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Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl α2→3 linkages when compared with α2→6 bound sialic acids. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Divalent cations were not required for activity. Sialidase was inhibited by 2-deoxy-2,3-didehydro- N-acetylneuraminic acid at unusually high concentrations ( K 1, =0.38 mM), but not by 20 mM N-acetylneuraminic acid. These values are the highest yet reported for a sialidase. K m, V max, and turnover number of the purified sialidase, measured using 2′-(4-methylumbelliferyl)-α-D- N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min −1, and 2,700 s −1, respectively. typhimurium sialidase was positively charged (pI≥9.0). Unlike all previously investigated sialidases, S. Overproduced enzyme was purified to near electrophoretic homogeneity after 66-fold enrichment using conventional preparative techniques. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Taken together the data show the dual role of sialic acid catabolism in nutrition and cell surface modulation.Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. coli (iii) sialylation of the lipooligosaccharide (LOS) epitope recognized by monoclonal antibody 3F11 is dependent on an environmental source of sialic acid (iv) a nanA mutant hypersialylates its LOS sialyl acceptor, corresponding to an apparent increased fitness of the mutant in the infant-rat model and (v) expression of the LOS sialyl acceptor is altered in cells grown without exogenous sialic acid, indicating the direct or indirect effect of sialic acid metabolism on LOS antigenicity. influenzae aldolase mutants showed that: (i) HI0142 is essential for sialic acid degradation (ii) the products of the open reading frames (ORFs) flanking HI0142 (HI0140, 41, 44 and 45) are likely to have the same functions as those of their counterparts in E. Phenotypic investigation of the resulting H. coli sialic acid aldolase structural gene, nanA, was subcloned and mutagenized by insertion of a kanamycin resistance cassette. We addressed these questions with Haemophilus influenzae type b, which metabolizes relatively few carbohydrates, using the infant-rat infection model. Despite the known ligand and antirecognition functions of the sialic acids, the contribution of their catabolism to infection or host colonization has never been directly investigated. In Escherichia coli, the breakdown of these sugars is catalysed by gene products of the nan ( N acyl neuraminate) operon other microorganisms may use a similar catabolic strategy. Many bacterial commensals and pathogens use the sialic acids as carbon and nitrogen sources.
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