The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Purification and Properties of Cloned Salmonella typhimurium LT2 Sialidase with Virus-Typical Kinetic Preference for Sialyl α2→3 Linkages
Lois L. HoyerPeter RoggentinRoland SchauerEric R. Vimr
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1991 Volume 110 Issue 3 Pages 462-467

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Abstract

Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI≥9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbellifery1)-α-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5, 200 nmol min-1 and 2, 700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2, 3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (K1=0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. 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 enzyme also efficiently cleaved residues from glycoproteins and ganglio-sides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl α2→3 linkages and to have a basic pI.

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