditions, the Km values calculated for N-acetyl-d-mannosamine and pyruvate (condensation direction), and NeuAc (hydrolysis direction) were 7.7, 8.3 and 4.8 mM respectively. NeuAc synthesis by the pure enzyme was activated by Ca2+ and inhibited by Mn2+ and NeuAc, whereas the enzyme cleavage direction was inhibited by Ca2+, Mn2+ and pyruvate. The reaction products, NeuAc and pyruvate, and Ca2+ are able to regulate the direction of this enzyme (synthesis or cleavage of sialic acid) and, accordingly, to modulate colominic acid biosynthesis.
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July 1996
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Research Article|
July 01 1996
N-acetyl-d-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1
Miguel A. FERRERO;
Miguel A. FERRERO
1Departamento de Bioquímica y Biología Molecular, Universidad de León, Campus de Vezagana, 24007 León, Spain
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Angel REGLERO;
Angel REGLERO
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*To whom correspondence should be addressed.
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Manuel FERNANDEZ-LOPEZ;
Manuel FERNANDEZ-LOPEZ
1Departamento de Bioquímica y Biología Molecular, Universidad de León, Campus de Vezagana, 24007 León, Spain
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Roberto ORDAS;
Roberto ORDAS
1Departamento de Bioquímica y Biología Molecular, Universidad de León, Campus de Vezagana, 24007 León, Spain
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Leandro B. RODRIGUEZ-APARICIO
Leandro B. RODRIGUEZ-APARICIO
1Departamento de Bioquímica y Biología Molecular, Universidad de León, Campus de Vezagana, 24007 León, Spain
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Publisher: Portland Press Ltd
Received:
January 15 1996
Revision Received:
February 23 1996
Accepted:
March 01 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 157–165.
Article history
Received:
January 15 1996
Revision Received:
February 23 1996
Accepted:
March 01 1996
Citation
Miguel A. FERRERO, Angel REGLERO, Manuel FERNANDEZ-LOPEZ, Roberto ORDAS, Leandro B. RODRIGUEZ-APARICIO; N-acetyl-d-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1. Biochem J 1 July 1996; 317 (1): 157–165. doi: https://doi.org/10.1042/bj3170157
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