Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl α2→3 linkages

Lois L. Hoyer, Peter Roggentin, Roland Schauer, Eric R. Vimr

Research output: Contribution to journalArticlepeer-review

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 66-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-methylumbelliferyl)-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-acetylneuraminic 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 gangliosides, 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.

Original languageEnglish (US)
Pages (from-to)462-467
Number of pages6
JournalJournal of Biochemistry
Volume110
Issue number3
DOIs
StatePublished - Sep 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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