Abstract
The ‘small’ (43 kDa) sialidase of Clostridium perfringens is inhibited by low concentrations of mercury ions. For the investigation of possible functional roles of the enzyme's four cysteine residues at the amino acid positions 2, 282, 333 and 349, they were separately altered to serine by site-directed mutagenesis. The four mutant sialidases expressed in E. coli and purified by metal chelate chromatography were markedly reduced in specific activity when compared to the wild-type enzyme but with the exception of C282S exhibited similar KM-values indicating an unchanged mode of substrate binding. The substrate specificity was also conserved for C2S, C282S, and C333S. Only the C349S sialidase exhibited a higher relative activity with colominic acid and the α2,6-linked sialic acid of sialyllactose compared to the α2,3-linked isomer than the other mutants. Chemical modifications with the thiol-blocking reagents N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB) and HgCl2 had little effect on the C282S sialidase, e.g., 6% inhibition by 5 m M NEM compared to reductions in activity between 65 and 90% for the wild-type and other mutant enzymes, supporting the idea that among the enzyme's cysteines, Cys-282 has the highest structural or functional significance. The results also explain the higher mercury tolerance of Salmonella typhimurium and Clostridium tertium sialidases, which have the positions equivalent to Cys-282 altered to Val and Thr, respectively, indicating that the thiol group of Cys-282, despite being situated near the active site, is not involved in catalysis.
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References
Corfield AP, Michalski JC, Schauer R (1981) In Sialidases and Sialidosis (Tettamanti G, Durand P, DiDonato S, eds) Vol 4, pp 3-70. Milano: Edi Ermes.
Corfield AP, Schauer R (1982) In Sialic Acids–Chemistry, Metabolism and Function (Schauer R, ed) pp 5-50. Cell Biology Monographs Vol 10, Berlin: Springer Verlag.
Saito M, Yu RK (1995) In Biology of Sialic Acids (Rosenberg A, ed) pp 261-313. New York: Plenum Press.
Schauer R (1982) Adv Carbohydr Chem Biochem 40: 131-234.
Schauer R (1983) In Structural Carbohydrates of the Liver (Popper H, Reutter W, Köttgen E, Gudat F, eds.) pp 83-97. Boston: MTP Press Ltd.
Popoff MR, Dodin A (1985) J Clin Microbiol 22: 873-6.
Corfield T (1992) Glycobiology 2: 509-21.
Müller HE (1992) BiOforum 1-2: 16-22.
Roggentin P, Schauer R, Hoyer LL, Vimr ER (1993) Mol Microbiol 9: 915-21.
Roggentin T, Kleineidam RG, Schauer R, Roggentin P (1992) Glyconconj J 9: 235-40.
Roggentin P, Kleineidam RG, Schauer R (1995) Biol Chem Hoppe-Seyler 376: 569-75.
Messing J (1983) Methods Enzymol 101: 20-78.
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Methods Enzymol 185: 60-89.
Yannisch-Perron C, Vieira J, Messing J (1985) Gene 33: 103-19.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor: Laboratory Press.
Roggentin P, Rothe B, Lottspeich F, Schauer R (1988) FEBS Lett 238: 31-4.
Kruse S, Kleineidam RG, Roggentin P, Schauer R (1996) Protein Expression and Purification 7: 415-22.
Laemmli UK (1970) Nature 227: 680-5.
Potier M, Mameli L, Bélisle M, Dallaire L, Melan÷on SB (1975) Anal Biochem 94: 287-96.
Ellis KJ, Morrison JF (1982) Methods Enzymol 87: 405-26.
Veh RM, Michalski JC, Corfield AP, Sander M, Schauer R (1981) J Chromat 212: 313-22.
Reuter G, Schauer R (1994) Methods Enzymol 230: 168-97.
Ray WJ, Koshland DE (1961) J Biol Chem 236: 1973-9.
Siebert HC, Tajkhorshid E, von der Lieth CW, Kleineidam RG, Kruse S, Schauer R, Kaptein R, Gabius HJ, Vliegenhart JFG (1996). J Mol Model 2: 446-55.
Crennell SJ, Garman EF, Laver WG, Vimr ER, Taylor GL (1993) Proc Natl Acad Sci USA 90: 9852-6.
Crennell SJ, Garman EF, Philippon C, Vasella A, Laver WG, Vimr ER, Taylor GL. (1996) J Mol Biol 259: 264-80.
Heuermann D, Roggentin P, Kleineidam RG, Schauer R (1991) Glycoconj J 8: 95-101.
Traving C, Schauer R, Roggentin P (1994) Glycoconj J 11: 141-51.
Rothe B, Roggentin P, Frank R, Blöcker H, Schauer R (1989) J Gen Microbiol 135: 3087-96.
Akimoto S, Ono T, Tsutsui H, Kinouchi T, Kataok a K, Ohnishi Y (1994) Biochem Biophys Res Commun 203: 914-21.
Rothe B, Rothe B, Roggentin P, Schauer R (1991) Mol Gen Genet 226: 190-7.
Thompson JD, Higgins DG, Gibson TJ (1994) Nucleic Acids Res 22: 4673-80.
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Kruse, S., Pommerencke, J., Kleineidam, R.G. et al. Effect of cysteine modifications on the activity of the ‘small’ Clostridium perfringens sialidase. Glycoconj J 15, 767–775 (1998). https://doi.org/10.1023/A:1006907931365
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DOI: https://doi.org/10.1023/A:1006907931365