Bacterial sialidases

For routine, enzymic hydrolysis of sialic acids on a preparative or an analytical scale in our laboratory, the substrates are incubated with bacterial sialidases in 50 mM acetate buffer at pH 5.5 and 37° for times ranging between a few minutes and several hours, depending on the substrate.107 After incubation, the sialic acids liberated are separated by dialysis, ultrafiltration, or protein precipitation at 0-2°, and purified as will be described in the following Section. 

Application of sialidase inhibitors for medical use is still in a premature state. It is imaginable that inhibitors would be useful drugs in infections, caused by micro-organisms, that lead to extensive production of sialidase, for example, in gas edema.371 In the oral cavity, plaque formation and dental caries may be influenced by desialylation of salivary glycoconjugates,399 and bacterial sialidases may play a role therein. This process may be retarded by secretion of the inhibitor Neu2en5Ac in saliva at concentrations which, in some cases, were found to be close to the K value for sialidases.34... 

Crystals of pronase-released heads of the N2 human strains of A/Tokyo/3/67 [44] and A/RI/5+/57 were used for an x-ray structure determination. The x-ray 3-dimensional molecular structure of neuraminidase heads was determined [45] for these two N2 subtypes by a novel technique of molecular electron density averaging from two different crystal systems, using a combination of multiple isomorphous replacement and noncrystallographic symmetry averaging. The structure of A/Tokyo/3/67 N2 has been refined [46] to 2.2 A as has the structures of two avian N9 subtypes [47-49]. Three influenza type structures [50] have also been determined and found to have an identical fold with 60 residues (including 16 conserved cysteine residues) being invariant. Bacterial sialidases from salmonella [51] and cholera [52] have homologous structures to influenza neuraminidase, but few of the residues are structurally invariant. 

The majority of other modifications examined at the C-4 position of Neu5Ac2en involved A -alkylation or /V-acylation of 4-amino-4-deoxy-Neu5Ac-2en or modification of the guanidino substituent of Zanamivir. IV-Methylamino (22), A -al lyl (23), and Ar,Ar-dimethylamino (24) derivatives of amine 9 were effective inhibitors of NA from influenza A and B viruses (A) = 10 5-10 7M) though they showed diminished activity against a number of bacterial sialidases.57,65... 

The roles of enteric bacterial sialidase, sialate O-acetyl esterase and glycosulfatase in the degradation of human colonic mucin. Glycoconj J 10, 72-81... 

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. 

Since the tertiary structure of bacterial sialidases is similar to that found in viral sialidases (e.g. refs. [756,792-794], for a review see ref. [246]) and residues of the Asp-box have also been found, viral and bacterial sialidases can be considered to belong to one enzyme family. The few mammalian sialidases sequenced so far, i.e. from rat muscle [774] or hamster ovary cells [775], as well as trypanosomal sialidases (see in section 9.2.3), also show many structural features in common with viral and bacterial sialidases and are correspondingly members of this family. A DNA sequence of human origin showing features similar to microbial sialidase genes is also known [795]. However,... 

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].

Evidence for a horizontal sialidase gene transfer between bacteria has been obtained by comparison of the similarities of bacterial sialidases so far sequenced [246,660,768,799]. It was found that some of the sialidases are related in accordance with the phylogenetic distances of their producers, e.g. Micromonospora viridifaciens and Actinomyces viscosus... 

Presently, several laboratories are intensively studying the gene structures of try-panosomal tra j-sialidases, in order to understand the differences between this unique enzyme and conventional sialidases. In accordance with the latter sialidases, the trans-sialidase genes of T. cruzi [768,776,814-818] also contain several Asp-boxes, first found in bacterial sialidases (see section 9.2.2 and Table 18). In addition, the T. cruzi trans-... 

Certainly, not only one function can be attributed to bacterial sialidases, since their cellular localization varies much with the species. They may be intracellular, periplasmic, membrane-bound or excreted, and they vary in their properties such as complexity, molecular mass and substrate specificity (refs. [33,244,246,660,768], and section 9.2). However, they belong to one gene family. 

Since sialic acids are involved in so many important physiological processes in animals and man (section 10), the flooding of a tissue or the whole organism with bacterial sialidases is potentially disastrous, leading to acute and chronic diseases. 

An alternative approach to the synthesis of compounds which mimic the sialosyl cation transition state intermediate (4) centres on jV-functionalised piperidines like (54) [125]. It is proposed that, upon protonation, the structure (54) would be electronically equivalent to that of resonance contributor (55) of the transition state intermediate (4) which bears the positive charge on the anomeric carbon. Whilst no information is given regarding the activity of compounds such as (54) against influenza sialidase [125], modest inhibition of bacterial sialidases is reported. It remains to be seen what effect, if any, the incorporation of some form of glycerol side-chain in this new struc-... 

J. Ferrari, R. Harris, and T. G. Warner, Cloning and expression of a soluble sialidase fi om Chinese hamster ovary cells Sequence alignment similarities to bacterial sialidases, Glycobiology, 4 (1994) 367-373. 

---