THE STRUCTURE OF THE O-SPECIFIC POLYSACCHARIDE OF CITROBACTER O-16 CONTAINING GLYCEROL PHOSPHATE

The O-specific polysaccharide, obtained by mild acid degradation of Ci trobacter O16 lipopolysaccharide, consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, glycerol and phosphate in the ratios 2:2:2:1:1. Selective cleavage of the polysaccharide was carried out by Smith degradation, N-deacetylation-deamination and dephosphorylation with 48% hydrofluoric acid, which was accompanied by unexpected splitt ing of one of the glycosidic linkages. The structures of the oligosacc harides thus obtained were established using H-1- and C-13-NMR spectro scopy, including one-dimensional NOE, two-dimensional rotating-frame N OE, homonuclear and heteronuclear C-13,H-1 correlation spectroscopy, a nd, for the Smith degradation product, positive- and negative-ion-mode fast-atom-bombardment MS and MS/MS with collision-induced dissociatio n. On the basis of these data and the results of methylation analysis, it was concluded that the O-specific polysaccharide has the following repeating unit structure: [GRAPHICS] enzyme it is not made as a proen zyme. For the soluble enzyme form, the optimum pH for activity was 5.5 -6.0, and the apparent pi value of the protein determined by isoelectr ic focusing was 4.2. The enzyme cleaves arginine from the C-terminus o f the synthetic peptide benzoyl-Phe-Ala-Arg with K-m 335 mu M and V-ma x 282 mu mol.min(-1).mg protein(-1). Insect-cell-derived Kex1 Delta p processes alpha-factor-Lys-Arg, a known natural substrate, to mature a ctive alpha-factor in a manner similar to the membrane-associated full -length enzyme. This secreted form of the enzyme is a convenient sourc e for the isolation of substantial quantities of the pure enzyme for d etailed kinetic and structural studies.