Catalytic preference of Salmonella typhimurium LT2 sialidase for N-acetylneuraminic acid residues over N-glycolylneuraminic acid residues

Akira Minami; Sayaka Ishibashi; Kiyoshi Ikeda; Erika Ishitsubo; Takanori Hori; Hiroaki Tokiwa; Risa Taguchi; Daisuke Ieno; Tadamune Otsubo; Yukino Matsuda; Saki Sai; Mari Inada; Takashi Suzuki

doi:10.1016/j.fob.2013.05.002

Catalytic preference of Salmonella typhimurium LT2 sialidase for N-acetylneuraminic acid residues over N-glycolylneuraminic acid residues

FEBS Open Bio. 2013 May 29:3:231-6. doi: 10.1016/j.fob.2013.05.002. Print 2013.

Authors

Akira Minami¹, Sayaka Ishibashi, Kiyoshi Ikeda, Erika Ishitsubo, Takanori Hori, Hiroaki Tokiwa, Risa Taguchi, Daisuke Ieno, Tadamune Otsubo, Yukino Matsuda, Saki Sai, Mari Inada, Takashi Suzuki

Affiliation

¹ Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.

Abstract

In a comparison of sialidase activities toward N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we found that Salmonella typhimurium LT2 sialidase (STSA) hardly cleaved 4-methylumbelliferyl Neu5Gc (4MU-Neu5Gc). The k cat/K m value of STSA for 4MU-Neu5Gc was found to be 110 times lower than that for 4-methylumbelliferyl Neu5Ac (4MU-Neu5Ac). Additionally, STSA had remarkably weak ability to cleave α2-3-linked-Neu5Gc contained in gangliosides and equine erythrocytes. In silico analysis based on first-principle calculations with transition-state analogues suggested that the binding affinity of Neu5Gc2en is 14.3 kcal/mol more unstable than that of Neu5Ac2en. The results indicated that STSA preferentially cleaves Neu5Ac residues rather than Neu5Gc residues, which is important for anyone using this enzyme to cleave α2-3-linked sialic acids.

Keywords: 4MU, 4-methylumbelliferone; 4MU-Neu5Ac, 4-methylumbelliferyl N-acetylneuraminic acid; 4MU-Neu5Gc; 4MU-Neu5Gc, 4-methylumbelliferyl N-glycolylneuraminic acid; AUSA, Arthrobacter ureafaciens sialidase; Boc, tert-butoxycarbonyl; CPSA, Clostridium perfingens sialidase; DANA, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid; DMAP, 4-dimethylaminopyridine; DMB, 1,2-diamino-4,5-methylenedioxybenzene; Docking simulations; E. coli, Escherichia coli; HPLC, high-performance liquid chromatography; MDSA, Macrobdella decora sialidase; N-glycolylneuraminic acid; N.D., not detected; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; PBS, phosphate buffered saline; STSA, Salmonella typhimurium LT2 sialidase; Salmonella typhimurium LT2 sialidase; Sia, sialic acid; Sialic acid; Substrate specificity; THF, tetrahydrofuran; VCSA, Vibrio cholerae sialidase; rt, room temperature.