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 comp osed of alpha(2-8)-linked N-acetyl-D-neuraminic acid (NeuAc) residues. Recently, we have described that NeuAc synthesis in this bacterium oc curs through the action of NeuAc lyase (EC 4.1.3.3) [Rodriguez-Aparici o, Ferrero and Reglero (1995) Biochem. J. 308, 501-505]. In the presen t work we analysed and characterized this enzyme. E. coli K1 NeuAc lya se is detected from the early logarithmic phase of growth, is induced by NeuAc and is not repressed by glucose. The enzyme was purified to a pparent homogeneity (312-fold) using two types of hydrophobic chromato graphies (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 se quence are also established, has a native molecular mass, estimated by gel filtration, of 135 +/- 3 kDa, whereas its molecular mass in SDS/P AGE was 33 +/- 1 kDa. The enzyme was able to synthesize and cleave Neu Ac in a reversible reaction. The maximal rate of catalysis was achieve d in 125 mM Tris/HCl buffer, pH 7.8, at 37 degrees C. Under these cond itions, the K-m values calculated for N-acetyl-D-mannosamine and pyruv ate (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 reac tion products, NeuAc and pyruvate, and Ca2+ are able to regulate the d irection of this enzyme (synthesis or cleavage of sialic acid) and, ac cordingly, to modulate colominic acid biosynthesis.