Sialidase substrate specificity

Khedri, Z., Muthana, M. M., Li, Y., Muthana, S. M., Yu, H., Cao, H., and Chen, X., Probe sialidase substrate specificity using chemo-enzymatically synthesized sialosides containing C9-modified sialic acid. Chem. Commun. 2012, 48 (27), 3357-3359. 

Only a few bacterial and viral sialidases have been purified to high purity or even to protein homogeneity."0 Complete purification of sia-lidase on a preparative scale from the culture filtrate ofC. perfringens was achieved111 by using poly(acrylamide) gel-electrophoresis as the final purification step (see Section VI,1). It is necessary that such purified sialidases be available, as the presence of proteases or other gly-cosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). 

More is known as to the pH optimum (usually, pH 4.5-5.5), the requirement for ions, the substrate specificity, and kinetic data on siali-dases, as such results, extensively reviewed,55,"<1, H7 313 can also be obtained by use of crude or partially purified enzymes. The data obtained with impure enzymes must he interpreted with care, as the presence of various sialidases, of other glycosidases modifying the substrate during incubation, or of hypothetical activators and inhibitors may lead to large errors. Correspondingly, such studies require well characterized substrates, and identification of the enzyme reaction-produets. 

Table V. The Substrate Specificity ot Sialidases, and Their Inhibition by Neu2en5Ac... 

The substrate-specificity studies described in this Subsection show striking similarities between the sialidases from various sources, but also characteristic differences with regard to the N- and O-substitu-tions of Neu, the sialic acid glvcosidic linkages, and the oligosaccharide, glycoprotein, or ganglioside nature of the substrates.55... 

F. Vandekerckhove, S. Schenkman, L. Pontes de Carvalho, S. Tomlinson, M. Kiso, M. Yoshida, A. Hasegawa, and V. Nussenzweig, Substrate specificity of the Trypanosoma cruzi trans-sialidase, Glycobiology 2 541 (1992). 

Another multifunctional bacterial enzyme that has been reported recently is Pasteurella multocida sialyltransferase (PmSTl or tPm0188Ph) (67, 68). It has four different activities, including an a2,3SiaT, a2,6SiaT, a2,3-sialidase, and trans-sialidase activities. PmSTl is a highly active sialyltransferase with broad donor and acceptor substrate specificity therefore it is a powerful tool... 

High-Throughput Substrate Specificity Studies of Sialidases Using Chemoenzymatically Synthesized pNP-Tagged Sialosides... 

Scheme 7. High-throughput substrate specificity assays for sialidases...

Due to its wide substrate specificity but very limited linkage specificity, the trans-sialidase from T. cruzi has been used for the sialylation of oligosaccharides, yielding Neu5Ac(a2-3)Gal sequences, in glycotechnological experiments [554], discussed in detail in section 6.3. 

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. 

C. Oehler, J. Kopitz, and M. Cantz, Substrate specificity and inhibitor studies of a membrane-bound ganglioside sialidase isolated from human brain tissue, J. Biol. Chem., 383 (2002) 1735-1742. 

Notes and discussion. A major drawback of enzymatic sialylation with sialyl-transferases is the strict acceptor specificity of these enzymes. This synthesis addresses this limitation by showing a widely applicable transfer of sialic acid (NeuAc) from a donor substrate of the sequence NeuAca-(2 —3)Gal-ORi to virtually any galactose acceptor (Gal-OR2). This method uses the less substrate specific glycosidase from Trypanosoma cruzi. The problem with this method is that the product is made at the expense of another sialoside, used as the donor substrate, and as NeuAc transfer is a reversible process, it is difficult to drive the equilibrium in favour of the desired sialoside. For this reason the sialoside donor substrate is regenerated in situ by an a-(2 3)-sialytransferase enzyme, thus enhancing the production of the desired product. The specificity of the sialyltransferase ensures that only the galactose byproduct, formed from the sialyl donor, is re-sialyated as the Gal-OR2 acceptor substrate is a poor substrate. Due to the broad specificity of the trans-sialidase, many a-NeuAc-(2 3)-Gal-OR sequences can be synthesised by... 

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