Vibrio cholerae sialidase

Scheme 9.11 Enzymatic transsialylation of four MPEG-DOXyl glycosides with sialidases from Vibrio cholerae and Salmonella typhimurium. V. cholerae sialylated in position 6 of the terminal monosaccharide units with yields of 12-17%, whereas S. typhimurium sialylated position 3 of the same monosaccharide units (yields 14-24%).

Figure 12. Time course of NeuAc release from liposome-associated ganglioside Gn,a by the action of Vibrio cholerae sialidase...

In a study from Vibrio cholerae, a 1000-fold drop in inhibitory potency was observed for 42 relative to Neu5Ac2en, indicating that the acyl group contributes heavily to binding.79 Interestingly, similar modification in the 4-amino-4-deoxy-Neu5Ac2en to give compound 43 and applied to influenza virus sialidase, inhibition was reduced only 10-fold relative to its /V-acyl counterpart 9.65... 

J. C. Wilson, R. J. Thomson, J. C. Dyason, P. Florio, K. J. Quelch, S. Abo, and M. von Itzstein, The design, synthesis and biological evaluation of neuraminic acid-based probes of Vibrio cholerae sialidase, Tetrahedron Asymmetry, 11 (2000) 53-73. 

E. Schreiner, E. Zbiral, R. G. Kleineidam, and R. Schauer, 2,3-Didehydro-2-deoxysialic acids structurally varied at C-5 and their behaviour towards the sialidase from Vibrio cholerae,... 

For instance a-fucosides and a-sialosides, which are both difficult to produce by means of classical carbohydrate chemistry, have been synthesized using the corresponding nitrophenyl glycoside donors by applying pig liver a-fucosidase [59] and the sialidase from Vibrio cholerae [60], respectively. 

M. Engstler, R. Schauer, M. A. Ferrero-Garcia, A. J. Parodi, T. Storz-Eckerlin, A. Vasella, C. Witzig, and X. Zhu, N-(4-Nitrophenyl)oxamic Acid and Related N-Acylanilines Are Non-competitive Inhibitors of vibrio cholerae sialidase but do not inhibit trypanosoma cruzi or trypanosoma brucei trans-sialidases, Helv. Chim. Acta., 77 (1994) 1166-1174. 

Vasella, A., and Wyler, R., Synthesis of a phosphonic acid analogue of 7V-acetyl-2,3-didehydro-2-deoxyneuraminic acid, an inhibitor of Vibrio cholerae sialidase, Helv. Chim. Acta, 74, 451, 1991. 

Seven different bacterial sialidases, including six commercially available sialidases from Arthrobacter ureafaciens, Clostridium perfingens. Streptococcus sp. IID, Vibrio cholera. Salmonella typhimurium, and Streptococcus pneumoniae, as well as PmSTl which also possesses sialidase activity (20), were used as model systems to test the application of the sialoside library and the 96-well plate based high-throughput colorimetric screening method. 

Thiem and Sauerbrei examined this concept to determine whether various sial-idases could be used synthetically [52]. The rate of condensation, or reverse hydrolysis, was found to be negligible. However, the product hydrolysis rate was competitive with the rate of transsialylation to an alcohol acceptor. In an attempt to minimize product loss, reactions were stopped after 65-75% of the starting material had been consumed. Interestingly, a mixture of a-2,3 and a-2,6 regioisomers was obtained for reactions with an immobilized Vibrio cholerae sialidase (Scheme 41). In all cases, the a-2,6 isomer predominated, probably because of a combination of faster hydrolysis of the a-2,3 products (123 —> 1 + 125) and lesser steric hinderance of the primary alcohol. Variations in the donor/acceptor ratio (1 7 optimized) had an effect on both reaction yield and regioselectivity, although most of the examples afforded only 2-3 1 a-2,6/a-2,3 product ratios in 14-20% overall yield. 

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-... 

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].

The set of variously iV-acylated 2,3-unsaturated sialic acids 70 was synthesized to probe the activity of the sialidase of Vibrio cholerae, the causative agent of cholera. Syntheses of a range of fluorinated analogues of the methyl glycoside of 4-(2,4-dihydroxybutryoyl)amino-2,6-dideoxy-D-mannopyranose, the monosaccharide determinant of Vibrio cholerae are covered in Chapter 8. 

Endosialidases are not unique among sialidases in combining a p-propeller with other structures. Many other sialidases contain additional domains like p-barrels, jelly rolls, or immunoglobulin modules [116] which are placed N-terminally, C-terminally, or even inserted into the p-propeller. N-terminal lectin-like domains have been identified in leech sialidase L [117] and Vibrio cholerae sialidase [118]. Interestingly, in the V. cholerae sialidase a second p-structure domain is inserted... 

Luo Y, Li SC, Chou MY, Li YT, Luo M (1998) The crystal structure of an intramolecular trans-sialidase with a NeuAc alpha2->3 Gal specificity. Structure 6 521-530 Moustafa I, Connaris H, Taylor M, Zaitsev V, Wilson JC, Kiefel MJ, von Itzstein M, Taylor G (2004) Sialic acid recognition by Vibrio cholerae neuraminidase. J Biol Chem 279 40819 0826... 

Some time ago A -acyl anilines were tested and found to inhibit the sialidase from Vibrio cholerae however, they did not show any effect with TcTS [62], There is some similarity in structural features between Neu5Ac and compound 106 (Fig. 19), which showed Ki = 0.33 mM for TcTS, measured by hydrolysis of MU-Neu5Ac [63]. 

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). 

The crystal structures of two large sialidases have been determined the 83 kDa Vibrio cholerae sialidase [17] and the 68 kDa M. viridifaciens sialidase [19], both shown in Figure 2. The V. cholerae enzyme revealed a central catalytic P-propeller domain, flanked by two additional domains with similar topologies, resembling that of the lectins. The first lectin domain is at the N-terminus, and the second is inserted between the second and third sheets of the P-propeller. The lectin domains bind on as yet unidentified carbohydrate, but their structure suggests a role for these domains in the small intestine where the secreted sialidase may need to grasp the cell surface to perform its catalytic function without being swept away. 

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