Clostridium perfringens sialidase

Ajisaka et al. examined different sialidase somces and found that Newcastle disease virus (NDV) sialidase afforded predominantly the a-2,3 regioisomers, while Arthrobacter ureafaciens and Clostridium perfringens sialidases, in addition to the Vibrio cholerae sialidase examined by Thiem, favored the a-2,6-linked products [53]. Unfortunately, the reaction yields did not improve for the new enzymes, varying from 0.8% to 3.6% isolated yield. In the case of NDV sialidase, the high selectivity for a-2,3-sialosides stemmed from a large a-2,6/a-2,3 hydrolysis ratio. Hydrolysis of the a-2,6 products was found to be 28 times faster than the a-2,3 isomers. Inter-... 

An interesting electrochemical method for the determination of bound sialic acid has been developed, making use of a potentiometric four-channel thick-film sensor [236]. The sialidase sensor consists of a bilayer of a membrane containing Clostridium perfringens sialidase immobilized in a poly(vinyl acetate)-polyethylene copolymer, which is placed on top of an fT -selective poly(vinyl chloride)-poly(vinyl acetate) indicator membrane. The enzyme-induced release of bound sialic acid leads to a concomitant decrease in pA a of the carboxyl function of sialic acid. This decrease affords a local pH change inside the sialidase-containing sensor membrane, which is monitored by the H -selective indicator membrane. The pH optimum of the sialidase sensor was pH 4 for sialyllactose, mucin and colominic acid. 

In the first primary structures of microbial sialidases, obtained by cloning and sequencing of the respective genes from Clostridium perfringens [769], Vibrio cholerae [770], Clostridium sordellii [77 ] and Salmonella typhimurium [772], an amino acid sequence motif was detected, which is repeated four-fold in each protein S-X-D-X-G-X-T-W [773]. This motif, named the Asp-box, was found in all 16 sialidases of animals, trypanosomes, and bacteria, which have so far been sequenced (see refs. [660,768] and Table 18). In viral sialidases, however, the motif was rarely detectable (e.g. only the sialidase from N9 influenza A virus strain exhibits the complete motif [786] and has probably undergone mutational alterations). 

Fig. 18. Amino acids essential for bacterial sialidase action. The positions of the amino acid residues interacting with different parts of the Neu5Ac molecule or forming a hydrophobic pocket (by Leu, Trp and Met indicated by a dotted line at the left side of the sialic acid molecule) are shown. Vc, Vibrio cholerae sialidase [791] St, Salmonella typhimurium sialidase [790] and Cp, Clostridium perfringens sialidase [R.G. Kleineidam, personal communication].

Folch upper phase [Folch etal., 1957]) is taken to dryness and dissolved in 50 xnM acetate buffer, pH 5.5,1 mM CaCl2, and incubated overnight with sialidase from Clostridium perfringens (10 units/liter) (G. Schwarzmann, personal communication). 

Another feature common to all nonviral sialidases is the so-called Asp-box, a motif (S/T-X-D-[X]-G-X-T-W/F) that repeats one to five times along the sequences. Each Asp-box forms a clamp-like loop and they occur at topologically equivalent positions on the outside of the structure between the third and fourth p-strand of a propeller blade [12, 114, 115]. For example, in the sialidase Nani of Clostridium perfringens four Asp-boxes are located in the blades one to four (PDB ID 2BF6 [115]). However, in endosialidases only two Asp-boxes have been found in the first and fourth blade of the propeller [12], with the sequences S-G-D-D-G-Q/ K-T-W and S-X-D-X-G-X-X-W that are conserved in aU so far known endosialidases. Interestingly, in endoNF the p-barrel domain is inserted between the third and fourth p-sheet of the second blade, thereby replacing a potential Asp-box. Since the Asp-boxes are located on the back side of the propeller and remote from the active site, any functions other than structural folds have not been found as yet for these motifs. 

Newstead SL, Potter JA, Wilson JC, Xu G, Chien CH, Watts AG, Withers SG, Taylor GL (2008) The structure of Clostridium perfringens Nani sialidase and its catalytic intermediates. J Biol Chem 283 9080-9088... 

The use of sialidases for the preparation of acylneuraminic acids has several advantages over the relatively destructive acid hydrolysis techniques. The hydrolysis is carried out under milder conditions of temperature and pH. Low temperatures (0-4 °C) can be employed and even on prolonged incubation (24-48 h) the destruction of released acylneuraminic acids is usually below 5%. The sialic acids are released into aqueous solution at pH 5-6, where they are stable for the duration of the incubation. The use of sialidases is widespread, and several bacterial preparations are available in partially purified form well suited for the experiments outlined here. The most widely available sialidases are those from Vibrio cholerae, Clostridium perfringens and Arthrobacter ureafaciens, and these have sufficiently high specific activities to be used in preparative work. Details of the properties and specificities of these and other sialidases are given in chapter I and in reviews by Drzeniek (1972, 1973) and Corfield et al. (1981). 

Fig. 5. The effect of naturally occurring N-acyl substitution on sialidase activity. A time curve of Neu5Acyl release from sialyl a(2-3)-lactose by Arthrobacter ureafaciens sialidase (A A, 1 mU) and from sialyl a(2-6)-N-acetylgalactosamine by Clostridium perfringens enzyme (O 3.2 mU). The substrates contain either N-acetyl- (O A) or N-glycolyl- ( , A) sialic acids at 1 mM final concentration.

Sialidase from Clostridium Perfringens (Wild Type and Mutants)... 

The presence of a number of CIDNP-sensitive amino acids on the protein surface can enable the detection of conformational alterations resulting from single site mutations. This has been demonstrated for wild-type forms and various mutants of EcorL and of the sialidase of Clostridium perfringens [5, 7], Sialidase of C. perfringens is larded with CIDNP-responsive amino acid residues. Therefore, these residues have been used as valuable sensors in NMR and modelling studies in which CIDNP-spectra of the sialidase wild type and various mutants are compared and correlated with modelled structures. 

Roggentin, P., Rothe, B., Lottspeich, F., and Schauer, R., 1988, Cloning and sequencing of a Clostridium perfringens sialidase gene, FEBS Lett. 238 31-34. 

Roggentin, T., Kleineidam, R. G., Schauer, R., and Roggentin, P., 1992, Effects of site-specific mutations on the enzymic properties of a sialidase from Clostridium perfringens, Glycoconj. J. 9 235-240. 

Barton, N. W., Lipovac, V., and Rosenberg, A., 1975, Effects of strong electrolyte upon the activity of Clostridium perfringens sialidase towards sialyllactose and sialoglycolipids, J. Biol. Chem. 250 8462-8466. 

Cassidy, J. T., Jourdian, G. W., and Roseman, S., 1965, The sialic acid. VI. Purification and properties of sialidase from Clostridium perfringens, J. Biol. Chem. 240 3501-3506. 

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