N-acetyl-D-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1.
Colominic acid is a capsular homopolymer from Escherichia coli K1 composed of alpha (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 degrees C. Under these conditions, the K(m) 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.
Ferrero
M A
MA
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
Reglero
A
A
Fernandez-Lopez
M
M
Ordas
R
R
Rodriguez-Aparicio
L B
LB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis*; D011773:Pyruvates / Q000494:pharmacology; D019289:Pyruvic Acid; D017421:Sequence Analysis; D012794:Sialic Acids / Q000378:metabolism*; D013997:Time Factors
D000596:Amino Acids; D002412:Cations; D011134:Polysaccharides; D011773:Pyruvates; D012794:Sialic Acids; D008345:Manganese; D019289:Pyruvic Acid; C014420:colominic acid; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D019158:N-Acetylneuraminic Acid; D000645:Ammonium Sulfate; D002118:Calcium